C1 Domains: Structure and Ligand-Binding Properties - Chemical

Review pubs.acs.org/CR

C1 Domains: Structure and Ligand-Binding Properties Joydip Das* and Ghazi M. Rahman Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, 521 Science and Research Building 2, Houston, Texas 77204, United States 8. C1 Domain Ligands as Inhibitors 9. Summary and Future Perspectives Author Information Corresponding Author Notes Biographies Abbreviations References

1. INTRODUCTION C1 domains are small zinc-binding structural units of approximately 50 amino acid residues, originally discovered as lipid-binding modules in protein kinase C (PKC) isoforms. Although PKCs were discovered in 1977,1,2 the C1 domains were first identified nine years later in 1986 while sequencing full-length PKC.3,4 Also termed as cysteine-rich or zinc finger domain, this domain however neither binds to DNA, like the conventional zinc fingers, nor possesses structure similarity. In 1997, a uniform nomenclature was adopted terming this domain as C1 domain and its multiple copies as C1A, C1B, C1C, and so on.5 The role of the C1 domain in membrane translocation and activation of PKCs has been well-studied. PKCs belong to the superfamily of serine threonine kinases that play a central role in intracellular signal transduction and regulate divergent cellular functions, such as cell growth, cell differentiation, metabolism, and apoptosis by phosphorylating target proteins.6,7 PKCs have been categorized into three different classes, conventional (α, βI, βII, γ), novel (δ, ε, θ, η), and atypical (ζ, ι/ λ). While conventional and novel PKCs are activated by diacylglycerol (DAG) or phorbol ester (Figure 1), atypical PKCs are not sensitive to these activators. Endogenous DAG acts as the second messenger, but its affinity for the C1 domain is 3 orders of magnitude less than the phorbol esters, the tumor-promoting diterpene derivatives from plants of the family Euphorbiaceae. Both conventional and novel PKCs have four major domains, termed C1 through C4. C1 and C2 are regulatory domains; C3 is an ATP binding domain, and C4 is the catalytic domain. The combined C3 and C4 are also termed the kinase domain. In addition, there is a small domain, called the pseudosubstrate domain present at the N-terminus. This domain occupies the substrate-binding domain in the inactive state. The domains are interlinked by five variable regions termed V1 through V5 (Figure 2 A,B). The C1 domain consists of a tandem repeat of highly conserved cysteine-rich subdomains (C1A and C1B) approximately 50 amino acids in length and binds with the activator DAG/phorbol ester.

CONTENTS 1. Introduction 2. Sequence of C1 Domains 3. Structure of C1 Domains 3.1. Ligand-Bound Structure of C1 Domains 3.2. Comparison of Various C1 Domain Structures 3.3. Topology of C1 Domain in the Full-Length Protein 4. Ligand Binding Properties of C1 Domains 4.1. Natural Compounds 4.1.1. DAG and Phorbol Ester 4.1.2. Prostratin and DPP 4.1.3. Gnidimacrin 4.1.4. Bryostatins 4.1.5. Indo- and Benzolactams 4.1.6. Ingenol Esters 4.1.7. Aplysiatoxin 4.1.8. Mezerein 4.1.9. Resiniferatoxin 4.1.10. Daphnoretin 4.1.11. Iridal 4.1.12. Anthracycline Derivatives 4.1.13. Calphostin C 4.1.14. Sphingosine 4.1.15. Retinoids 4.1.16. Polyphenols 4.2. Synthetic Compounds 4.2.1. DAG Lactones 4.2.2. Isophthalic Acid Derivatives 4.2.3. Bidentate Ligands 4.3. Ligand Binding Affinity of Isolated C1 Domains and Full-Length Proteins 4.4. Methods for Binding Affinity Measurements 5. Nonequivalency of C1A and C1B in Ligand Binding 6. C1 Domains and Phospholipid 7. Structural Basis of Ligand Binding

© 2014 American Chemical Society

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Received: November 30, 2012 Published: November 6, 2014 12108

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Recent review articles on C1 domains primarily highlight its role in the activation of the related signaling pathways and its importance as a target for natural and synthetic ligands.8,34−36 The present article provides a comprehensive structural analysis of various C1 domains, and their binding affinity for various natural and synthetic ligands, and it discusses structural basis of ligand-binding, and the future perspectives of research on C1 domains.

2. SEQUENCE OF C1 DOMAINS All C1 domains consist of 50 or 51 (for PKCεC1A, PKCηC1A, and DGKC1B) amino acids and have a common structural motif, HX12CX2CXnCX2CX4HX2CX7C, where C and H are cysteine and histidine, respectively; X represents other residues; and n is either 13 or 14 (for PKCεC1A, PKCηC1A, and DGKC1B).37 Across all PKC C1 domains the residues in consensus positions 1, 3, 14, 17, 23, 27, 28, 31, 34, 39, 42, and 50 are identical (Figure 4). For C1A of PKCε and η, the last seven consensus positions are 28, 29, 32, 35, 40, 43, and 51, respectively, due to one extra amino acid in their sequences. Among the identical residues, six cysteines at consensus positions 14, 17, 31, 34, 42, and 50, and two histidines at consensus positions 1 and 39 (for C1A of PKCε and η, cysteines are at positions 32, 35, 43, and 51, and histidine is at position 40) constitute two zinc coordinating sites.38 Site directed mutagenesis of residues at 1, 14, 17, 31, 34, and 42 in PKCδC1B cause disruption of the C1 structure due to lack of zinc coordinations which completely abolishes affinity for phorbol esters,37 although mutation of H-39 and C-50 still quite significantly retains the affinity for phorbol ester.39 Additionally, all C1 domains have one phenylalanine, two glycines, and one glutamine at consensus positions 3, 23, 28, and 27, respectively (for C1A of PKCε and η these consensus positions are 3, 23, 29, and 28, respectively). Glycine at position 28 is however replaced by an aspartic acid in DGKC1B. F-3, G23, G-28, and Q-27 play a crucial role in binding with activators for both conventional and novel PKC C1 domains as evident from mutational and binding studies. The F-3 is replaced by a leucine in PKDC1A and by a tryptophan in DGKC1B and DGKαC1A. Mutation of F-3 and Q-27 in PKCδC1B with glycine completely abolishes the binding of phorbol ester but retains the affinity for bryostatin 1. However, introduction of a tryptophan at Q-27 abolishes the affinity for both phorbol ester and bryostatin 1.37 Interestingly, mutation of F-3 with either an aromatic or aliphatic hydrophobic residue still retains phorbol ester binding.37 Thus, Q-27 and F-3 are important for phorbol ester binding.37,40 Comparing the sequence at positions 11−13, it is observed that all typical C1 domains have a consensus motif of P11T12X13 (where X is phenylalanine), except for the C1A of PKCε and PKCη, which have tyrosine at the homologous position. An aromatic hydrophobic residue at position 13 in the consensus C1 domain plays a favorable role in activator binding.37 In PKCδC1B, mutation of phenylalanine at position 13 to either tyrosine or lysine does not change significantly its binding affinity toward phorbol ester either in presence or absence of PS.41 Same mutation in the full-length PKCδ in the cell lysate also does not have any significant change in the phorbol ester binding affinity.37 The proline at position 11 is replaced by an arginine in atypical C1. Mutation of P-11 with glycine significantly reduces the affinity of PKCδC1B for phorbol ester.37 In comparing the sequences of C1A and C1B, all conventional and novel PKC C1As consist of additional

Figure 1. Chemical structure of DAG, sn-1, 2-dioleoylglycerol (DiC18) (top) and phorbol 12-myristate 13-acetate (TPA) (bottom).

Atypical PKCs have a single C1 domain that does not bind to DAG/phorbol ester. Instead, these C1 domains bind to ceramide and are involved in protein−protein interactions.8 In novel PKC isoforms, the C1 domain binds DAG with an affinity high enough to recruit them to the membranes. In conventional PKC isoforms, the affinity of the isolated C1 domain for DAG is lower by about 2 orders of magnitude than the C1 domains of novel PKC C1 domains, thus requiring the coordinated binding of C1 domain and a Ca2+-regulated C2 domain for membrane translocation and activation.9 In the inactive state of the enzyme the pseudosubstrate domain at the N-terminus occupies the substrate binding site at the catalytic domain thereby preventing the phosphorylation of the substrate. After assuming an active conformation by phosphorylation, the C2 and C1 domains anchor the conventional PKCs from the cytosol to the membrane where endogenous DAG binds to the C1 domain. Membrane penetration and DAG binding pull the pseudosubstrate region from the active site in the kinase domain, resulting in enzyme activation and subsequent phosphorylation of the target proteins10−12 (Figure 3). This is how binding of DAG to the C1 domain activates PKC, thus triggering a series of interlinked signaling pathways that ultimately translates the signal into a biological response. PKCs have been implicated in many disease states which include cancer,13,14 diabetes,15 ischemic heart disease,16,17 acute and chronic heart disease,18 heart failure,19 stroke,20 lung21 and kidney complications,22 various dermatological conditions including psoriasis,23 different autoimmune conditions,24,25 bipolar disorders,26 Parkinson’s disease,27,28 dementia,29 Alzheimer’s disease,30,31 and pain.32 One of the major goals of studying C1 domains is to develop potent and selective ligands to regulate PKC signaling in these disease states. There are about 60 different C1 domains known to date. C1 domains that bind and respond to DAG/phorbol ester are termed as typical, and those that do not respond to DAG are termed as atypical. A C1 domain can also be termed a PKC or non-PKC C1 domain based on the parent protein’s activity. Most of the C1 domain containing non-PKC proteins acts as lipid kinases and scaffolds, except PKD which acts as a protein kinase.33 A complete list of the cellular roles of C1 domain containing proteins is shown in Table 1. While novel and conventional PKCs, PKDs, and DGK (β, γ) contain two copies, DGKθ (a lipid kinase) contains as many as three C1 domains. The domain structure of the C1 domain containing proteins is shown in Figure 2. 12109

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Figure 2. (A) Schematic representation of DAG responsive C1 domain containing proteins. For conventional and novel PKCs, four distinct domains are C1, C2, C3 and C4 and five variable regions are V1, V2, V3, V4, and V5. PS is the pseudosubstrate domain. Other known non-PKC C1 domain containing proteins consist of TD, transmembrane domain; PH, plekstrin homology domain; SH, Src homology domain; Ras, reticular activating system; Rac, Ras-related C3 botulinum toxin substrate; Rac-GAP, Rac GTPase-activating protein domain; REM, Ras exchange motif; GEF, guanine nucleotide exchange factor; EF hands, a helix-loop-helix structural domain; GEF, guanine nucleotide exchange factor; CC, coiled-coil domain; CH, citron homology domain; PB, p21 GTPase binding domain; MHD, munc homology domain. (B) Schematic representation of non DAG responsive C1 domain containing proteins. Atypical PKCs possess three highly conserved regions (C1, C3, and C4) and five variable regions (V1, V2, V3, V4, and V5). PS is the pseudosubstrate domain and the PB1 is Phox and Bem1 domain. Other known non-PKC C1 domain containing proteins consist of PH, plekstrin homology domain; SH, Src homology domain; DAGK, Sn-1, 2-diacylglycerol kinase; Ras, reticular activating system; Rac-GAP, Rac GTPase-activating protein domain; REM, Ras exchange motif; Rho, Rhomboid, a transmembrane serine protease; RBD, Rho binding domain; NBD, nucleotide binding domain; SAM, sterile alpha motif domain; AR, acidic-rich (Ac) region; SARAH, Sav/Rassf/Hpo domain.

conserved residues including consensus A-5, Q-10, S-15, F-20, and W-22, whereas all the conventional and novel PKC C1Bs consist of other conserved residues including consensus Y-8, H16, G-18, M-36, N-37, V-38, and V-46. All typical PKC C1As have phenylalanine at consensus position 20 whereas C1B has leucine in that position. Atypical PKC C1 domains have an

arginine at that position. C1A of PKCα, PKCβ, PKCε, and PKCη has one extra cysteine, which is not present in their C1B domains. The exact role of the extra cysteine residues has not been determined. On the other hand, DGKιC1A, DGKζC1A, and RASSF1C1 have one cysteine less as compared to regular six cysteines.42 Conventional PKCs contain a consensus Y-22 12110

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Figure 3. Simplified scheme showing the activation process of Ca2+-sensitive PKC. In the inactive state the C2 domain binds to cytosolic calcium while interacting with C3−C4 domain. In the next step, the calcium-bound C2 domain anchored to the phosphatidylserine (PS) of the membrane, which pulls out the C1 domains away from the catalytic domain. Then the C1A/C1B domain first binds to diacylglycerol (DAG) which eventually pulls out the pseudosubstrate motif from the substrate binding pocket and thereby activates the PKC. Binding affinity of DAG for C1A and C1B varies from isotype to isotype. For PKCα, DAG has higher affinity for C1A than C1B.

PKCδC1B complexed with phorbol 13-acetate (PDB ID: 1PTR).45 Subsequently, the solution structures of isolated Raf1C1 (PDB ID: 1FAQ), PKCθC1A (PDB ID: 2ENN), PKCθC1B (PDB ID: 2ENZ), PKCδC1A (PDB ID: 2YUU), PKCγC1A (PDB ID: 2E73), and PKCγC1B (PDB ID: 1TBN) were determined. Recently reported high resolution crystal structures of PKCδC1B (PDB ID: 3UEJ) and PKCθC1B (PDB ID: 4FKD) provide insights into the orientations of various residues in these domains.40,46 The crystal structures of C1containing full-length β2-chimaerin, 47 Vav1, 48−50 and PKCβII51,52 provided insights into the topology of C1 domains in these proteins and their mechanisms of activation. Interestingly, not a single C1A domain has been crystallized, and this subdomain is not resolved in the full-length protein structure of PKCβII. As for the activator-bound structures, only one such structure has been reported to date.45 A list of all the known C1 domain structures and the corresponding PDB ID are shown in Table 1.

whereas all other PKC C1Bs contain a tryptophan in that position. The tryptophan versus tyrosine switch at this position controls the membrane affinity and localization of conventional and novel PKCs. C1B domains of novel PKCs translocate and bind to the DAG-containing membrane with higher affinity due to the presence of W-22. Conversely, the C1B domains of conventional PKCs with Y-22 require coordinated binding of C1 and the calcium-regulated C2 domain for the membrane translocation.9 Furthermore, while Y-22 at this switch position causes the cytosolic localization of the C1B domain under an unstimulated condition, W-22 localizes it to the Golgi.9 This study provides a molecular explanation for the difference between the conventional and novel PKCs for their DAGinduced activation and cellular localization. Among the conventional PKC C1A domains, 44 out of 50 residues are common (showing 88% sequence similarity), whereas among C1B domains, only 76% (38 out of 50 residues are identical) are common. Similarly, among the atypical C1 domains the sequence similarity is very high (72%) with 36 residues out of 50 being identical. Despite high sequence similarities among different C1 domains, some are DAGsensitive, and some are not, which implies the importance of specific residues of C1 domains for ligand and membrane binding.

3.1. Ligand-Bound Structure of C1 Domains

The crystal structure of PKCδC1B complexed with phorbol 13acetate (PDB ID: 1PTR) revealed the presence of two sets of three cysteines and one histidine that form two Zn2+ ion coordination sites at the end of the β sheets (Figure 5A). Two long β-sheets (20% β-sheet) formed a V-shaped activator binding groove, and there was a short α-helix (4% α-helix) at the C-terminal end. Ligand association does not result in a significant conformational change, as binding only leads to a 0.4 Å displacement of the β-sheets and rmsd of 0.3 Å of the main chain. Phorbol 13-acetate forms five hydrogen bonds with the protein residues in the activator binding groove; two hydrogen bonds with Gly-253 (C4 hydroxyl designated as O4 of the

3. STRUCTURE OF C1 DOMAINS PKCαC1B was the first C1 domain whose structure was determined in 1994.43,44 The structure, determined by NMR analysis, revealed that the overall topology of this domain is a globular fold with two separate Zn2+-binding sites. Comprehensive information on C1 domain−ligand interactions was gained in the following year from the crystal structure of 12111

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Table 1. Known C1 Domains and Their Function and Structurea protein family and subtypes

no. of C1 domain

function

Typical (Binds to DAG/Phorbol Esters) PKC(α, βI/ βII, γ) 2

protein kinase

PKC(δ, θ, ε, η)

2

protein kinase

PKD(1, 2, 3) DGK(β, γ)b Chimaerin(α1, α2, β1, β2)

2 2 1

protein kinase lipid kinase Rac-GAP

known C1 structure and PDB codes αC1B: 2ELI PKCβII: 3PFQ γC1A: 2E73 γC1B: 1TBO/1TBN δC1A: 2YUU δC1B: 1PTQ/1PTR δC1B: 3UEJ, 3UGD, 3UGI, 3UGL θC1A: 2ENN θC1B: 2ENZ, 4FKD

Chimaerin α1C1: 3CXL Chimaerin βII C1: 1XA6 Munc-13-1: 1Y8F

Munc-13(1, 2, 3) 1 RasGRP(1, 4) 1 RasGRP-3 1 MRCK(α, β, γ) 1 Atypical (Does Not Bind to DAG/Phorbol Esters) aPKC(ζ, ι/λ) 1 RasGRP-2 1 DGK(α, δ, η, ε, ζ, ι) 2 DGKθ 3 Vav(1, 2, 3) 1

protein kinase Ras/Rap-GEF lipid kinase lipid kinase Rac/Rho-GEF

Raf-1 KSR(1,2) Dbl Lfc ROCK(1, 2) Citron-N Citron-K AKAP13 ARHGAP29 ARHGEF2 GMIP HMHA1 MYO9A MYO9B PDZD8 RACGAP1 RASSF1 RASSF5 STAC STAC2 STAC3 TENC1

protein kinase scaffold Rho-GEF protein kinase Rho effector protein kinase Rho guanyl-nucleotide exchange factor RhoGTPase activator Rho guanine nucleotide exchange factor Rho GTPase-activator GTPase activator GTPase activator Rho GTPase activator intracellular signal transduction Rac GTPase effector potential tumor suppressor, death receptor-dependent apoptosis potential tumor suppressor, lymphocyte adhesion neuron-specific signal transduction intracellular signal transduction neuron-specific signal transduction cell motility, proliferation and phosphatase activity

1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1

scaffold Ras-GEF Ras/Rap-GEF protein kinase

DGKδ C1B: 1R79 VAV1 C1 human: 3KY9 VAV1 C1 mouse: 2VRW Raf-1 C1: 1FAQ/1FAR KSR-1 C1: 1KBE/1KBF ROCK2 C1: 2ROW

RASSF5 C1: 1RFH/2FNF

STAC3 C1: 2DB6

Protein sequence and function information can be found in UniprotKB database. bOnly the C1A of DGKβ and γ are typical, whereas their corresponding C1B domains are atypical.

a

hydrogen bonds that are replaced by bonds with oxygen atoms from positions C3, C4, and C20 in the phorbol ring. Hydrophobic interactions occur with residues 239−242 (9− 12 in δC1B) and 251−254 (21−24 in δC1B) in the wall of the groove.45 The crystal structure of PKCδC1B also provides insights into the mechanisms of membrane association, a key step in the PKC activation process. A cluster of hydrophobic residues form the top third of the wall of the binding pocket, while the middle region is composed mainly of positively charged residues. Binding of ligands caps the hydrophilic pocket, thereby creating

phorbol ester forms hydrogen bond with the oxygen atom of the backbone carbonyl whereas C3 carbonyl designated as O3 forms a hydrogen bond with the nitrogen atom of the backbone amide), two hydrogen bonds with Thr-242 (C20 hydroxyl designated as O20 forms two hydrogen bonds with the nitrogen and oxygen atoms of the backbone amide), and one hydrogen bond with Leu-251 (O20 forms a single hydrogen bond with the oxygen atom of the backbone carbonyl).45 Gly253, Thr-242, and Leu-251 of full-length mouse PKCδ correspond to G-23, T-12, and L-21 of δC1B. In the absence of phorbol 13-acetate, water molecules fill the groove and form 12112

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Figure 4. Sequence alignment of typical and atypical C1 domains. All the sequences belong to human except the PKCλ, which is from Xenopus tropicalis. The sequences are aligned within a frame of 1−50, although some C1 domains have more than 50 residues. The zinc binding residues are shown in pink, highly conserved residues are shaded in gray, the activator binding residues PTF/PTY at positions 11−13 are shaded in yellow, and the W/Y at position 22 is in cyan. Conserved F of typical C1A and L of typical C1B at position 20 are shaded in green. The arginine residues (R) that line the rim of the homologous activator binding in atypical C1 domains are shown in red. Additional cysteine residues (C) in the C1A domains of PKCα, β, ε, and η are shown in blue.

in the alcohol/anesthetic binding site in proteins and confirmed that both van der Waals interaction and hydrogen bonding are essential for binding.

a continuous hydrophobic surface that facilitates insertion of the C1 domain into membranes. Unliganded C1 domain does not satisfy necessary hydrogen bonding potential between the membrane and the groove. The positively charged residues interact with negatively charged phospholipids at the membrane, thus explaining the preference for acidic phospholipids as cofactors for ligand binding. The bottom half of the C1 domain contains the Zn2+-coordinating sites. The zinc ions bind to cysteine and histidine residues to maintain proper folding.45 Very recently the structures of PKCδC1B complexed with an alcohol (cyclopropylmethanol) (PDB ID: 3UGL) and an ether (methoxymethylcycloprane) (PDB ID: 3UGI) at a 1.36 Å resolution have been reported.46 In these structures the cyclopropane rings of both agents displace a single water molecule in a surface pocket adjacent to the phorbol binding site, making van der Waals contacts with the backbone and/or side chains of residues Asn-237 to Ser-240. Two water molecules anchored in a hydrogen-bonded chain between Thr-242 and Lys-260 impart elasticity to one side of the binding pocket. The cyclopropane ring takes part in π-acceptor hydrogen bonds with Met-239’s amide (Figure 5B). A hydrogen bond between the cyclopropanemethanol’s hydroxyl group and the oxygen atom of the hydroxyl of Tyr-236 (Y-6 in mouse δC1B) is crucial. A similar study with the crystals of Tyr236-Phe mutant (PDB ID: 3UEY) did not show any binding, confirming the role of hydrogen bond interactions in alcohol binding. These structures defined the roles of water molecules

3.2. Comparison of Various C1 Domain Structures

The overlaid structures of various C1 domains are shown in Figures 6 and 7. A comparison of the C1A and C1B structures of the PKC family reveals the overall similarity of the structures. In all these structures there are two rims, the upper portions of which are associated with the membrane. The ligand binds into the pocket formed by the two rims. Measurement and comparison of the pocket volume and surface area of the C1B domain of conventional α, βII, and γ indicate that the volume/surface area for γ is much smaller than the C1B domain of the other two.42 In contrast, the volume/surface area of the C1B domains of novel isotypes δ and θ are comparable,42 and the structures are almost superimposable with one another (Figure 6). Their C1A and C1B domains, however, are not completely superimposable. The volume/surface area of the δC1A pocket is larger than the δC1B domain, whereas the opposite is true for θC1A and θC1B. The volume/surface area of γC1A and γC1B are comparable. In contrast to PKC C1, the structures of the non-PKC C1 domains show large differences among themselves. In particular, the structures of KSRC1 and RAF1C1 are different from that of the PKC C1 structures in that the groove-forming rims are dissimilar (Figure 7). The question of whether these 12113

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Figure 5. Ligand-bound structure of C1 domain. (A) Crystal structure of PKCδC1B bound with phorbol 13-acetate (PDB ID: 1PTR).45 PKCδC1B possesses a V-shaped activator binding groove. Phorbol 13-acetate forms five hydrogen bonds in the activator binding groove: two bonds with Gly253 (O4 formed hydrogen bond with oxygen atom of backbone carbonyl whereas O3 with the nitrogen atom of backbone amine group), two bonds with Thr-242 (O20 formed two hydrogen bonds with nitrogen and oxygen atom of backbone amine and carbonyl groups respectively), and one bond with Leu-251 (O20 formed single hydrogen bond with oxygen atom of backbone carbonyl group). Each zinc atom (shown as pink balls) coordinates with three cysteines and one histidine. (B) The crystal structure of PKCδC1B complexed with cyclopropanemethanol (CPM) (PDB ID: 3UGL).46 The hydroxyl group of cyclopropanemethanol (CPM; cyan carbons) binds to the protein in a pocket created by Tyr-236, Asn-237, Tyr238, Met-239, and Ser-240 and forms hydrogen bond with the Tyr-236 (2.8 Å).

Figure 6. Comparison of the (A) C1B domains of PKCδ (PDB ID: 1PTQ) and PKCθ (PDB ID: 2ENZ), (B) C1A domains of PKCδ (PDB ID: 2YUU), PKCθ (PDB ID: 2ENN), PKCγ (PDB ID: 2E73) and C1B domain of PKCδ (PDB ID: 1PTQ), and (C) C1B domains of PKCα (PDB ID: 2ELI), PKCβII (PDB ID: 3PFQ), and PKCγ (PDB ID: 1TBN).

differences in structure affect the ligand binding properties is discussed in section 7. A comparison of the crystal structures of δC1B and θC1B reveals remarkable similarity in their overall structures. The backbones of these two structures are almost superimposable with each other. While the orientations for most of the residues

of both the structures are almost identical, the orientation of the single tryptophan is quite different in these two structures (Figure 8). In a representation of the structure where the activator-binding loops are positioned toward the membrane, Trp-253 (W-22 in θC1B) of PKCθ is oriented toward the membrane while the homologous Trp-252 (W-22 in δC1B) of 12114

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Figure 7. Comparison of the structures of DAG insensitive non-PKC C1 domains with PKCδC1B (blue, PDB ID: 1PTQ). (A) Overlaid structures of KSR1 C1 (PDB ID: 1KBE), Munc13.1 C1 (PDB ID: 1Y8F), RAF1 C1 (PDB ID: 1FAQ) and (B) overlaid structures of RASF5 C1 (PDB ID: 1FRH), ROCK2 C1 (PDB ID: 2ROW), and STAC3 C1 (PDB ID: 2DB6).

Figure 8. Orientation of consensus tryptophan in PKCδC1B, PKCθC1B, and Munc13.1 C1. (A) Trp-252 of PKCδC1B (PDB ID: 1PTQ), Trp-253 of PKCθC1B (PDB ID: 4FKD), and Trp-588 of Munc13.1C1 (PDB ID: 1Y8F) at consensus position 22 in the overlaid structure are shown in blue, green, and red, respectively. Trp-252 is oriented away from the membrane, whereas Trp-253 is oriented toward the membrane. Trp-588 of Munc13.1C1 is projected inside the activator binding pocket. (B) Possible π/π stack interaction between Trp-252 and His-269 and cation−π interaction between Trp-252 and Lys-271 in PKCδC1B and (C) Possible cation−π interaction between His-270 and Arg-272 in PKCθC1B.

with His-270 at a distance of 3.81 Å.40 The same “away from the membrane” orientation of the tryptophan in both unliganded and ligand-bound δC1B and the minimal conformational difference (rms = 0.3 Å)45 between these structures indicate that this orientation of tryptophan is preferred one at least in the in vitro environment. It is however not known if the orientation of the tryptophan is flipped toward the membrane at the membrane interface in an in vivo environment. The orientation of the homologous tryptophan in the δC1A, θC1A, and γC1A is toward the membrane as seen in θC1B, and the orientation of tryptophan in β2 chimerin is away from the membrane as seen in δC1B. Interestingly, the homologous Trp588 in Munc13.1 occludes the DAG binding site,53 thereby causing lower DAG binding affinity.

PKCδ is oriented downward away from the membrane.40 Because of this particular orientation of Trp-253 in PKCθ, its interactions with nearby residues are distinctly different from that of PKCδ. In PKCδ, the indole ring of homologous Trp-252 is oriented in parallel to the imidazole ring of His-269 at a distance of 4.13 Å, possibly involving π-stacking interactions (Figure 8B). In PKCθ, however, similar interactions between the Trp-253 and His-270 are absent because these two rings are far apart (>10 Å) from one another (Figure 8). The ε-NH2 of Lys-271 in PKCδ, which is protonated at physiological pH, is located at a distance of 5.12 Å from the imidazole ring of His269 and at a distance of 5.49 Å from the indole ring of Trp-252. It is therefore possible that His-269 is involved in cation−π interactions with both rings. In PKCθ, however, this lysine is replaced by Arg-272, which is involved in cation−π interactions 12115

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Figure 9. Topology of C1 domains in the full-length proteins and its interaction with other domains. (A) PKCβII (PDB ID: 3PFQ), (B) β2chimerin (PDB ID: 1XA6), and (C) VAV1 (PDB ID: 3KY9).

β2-chimaerin to phorbol-ester-induced translocation relative to PKCs. The C1 domain in β2-chimaerin interacts with endoplasmic reticulum/Golgi proteins p23/Tmp21.56,57 p23/ Tmp21 acts as a C1 anchoring protein, which upon binding mediates the perinuclear translocation of β2-chimaerin.58 Deletion of the C1 domain in β2-chimaerin completely abolishes this translocation.57 Additionally, β2-chimaerin fails to distribute in the perinuclear compartment in cells deficient of p23/Tmp21, indicating that this protein is crucial for the compartmentalization of β2-chimaerin58 critical for its role in proliferation and migration of smooth muscle cells. Studies also suggest that Glu-227 (E-15 in C1) and Leu-248 (L-36 in C1) in the C1 domain are responsible for p23/Tmp21 anchoring. A single point mutation in Glu-227 or Leu-248 slightly increases the perinuclear translocation, but a double mutation including both of these positions completely abolishes the perinuclear translocation.58 In Vav1,49 a guanine nucleotide exchange factor for Rho family GTPase, C1 domain is involved in the intramolecular network of contacts between the PH-C1 unit and the DH domain and stabilizes the conformation of a critical α-helix within the DH domain thereby facilitating the displacement of the guanine nucleotide from GTPase (Figure 9C). While the overall structural features of this C1 domain are very similar to that of the δC1B, the topology of this domain in the full-length protein is somewhat different from PKCβII and β2-chimaerin. It is, however, difficult to compare the relative topology and dynamics of these proteins while in action based on only a crystal structure. The structures of the isolated C1 domains discussed here are determined either in solution by NMR or in crystals by X-ray crystallography. Although there could be slight variation in the structures depending on the method of its determination, this point has been ignored while comparing different C1 domain structures. One of the differences between the NMR and X-ray structures is that solution NMR data represent an average of semirandomly oriented molecules in solution whereas diffraction data represent an average over molecules arranged in a periodic crystal lattice.59,60

In summary, there are subtle differences among the C1 domain structures although the overall structures are similar. 3.3. Topology of C1 Domain in the Full-Length Protein

PKCβII, β2-chimaerin, and Vav.1 are the only C1 domaincontaining proteins whose full-length structures are known. The 4 Å structure of PKCβII (PDB ID: 3PFQ), published in 2011,51 is one of the most awaited structures in the PKC research field. Although the electron density for the pseudosubstrate domain, the C1A domain, and the V3 linker region were not visible in the structure, it has provided insights into how the C1 domain interacts with other domains to initiate the activation process. In the crystal structure of PKCβII (Figure 9A) the C1B domain is located close to the kinase domain rather than the C2 domain. One of the most important features of this structure is that the C1B domain interacts intramolecularly with the residues 619−633 of C-terminal tail, and Phe-629 of the conserved NFD motif of the catalytic domain.51 The 616−633 region of the C tail is one that alternatively plays an important role in stabilizing the active site of the kinase domain. In the absence of DAG, PKCβII undergoes at least two modes of autoinhibition. First, the involvement of the pseudosubstrate region blocks the substrate binding cleft of the catalytic region, and second, the DAG binding site in the C1B domain clamps to the NFD motif of the C tail.54,55 DAG engagement of the C1B domain upon membrane translocation unclamps the NFD motif from the C1B domain and, thus, makes the kinase fully activated for its function. Although not visible in the structure, it has been postulated that the C1A domain is aqueously exposed and first binds to the membrane DAG upon initial activation of the C2 domain by calcium. This disrupts the interactions between the N-terminal pseudosubstrate region and the C-terminal region followed by disruption of the tethering between C1B and the NFD motif.51 This event initiates the C1B insertion in the lipid bilayers through docking of membrane DAG, thereby activating PKCβII. The C1 domain in β2-chimaerin is positioned between the SH2 and RacGAP domains and interacts extensively with the N-terminus of SH2 and RacGAP domains and the interdomain linkers (Figure 9B). Like the C1B domain of PKCβII, the C1 domain of β2-chimaerin is buried, contacting the catalytic RacGAP and other regions. This makes the domain inaccessible to DAG/phorbol ester when the protein is folded in an inactive conformation, and this may explain the reduced sensitivity of

4. LIGAND BINDING PROPERTIES OF C1 DOMAINS Binding of endogenous DAG to the C1 domain followed by membrane insertion are the two important events in the PKC activation process. Upon activation, PKC isozymes translocate 12116

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Table 2. Binding Affinity of Phorbol-Ester- and DAG-Based C1 Activators Determined by Radioactive PDBu Binding Aassay, Isothermal Calorimetry (ITC), and Surface Plasmon Resonance (SPR) Methodsa C1 domains PKCαC1A PKCαC1B PKCα PKCβC1A PKCβC1B PKCβI/βII PKCγC1A PKCγC1B PKCγ PKCδC1A PKCδC1B PKCδ PKCεC1A PKCεC1B PKCε PKCθC1A PKCθC1B PKCθ PKCηC1A PKCηC1B PKCη

PDBu,b ([3H]PDBu binding assay), Kd, nM76,77,215123,212 >300077/1.1215/0.4 ± 0.11201 46.777/5.3215/7.4215/3.4 ± 0.12201 60215/0.15215/3078 >300078 1.378 3.9215/9.5215/0.1478 65.877/1.5123 16.977/1.2215 18215/0.37215/2.4215/6.878 30076/51.9215/0.34 ± 0.08213/2.04 ± 0.24201 1.0 ± 0.176/0.53215/0.33 ± 0.05201 4.0215/0.7178 5.676 0.8176/1.5215 18.0215/0.6378 900215/>200215 3.4215/0.72215

PDBu (ITCc), Kd, nM76,77 21.4 ± 5.0

77

TPAd (SPRe), Kd, nM76,77

DiC8f (ITCc), Kd, nM76,77

DiC18g (SPRe), Kd, nM76,77,214

121.0 ± 26.277 6.3 ± 1.577

10.2 ± 3.177 0.008232

3.6 ± 0.577 2700 ± 710.077 36 ± 0.9228

51.9 ± 17.077 9.3 ± 3.877

1.9 ± 0.377 1.7 ± 0.577

10.4 ± 5.077 8.9 ± 3.177

5.5 ± 1.077 5.2 ± 0.677

85 ± 2776

58 ± 26

360 ± 2176 40 ± 476

74 ± 1076 23 ± 976

14 ± 376 5.2 ± 176

38 ± 576 110 ± 4076

30 ± 276 7800 ± 180076 6.6 ± 1.0209 11 ± 176 52 ± 1076 2.0 ± 0.376 1900 ± 100123 26 ± 3123 20 ± 4214

76

4.3215 0.91215/0.45215 0.5878

a For SPR, ligand is vesicle incorporated. For ITC, there were no vesicles. bPhorbol 12,13-dibutyrate. cIsothermal calorimetry. dPhorbol 12-myristate 13-acetate. eSurface plasmon resonance. fsn-1, 2-Dioctanoyl glycerol. gsn-1, 2-Dioleoyl glycerol.

to new distinct intracellular sites. The translocation sites for different PKC isotypes are different, and different ligands can affect the localization and its kinetics.61−64 For example, while PKCα translocates to the plasma membrane or to adhesion foci,65 PKCδ translocates to the plasma membrane, nuclear membrane, and mitochondrial membrane.64,66,67 In the following section, several natural and synthetic compounds are discussed for their binding to C1 domain, the membrane translocation property, and isoform selectivity. Binding affinity of a ligand for a C1 domain or a full-length protein is represented by a dissociation constant (Kd) or an inhibitory dissociation constant (Ki). Binding affinity values for phorbol esters and DAGs for isolated C1 domains and the fulllength PKCs are shown in Table 2. Similarly, the binding data for non-PKC C1 domains and the corresponding full-length protein are presented in Table 3. Table 4 shows the binding affinity of various natural and synthetic compounds with PKC and other non-PKC related C1 domains. A representative chemical structure for each class of ligands is shown to aid in the discussion.

Table 3. Binding Affinity of Phorbol-Ester- and DAG-Based Activators with Purified Non-PKC C1 Domains Determined by Radioactive PDBu Binding Assay, Isothermal Calorimetry, and Surface Plasmon Resonance

4.1. Natural Compounds

4.1.1. DAG and Phorbol Ester. sn-1,2-Diacylglycerol (DAG; Figure 1) is one of the central second messengers in the cell that binds to the typical C1 domains. The tetracyclic diterpenoids, phorbol esters, obtained from plant sources mimic the actions of DAG68,69 and show at least 3 orders of magnitude higher binding affinity compared to DAGs,70,71 making them the most widely used molecules in the study of PKC and related proteins. Nevertheless, their potential as drug candidates is limited because of their toxicity and tumorpromoting properties. Moreover, synthesis of these compounds poses a significant challenge because of the presence of several asymmetric centers in these molecules. Both phorbol ester and DAG cause a dramatic increase in the affinity of PKC for

purified non-PKC C1 domains

PDBu,a Kd, nM

OAG,b Ki, nM

Unc13.1C181 Munc13.1C180 α1-chimerinC182 α1-chimerin173 β2-ChimerinC183 β2-Chimerin56 Ras-GRP1C1107 Ras-GRP1107 Ras-GRP3C183 Ras-GRP384 MRCKαC199 MRCKβC199 PKD1C1B85 PKD2C1B85 PKD3C1B85 PKD3C1A85 hDGKβC1A86 rDGKγC1A86 rDGKγ86

1.31 ± 0.20 5.0 2.0 0.17 4.49 ± 0.09 1.9 ± 0.02 0.58 ± 0.08 0.49 ± 0.09 1.52 ± 0.37 1.53 ± 0.33 10.3 ± 2.0 17 ± 1.2 0.37 ± 0.03 0.32 ± 0.09 0.28 ± 0.04 1.62 ± 0.03 14.6 3.6 4.4

378 ± 20

a Phorbol 12,13-dibutyrate. Dioctanoyl glycerol.

b

DiC8,c Ki, nM

144 ± 26

2820 ± 260 6960 ± 300 159 ± 28 154 ± 11 168 ± 29 44.1 ± 8.5 557

1-Oleoyl-2-acetyl-sn-glycerol.

c

sn-1,2-

membranes, serving as “molecular glue” to recruit PKC to membranes.72 Phorbol ester binds to the C1 domain in a similar qualitative manner to DAG,73 but they penetrate the membrane differently70 and the affinity of the membranecontaining phorbol ester is 2 orders of magnitude higher than that of membrane-containing DAG.9 However, the major 12117

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Table 4. Binding Affinitya (Ki, nM) of Non-DAG/PE Ligands to PKC and Non-PKC C1 Domains and the Full-Length Proteins

a

Multiple values indicate values reported by different groups. bAngelate. cBenzoate. dApparent dissociation constant, kd′. eValues are in μM.

esters translocate the protein to the nuclear membrane. The Cho group,74 on the other hand, observed that short chain hydrophilic DAG analogs translocate PKCδ to the plasma membrane. These observations strongly suggest that the mechanisms of DAG- and phorbol-ester-induced PKC activation are different. In the DAG-induced activation of PKCα, its membrane binding is initiated by the Ca2+-dependent adsorption of the C2 domain to the anionic membrane surface. Phosphatidylserine (PS) binding to the cationic β-groove of C2 domain may also be important for localization and activation.75 Binding of a PS molecule in the membrane to the Ca2+-binding loops of the C2 domain causes a local conformational change that results in the unleashing of the C1A domain, which then penetrates the membrane and binds to DAG. This movement of the C1A domain not only enhances the membrane-binding energy but also removes the pseudosubstrate from the active site, leading to enzyme activation (Figure 3).76,77 In contrast to DAG, the phorbol-ester-mediated membrane translocation and activation mechanism for PKCα is somewhat different given the opposite binding affinity of its C1A and C1B domains for DAG and phorbol ester.77,78 In the phorbol-ester-induced activation,45,70,79 it has been suggested that phorbol ester induces an irreversible insertion of the protein into the membrane and produces kinase activity, which is calcium independent. The kinase activity is however dependent on the lipophilicity of the phorbol esters and the PS concentrations.64,70 In addition to

differences in this process are that membrane recruitment and activation initiated by DAG are short-lived because DAG is rapidly metabolized, in contrast to phorbol esters that are not readily metabolized and result in constitutive activation of PKC.6 Most of the phorbol esters are tumor promoters and show antinutritional and toxic effects even at very low concentrations. Structurally, they are more complex than the DAGs in having eight asymmetric centers as compared to only one in DAG. DAGs with two carbonyls and one hydroxyl group have the potential for hydrogen bond interactions with the C1 domain residues. On the other hand, phorbol esters have three carbonyl and three hydroxyl groups for such interactions. While the four rings in phorbol esters render rigidity in the molecule and the possibility of van der Waals interactions with the protein, the long chains in DAG are flexible. As a consequence, the immobilization of the glycerol upon binding is entropically unfavorable. All of these factors contribute to the higher binding affinity of phorbol esters compared to DAGs70,71 for PKC C1 domains. C1A and C1B subdomains of PKCs and other C1 domain containing proteins show variable binding affinities for phorbol ester and DAG, details of which are discussed in section 5. This differential binding of DAG and phorbol ester is also reflected in the translocation properties of the full-length protein. For example, Wang et al.64 observed that, in the case of PKCδ, the more hydrophobic phorbol esters tend to translocate the protein to the plasma membrane, whereas hydrophilic phorbol 12118

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this, Bazzi and Nelsestuen described the ability of PKC to form another membrane-associated state, which is calcium-dependent and reversible upon calcium chelation.79 The large number of hydrophobic residues in the phorbol-ester-binding site favors the membrane insertion, and binding of phorbol ester in the activator-binding groove satisfies the hydrogen bonds, which makes the membrane insertion energetically favorable.45 A systematic study for each PKC isoform for their DAG- and phorbol-ester-induced activation mechanism is required to fully understand the molecular basis of these differential activation mechanisms. Phorbol esters also bind to the non-PKC C1 domain, such as Munc13.1, 80 Unc13.1, 81 chimerins, 82,83 Ras-GRP3, 83,84 PKD3,85 and DGK86 with affinity in the high nanomolar range (Table 3). Similar to the PKC C1 domains, the binding affinity of DAG is less as compared to the phorbol esters. 4.1.2. Prostratin and DPP. Prostratin (12-deoxyphorbol13-acetate) and DPP (12-deoxy phorbol-13-benzoate) (Figure 10) are non-tumor-promoting phorbol ester analogs. Studies on

Figure 12. Chemical structure of bryostatin 1.

low concentrations (0.1−1 nM) it downregulated PKCδ to a similar extent to that of TPA, but at high concentrations (100− 1000 nM) it protected PKCδ from TPA-induced downregulation. The dose−response curve of PKCδ and bryostatin 1 is U shaped with the maximal effect occurring at 1 nM and less activation at >10 nM.94 This phenomenon of less activation at higher concentration is also observed in an in vivo system where excessive bryostatin 1 treatment blocks learning altogether rather than enhancing memory.97 While both phorbol ester and bryostatin 1 show strong binding affinity for PKCs, several pharmacological differences have been noted among the PKC isoforms in terms of their translocation and downregulation properties as compared with phorbol esters.94,96,98−100 Unlike TPA, which is a tumor promoter, bryostatin 1 shows antiproliferative effects and often suppresses or reverses the effects of TPA.95,101,102 In CHO-K1 cells, TPA translocated PKCδ to the plasma membrane, whereas bryostatin 1 mainly translocated it to the nuclear membrane.67 While TPA-induced PKC translocation occurs by binding to the C1B subdomain, both C1A and C1B contribute to the bryostatin 1-induced PKC translocation.90,94 Studying with the chimeric constructs between PKCα and PKCδ, Lorenzo et al. proposed that the C1A and the catalytic domains are involved in protection from downregulation of PKCδ induced by bryostatin 1 or byrostatin 1 plus TPA.94,98 Bryostatin 1 competes with the phorbol-ester-binding site of PKCα with very high affinity (1.35 nM),103 although its selectivity for different C1 domains is very poor (Table 4).104 It also binds to C1 domains of PKD,105,106 RasGRP,100,107,108 and β-chimaerin109 with high nanomolar affinities (Table 4). The Wender group110,111 and the Keck group112−114 synthesized a variety of bryostatin analogs called “bryologs” and studied their structure−activity relationship and isoform selectivity. The affinity of some of these analogs for PKCs varies greatly within the range 0.25 nM to >10 μM.103 A recently developed bryostatin 1 analog called “picolog” shows higher potency in the growth inhibition of MYC-induced lymphoma, which is dependent on PKC activation.115 4.1.5. Indo- and Benzolactams. Indolactams are indole alkaloids of teleocidin family. They were first isolated from Streptomyces mediocidicus116 and showed tumor promoting activities117,118 or tumor inhibiting activities119−121 depending on the cell lines. These compounds contain a core structure of indolactam V (Figure 13), and the mechanism of their tumor promoting activity is believed to be by activating PKC. Benzolactams are analogs in which the indole ring is replaced by a benzene ring. Extensive structure−activity relationship of indo- and benzolactam derivatives has been studied for identifying isotype selective PKC activators.122−124 In the indolactam series, introduction of a lipophilic chain at C6 or C7 increased

Figure 10. Chemical structure of prostratin.

the PKCδ reveal that, unlike the phorbol ester TPA, these compounds are not selective to the C1B domain. They display different binding and pharmacology as compared to phorbol esters.87−89 The ED50 of prostratin and DPP for the membrane translocation of PKCδ are found to be 4100 nM and 188 nM, respectively.90 4.1.3. Gnidimacrin. This is a daphnane-type diterpene (Figure 11) that displays potent antitumor activity at concentrations 0.1−1 nM. It inhibits PDBu binding to K562 cells and acts as a PKC activator at a concentration of ∼3 nM.91,92

Figure 11. Chemical structure of gnidimacrin.

4.1.4. Bryostatins. These are highly oxygenated macrocyclic lactones produced by a bacterial symbiont of the bryozoan Bugula neritina93 and show very unique pharmacology.94 The chemical structure of bryostatin is extremely complex in having 11 asymmetric centers. Out of about 20 naturally occurring bryostatins, bryostatin 1 (Figure 12) is extensively studied for its binding and pharmacological effects on PKCs. Although bryostatin 1 downregulates most PKC isozymes similar to TPA, it regulates PKCδ differently.95,96 At 12119

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keratosis. The Blumberg group137 has thoroughly characterized this compound for their interaction with different PKC isoforms. In the in vitro binding assays ingenol-3-angelate did not show any selectivity for its binding to various PKC isoforms, all showing Ki values in the range 0.1−0.4 nM, although in the kinase assay it caused higher activation to PKCδ than PKCα. Compared to the phorbol ester TPA, it shows differences in membrane insertion and translocation properties,137,141,142 which have been attributed to differences in their lipophilicity.137 The lower lipophilicity of ingenol-3-angelate caused lower stabilization of the PKC-ligand-membrane complex resulting in partial agonism property of this compound. This study also provided a strategy to develop PKC inhibitors based on C1 domains as discussed in section 8. Another compound of this series, ingenol-3-benzoate, has been studied extensively for its binding to different C1 domains, and moderate differences in the binding affinity between the C1A and C1B subdomains of α, δ, and ε were observed.123 4.1.7. Aplysiatoxin. This is a cyanotoxin derived from marine mollusk Stylocheilus longicauda143 and contains spiroketal moiety in its chemical structure (Figure 15). It

Figure 13. Chemical structure of indolactam-V (left) and 9-decylbenzolactam-V8 (right).

potency,125,126 whereas the presence of a hydrophilic group in the chain reduced PKC activity.121 A long chain at C7 show slightly improved selectivity for the C1B domain of novel PKCs. Substitution of the proton of the NH in the indole ring with an n-hexyl group increases the selectivity for C1B domains of novel PKCs over conventional PKCs.127,128 One of the indolactam derivatives, (−)-octyl indolactam V, in which the octyl group is at C7, identified differential roles of the C1A and C1B in the membrane translocation of PKCδ and PKCα in NIH 3T3 cells. Whereas C1B plays a predominant role for PKCδ translocation, both C1A and C1B play equivalent roles for PKCα.90,129 For the benzolactam derivatives, introduction of a decyl chain at C7 shows an 8-fold selectivity for C1B domains of novel PKCs over the conventional PKCα.130 In contrast, a decyl chain at C8 improves about 4-fold selectivity for conventional PKCα and PKCβ as compared to novel PKCδ and PKCε.121 Using synthetic peptides of C1 domains, the Irie group131,132 however observed slightly higher affinity for novel PKCs than the conventional PKCs, with the decyl group either at C8 or C9. Introduction of the decyl group at position 9 (Figure 13) however improved its PKC activation property as compared to the benzolactam V8 without this group.120 The binding affinity of 9-decyl benzolactam V8124 with different PKCs is shown in Table 4. A compound in the benzolactam series, 8-octyl-benzolactam-V9, translocated PKCε and PKCη from cytoplasm to plasma membrane at 1 μM, whereas other PKCs did not respond to this compound even at 10 μM.128 Several indo- and benzolactam compounds also bind to the C1 domain of RasGRP with nanomolar affinity.133 4.1.6. Ingenol Esters. Ingenol esters belong to the ingenane family of macrocyclic diterpenes extracted from E. peplus134 or E. antiquorum.135,136 In general, these compounds show cytotoxic, proinflammatory, and cell-differentiating activity. Structurally, these compounds are similar to phorbol esters although they have some differences; for example, the sixmembered ring of phorbol is replaced by a seven-membered ring in ingenols, and the number and position of hydroxy, carbonyl, and carboxy ester groups of the seven-membered ring of phorbol are different in ingenols.137−139 One of the compounds of this group, ingenol-3-angelate (Figure 14), shows promise for the treatment of skin cancer140 and has been approved by FDA and EMA in 2012 for the treatment of actinic

Figure 15. Chemical structure of aplysiatoxin.

activates PKC by binding to its C1 domain, and it is a tumor promoter.144,145 With PKCδ, it binds with a Ki of 3 nM and shows higher affinity for the C1B (Ki = 0.41 nM) than the C1A domain (Ki = 12 nM).146 Binding studies with the C1A and C1B domains of other PKC subtypes did not show better selectivity.147 Some of the recently developed146,148 analogs of aplysiatoxin are also called “aplog” which showed nontumor promoting activities like bryostatin 1 and translocated PKCδ to the nuclear membrane in CHO-K1 cells. 4.1.8. Mezerein. This is a highly toxic nonphorbol diterpene (Figure 16) isolated from the seeds of D. mezereum149−151 and acts as a second state tumor promotor. It activates PKC152 and binds to the C1 domains with varying affinities. With full-length PKCs it binds with nanomolar

Figure 14. Chemical structure of ingenol 3-angelate.

Figure 16. Chemical structure of mezerein (NSC 239072). 12120

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rabbit platelets in a concentration-dependent manner with an IC50 of 45.2 μM,160 indicating its binding to the C1 domain. 4.1.11. Iridal. These triterpinoids are obtained from Iris spp.162 Two iridals, NSC 631939 and NSC 631941 (Figure 19),

affinity, and conventional PKCs showed higher affinity than the novel PKCs.153 Mezerein binds to PKCδ with a Ki of 11.7 nM, with its C1B having higher affinity (Ki = 10.3 nM) than the C1A (Ki = 1378 nM).154 It has also been shown that the tumor promoting property of mezerein does not depend on the preferential binding to the C1B domain.90 In fact both C1A and C1B have been shown to play equivalent role in the membrane translocation of either PKCα or PKCδ when expressed in NIH 3T3 cells.90,129 Between PKCα and β2-chimaerin, mezerein shows about 17 times higher affinity toward PKCα (Ki = 0.27 nM). Thymeleatoxin, a mezerein derivative, shows 60 times higher affinity for PKCα (Ki = 0.29 nM) than β2-chimaerin.56 In yeast sample expressing different PKC isoforms, mezerein and its derivative daphnetoxin show differential activation of PKCα, PKCβ1, PKCδ, and PKCζ.155 4.1.9. Resiniferatoxin. This compound is isolated from Euphorbia resinifera156 and contains a core resiniferonol structure that has similarity with phorbol esters (Figure 17).

Figure 19. Chemical structure of iridals, NSC 631939 (top) and NSC 631941 (bottom).

activate PKCs and bind to RasGRP3 and PKCα with Ki values between 16 and 84 nM with modest selectivity.163 Relative to PDBu, these iridals show 15- and 6-fold selectivity for RasGRP3.164 These compounds induce translocation of RasGRP3 from the cytoplasm to fibrillar structures and the nuclear membrane like the phorbol ester, TPA, although with 30 times less potency than the latter. 4.1.12. Anthracycline Derivatives. Anthracyclines are natural compounds derived from Streptomyces bacterium.165 Widely used as anticancer agents, they exert their anticancer activity by several mechanisms such as intercalation into DNA, inhibition of topoisomerase II during DNA replication, and generation of iron-mediated free oxygen radicals that damage DNA, protein, or cell membrane. One of the anthracycline derivatives AD198 (Figure 20) binds the C1B domain of PKCδ,

Figure 17. Chemical structure of resiniferatoxin.

Resiniferatoxin contains an ester group at the C20 position. Whereas resiniferatoxin binds to PKCα with a Ki value of 1.49 μM, the affinity of its hydrolysis product, resiniferonol is about 40 times higher than the former.157 This again highlights the importance of the C20 hydroxyl of phorbol ester and related compounds in forming hydrogen bonds with the protein residues in the C1 domain groove. At 1−100 nM resiniferatoxin activates PKCα, but at 1 μM it activates other PKC isoforms as well.158 While in one study resiniferatoxin competed with phorbol ester in PKCβ, in another study it did not displace phorbol ester in the rat cytosol and membrane fraction of rat dorsal root ganglia, although it activated PKC.159 Binding of resiniferonol with different C1 domains indicates that it has higher affinity for the PKCαC1A than PKCαC1B.123 4.1.10. Daphnoretin. This compound (Figure 18) is isolated from the Chinese medicinal herb Wikstroemia indica

Figure 20. Chemical structure of anthracycline AD198.

full-length PKCδ, and β2-chimaerin with Ki values of 2.7, 2.5, and 1.5 μM, respectively.166,167 This compound also causes translocation of PKCα and PKCδ from cytosol to the membrane in 32D.3 cells.168 4.1.13. Calphostin C. A polycyclic aromatic perylenequinone, calphostin C (Figure 21) isolated from the fungus Cladosporium cladpsporioides,169 inhibits PKCs. The proposed mechanism is that calphostin C binds to the C1 domain and then inactivates the enzyme irreversibly through the production of free radical and destruction of the enzyme.170 The inhibitory mechanism is suggested to be light dependent.171 Further, a role of the C2 domain in this process has also been implicated.169,171,172 Calphostin C competes with PDBu in

Figure 18. Chemical structure of daphnoretin.

C.A. Mey.160,161 Daphnoretin activates platelet cytosolic PKC in a concentration dependent manner with an EC50 of 12.4 μM. It induces translocation of PKC from the cytosol to the membrane and downregulates intracellular PKC levels in the Hep3B cells.161 It also increases the membrane-associated PKC activity. Further, it inhibited [3H]PDBu binding to washed 12121

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Figure 24. Chemical structure of curcumin (left) and resveratrol (right).

Figure 21. Chemical structure of calphostin C.

indicate the inhibitory effect of curcumin on PKC activity,186,189,190 more recent investigations reveal that curcumin can either activate or inhibit PKC depending on the presence and absence of calcium and the nature of the lipid mixture in the assay system.188,191 In mouse skin, curcumin reduces TPAinduced membrane translocation of PKCα, PKCβ, PKCγ, PKCε, and PKCη.190 Studies with the C1B domain indicated that curcumin and its long chain derivatives bind to the C1B domain in a manner similar to that of phorbol ester, and some degree of selectivity can be achieved among δ, ε, and θ with the curcumin derivatives.192,193 Resveratrol’s primary source is grapes, and it is known to have many effects on multiple targets, thereby regulating various disease states.194 Both in vivo and in vitro studies provided evidence for the inhibition of TPA-induced PKCα activity by resveratrol.183−185 In vitro studies, however, indicated that other subtypes such as δ, ε, and ζ are unaffected.185 A recent study indicated that some resveratrol derivatives activated PKCα, but not PKCε at 100 μM concentrations.195,196

PKCα173 and binds to PKCδC1B with a Ki of 4.3 μM.81 Several derivatives have been synthesized, and the chemical modifications improved the IC50 values up to 0.4 μM.174,175 Besides PKCs, calphostin C also inhibits diacylglycerol kinase with an IC50 in the micromolar range.176 It binds to the C1 domains of n-chimerins Unc-13 with ED50 of 8.9 μM,81 and RasGRP with ED50 of 1.47 μM.81,107,173 4.1.14. Sphingosine. Like calphostin C, sphingosine (Figure 22) has also been shown to inhibit PKCs by interacting with the regulatory domain.177 It binds with the C1 domain of PKCα and n-chimaerin and competes with PDBu with an ED50 of 3 μM.173

Figure 22. Chemical structure of sphingosine.

4.1.15. Retinoids. Retinoids are vitamin A derivatives (Figure 23) involved in general growth and important

4.2. Synthetic Compounds

4.2.1. DAG Lactones. The Blumberg and Marquez groups at the National Cancer Institute undertook a massive research effort, synthesizing hundreds of cyclic DAG analogs to achieve higher affinity and selectivity for various C1 domains. This is based on the principle of reducing the entropy associated with the flexible glycerol moiety. Cyclization of the glycerol moiety to generate rigid DAG-lactone structure provides favorable entropy for binding while still retaining a simple chemical scaffold. DAG-lactones are activators of PKC and show antitumor activities in several cell lines.197 Several of these lactones bind to the C1 domains with high affinity comparable to phorbol esters but have much higher selectivity than the phorbol esters in terms of both binding affinities and membrane translocations. 198−201 One such compound, 130C037 (Figure 25), not only showed high affinity but also showed high selectivity.201 It binds to the isolated PKCδC1B with an affinity (Ki) of 1.8 nM, whereas its affinity for either

Figure 23. Chemical structure of retinoids, retinol (top) and retinoic acid (bottom).

physiological roles, such as vision, cell differentiation, etc. Retinoids, particularly retinoic acid, activate PKCδ.178,179 Apparent dissociation constants for the C1 domain of PKCα and retinoids were determined by fluorescence spectroscopy, which showed that retinoids bind to the PKCαC1A with nanomolar affinity.180,181 The PKCαC1B domain, on the other hand, binds poorly with the retinoids. Retinol, 14-hydroxyretro-retinol, anhydroretinol, and retinoic acid show binding with similarly high affinities to a given C1 domain (e.g., PKCαC1A, PKCδC1B, PKCμC1A, and C1B domains), with similar low affinities (e.g., PKCδC1A) or failure to bind (e.g., PKCαC1B) to other domains. Retinol also binds to the C1 domains of c-Raf and Vav.180 Retinoic acid has also been reported to bind with the C2 domain of PKCα.182 4.1.16. Polyphenols. Curcumin and resveratrol are the two dietary polyphenols (Figure 24) that have been shown to regulate PKC activity directly or indirectly.183−188 Curcumin’s natural source is the plant curcuma longas. While some studies

Figure 25. Chemical structure of DAG lactones, 130C037 (left) and DAG-indolactone-12r-E (right). 12122

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4.3. Ligand Binding Affinity of Isolated C1 Domains and Full-Length Proteins

PKCδC1A or for C1A or C1B of PKCα was in the micromolar range. In contrast, the affinity of PDBu for these four C1 domains was similar within a factor of 10. Compound 130C037 binds to full-length Ras activators RasGRP1 and RasGRP3 with high affinity (Ki, ∼4 nM). It binds with 8-fold weaker affinity to PKCε and with 90-fold weaker affinity to PKCα. Similar selectivity was observed in intact cells for membrane association or translocation as well as for activation on ERK phosphorylation downstream of Ras. Whereas 130C037 caused rapid translocation of RasGRP3, limited slow translocation of PKCε and no translocation of PKCα were observed in LNCaP cells.201 When the lactone ring in 130C037 was substituted with a heterocyclic ring via the α-arylidene moiety and an additional lipophilic-branched acyl chain as in compound DAGindolactone-12r-E (Figure 25), selectivity for RasGRP3 over PKCα was found to be 165-fold.199 4.2.2. Isophthalic Acid Derivatives. Recently developed isophthalate derivatives, such as HMI-1a3 (Figure 26), bind to

Generally, the affinity of a ligand for an isolated domain and the full-length protein could vary due to the existence of domain− domain interactions in the full-length protein resulting in structural changes at the ligand binding site. However, a comparison of the binding affinity of the isolated C1 domain and the corresponding full-length protein for a particular ligand reveals remarkably similar values (Tables 2−4). In general, for proteins that have two copies of C1 domains, the value of the high affinity copy is comparable with the value for the fulllength protein. For example, the Kd values for PDBu binding to PKCδC1B and PKCδ are 1 and 0.71 nM, respectively. Similarly, the Ki for DiC18 binding to PKCδC1A and PKCδ are 30 and 6.6 nM, respectively (Table 2). In the case of RasGRP1, the C1 domain and the full-length protein bind to PDBu with Kd of 0.58 and 0.49 nM, respectively (Table 3). This is also observed for many other ligands, such as aplysiatoxin, mezerin, bryostatin-1, indolactam-V, and 9-decyl benzolactam V8, that bind with PKCδ, as shown in Table 4. This similar nanomolar binding affinity of both the isolated domains and the full-length protein for a particular ligand indicates that the isolated C1 domain is conserved structurally and preserves the elements required for protein−ligand interactions, which are merely affected by the domain−domain interactions in the fulllength protein. 4.4. Methods for Binding Affinity Measurements

Figure 26. Chemical structure of the isophthalate derivative HMI-1a3.

The binding affinity (Kd or Ki) for various ligands and various C1 domains have been determined using various methods. [3H]PDBu binding and its displacement by other ligands, pioneered by the Blumberg laboratory,208 have been widely used for measuring the binding affinity. In this method, [3H]PDBu is usually incubated with the protein in the presence and absence of other ligands at various concentrations. After incubation, the protein is precipitated using polyethylene glycol (PEG). Kd and Ki are calculated from the bound and free [3H]PDBu. Although this method provides relatively accurate binding affinity measurements, high cost and limited availability of [3H]PDBu have somewhat restricted the use of this method. Among the nonradioactive methods, surface plasmon resonance (SPR), isothermal calorimetry (ITC), fluorescence spectroscopy, and ultracentrifugation have also been used although they are not without limitations. In SPR, change in refractive index near a sensor surface is measured during the protein−ligand interactions. The ligand is incorporated into lipid vesicles and is immobilized on a chip that forms the sensor surface of the flow cell. The protein solutions are injected in the aqueous solution through the flow cell.76,77,209 Kd for the protein and the vesicle-bound ligand is calculated from the change in the refractive indexes. ITC is a thermodynamic technique by which heat generated or absorbed during a bimolecular interaction is measured. This has been used for both the phorbol esters and DAGs in the absence of any lipids.76,77,209 This technique requires higher amounts of protein as compared to other techniques, and insolubility of the ligands can pose problems. Ultracentrifugation technique has been used to determine binding between protein and vesicle loaded ligands. Vesiclebound and unbound proteins are separated, and the bound protein is quantified using gel electrophoresis with different staining or activity measurements.72,207 Fluorescence resonance energy transfer (FRET) technique using a fluorescent phorbol

PKCα and PKCδ with submicromolar affinities and without much selectivity. These derivatives possess the same pharmacophore as the phorbol esters and bind to the C1B domain.202 Some of these derivatives promote neurite overgrowth in SH-SY5Y cells203 and induce cell elongation and cell cycle arrest in HeLa cells.204 Successful binding of these compounds led to the design of alkyl cinnamates as PKC ligands, although no activation properties were reported for these compounds.205 4.2.3. Bidentate Ligands. Bidentate ligands of variable linker length have been designed with the objective of targeting both C1A and C1B domains of PKC.206,207 This includes the dimers of benzolactams or naphthylpyrrolidones, which show binding affinity at the nanomolar range, but do not show selectivity among PKCs. The binding affinity for the benzolactam dimers depends on the spacer’s length and lipophilicity. With a spacer of 14 oligomethylene, the benzolactam dimer (Figure 27) shows 100 times higher affinity

Figure 27. Chemical structure of benzolactam dimer 2f.

than the momoner for PKCα.206 Studies with dimeric phorbol esters showed 3−4-fold higher binding with PKCβII as compared to the monomer, and it was found that both C1A and C1B contribute to the binding and activity.207 12123

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ester sapintoxin-D has also been applied to study the binding of phorbol ester and PKCα.210,211

The differential role of C1A and C1B in the translocation properties of the full-length protein is of particular interest. In human neuroblastoma cells, the isolated C1B domain of PKCα, PKCδ, PKCε, PKCη, and PKCθ are targeted to the Golgi complex whereas C1A localizes throughout the cell.218 The Met-267 (M-36 in θC1B) in PKCθ, which is conserved among the C1B domains, is critical for translocation. This residue is absent in C1A. Mutation of Met-267 in PKCθ and the homologous residue in PKCε to glycine completely abolishes Golgi translocation.218 In the case of PKD1, however, it is the C1A domain that is sufficient for its binding to the Golgi.219,220 In the NIH 3T3 cells, P11G mutation in δC1B does not disrupt overall folding of the C1 domain but causes about a 20-fold reduction in the membrane translocation compared to the wild type, whereas, for the homologous mutation in C1A, the reduction is minimal.221 Similar studies in PKCα did not detect much difference in translocation properties.129

5. NONEQUIVALENCY OF C1A AND C1B IN LIGAND BINDING One of the key features of the ligand binding properties of C1 domains is the differential binding affinity of C1A and C1B for a particular ligand (Tables 2−4). In general, the binding affinity of phorbol ester for the C1B subdomain of conventional PKC (α, β, γ) is higher than C1A. This is more prominent for α and β and less prominent for the γ isoform.74,76−78,212,213 On the other hand, from the limited binding data available on DAG binding, αC1A shows higher affinity for DiC18 than αC1B. For PKCγ, DiC18 binds to both C1A and C1B with almost equal affinity. Among the novel PKCs, C1B of δ and ε show higher affinity for PDBu than C1A. Additionally, more like PKCα, C1A of δ and ε show higher affinity for DiC18 than C1B. In the case of PKCθ, however, the affinity for both phorbol ester and DAG is higher for C1B than C1A,214 making it the only C1B of any PKC that shows higher affinity for DAG. For the binding of phorbol ester with various C1A and C1B domains, several discrepancies have been observed in the relative affinities of these domains (Table 2). These discrepancies could be due to the variation of different components in the binding assay. For example, in some of the binding assays the C1 domains were synthesized using a peptide synthesizer whereas in some assays the C1 domains were expressed and purified from E.coli. In some cases, the C1 domains were GST-bound, and in other cases they were not. Moreover, for synthetic C1 domains, Kd values at 4 and 30 °C were different.215 Several other ligands such as aplysiatoxin, mezerin, DAG lactone 130C037, indolactam-V, 9-decylbenzolactam V8, ingenol ester, resiniferonol, and retinol show higher binding affinity (Ki) for αC1A than αC1B. In PKCδ, however, these ligands bind to C1B with higher affinity than C1A. Like PKCδ, in PKCη some of these ligands bind to C1B with higher affinity than C1A (Table 4). The C1A and C1B domains in PKD1 have differential binding affinities for phorbol esters both in vivo and in vitro, with the C1B domain being primarily responsible for phorbol ester binding.85,216,217 For PKD3, however, contrasting results were reported by the same group.85,217 Most recent study from the same group indicated that C1B has higher affinity for phorbol ester than C1A, and C1A has higher affinity for a soluble DAG analog (DiC8) than C1B.85 The differential binding of C1A and C1B defines their roles in the membrane translocation and activation process of a particular PKC isoform. In DAG-induced activation process, C1A plays a predominant role for PKCα and PKCδ for their C1A has much higher affinity than C1B.74,77 In the phorbolester-mediated activation for PKCδ, however, Pu et al. showed that C1B plays a major role in the activation process of PKCδ.154 This indicates that a phorbol-ester- and DAGinduced mechanism of PKC activation could be different. For PKCγ, both C1A and C1B showed comparable binding affinity for DAG, and both participate in DAG induced activation.77 PKCε is similar to PKCγ in that both C1A and C1B play roles in the DAG induced activation.76 On the other hand, for PKCθ, the C1B domain plays a predominant role for its higher affinity to DAG.214

6. C1 DOMAINS AND PHOSPHOLIPID The hallmark of PKC activation in cells is its translocation and anchoring to the plasma membrane through the C1 domain.6 The activity of all PKC isoforms is regulated by phosphatidylserine (PS), an anionic phospholipid found exclusively on the cytoplasmic leaflet of membranes comprising approximately 15 mol % of the total lipid.222 In the absence of DAG, PKC displays little selectivity for PS compared with other monovalent anionic lipids. However, the presence of DAG causes PKC to bind PS-containing surfaces with an order of magnitude higher affinity than membranes composed of other anionic lipids. Similarly, in the absence of lipid the binding affinity of the C1B domain for phorbol ester is reduced 5-fold compared to that in the presence of lipid. This stimulation by PS is stereospecific for sn-1,2-phosphatidyl-L-serine. It had been suggested that this requirement arose both from unique membrane-structuring properties of PS in optimizing the membrane surface for PKC binding and from molecular determinants on PKC recognizing the shape of the PS molecule.223 The affinity of C1 domain of PKCδ for phorbol ester and related compounds was found to be 80 times higher in the presence of PS than in its absence. Similar increase in the affinity was also observed for the full-length PKCδ purified from insect cells.224 Kinetic studies with the C1B domain of PKCβ showed that the presence of 30 mol % anionic lipids enhanced its rate of association to the phorbol-ester-containing membrane by 1 order of magnitude. Another 2-fold enhancement was observed upon specific interaction with PS.225 This study also revealed that the affinity of the C1 domain for phorbol-ester-containing membrane is 2 orders of magnitude higher than that for diacylglycerol-containing membrane. The authors concluded that while membrane association of the C1 domain is highly sensitive to anionic lipid and relatively insensitive to the C1 ligand, membrane retention of the C1 domain is relatively insensitive to anionic lipid and highly dependent on C1 ligand.225 PS sensitivity is different for different C1 domains, which is in part governed by the electrostatic interactions between the positively charged residues in the corresponding C1 domain and the negatively charged PS. The C1 domain of PKCα, β, and δ exhibits strong preference for the PS compared to other lipids whereas the C1 domain of PKCγ and ε does not show such preferences.74,76,226−228 In vitro activity and membrane binding assays of PKCδ mutants showed that Glu-177 (E-19 in 12124

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mouse δC1A) in the conformationally restricted C1A domain plays a key role in kinase activation and membrane anchoring, which is dependent on PS.74 Consensus hydrophobic residues Leu-250 and Leu-254 (L-20 and L-24 in δC1B) in the binding cleft of δC1B critically influence the binding of DAG or phorbol ester, which is also dependent on PS concentration.41 Mutation of both of these leucines with aspartic acid completely abolishes the binding of the activators either in the presence or absence of PS. Studying variety of mutation in these positions, the authors concluded that the interaction between C1 domain and PS occurred through the hydrophobic residues present near the rim of the binding cleft.41 This study also showed that binding of branched DAG was less sensitive to PS but more sensitive to mutation of Trp-252 (W-22 in δC1B) with either a tyrosine of lysine.41 Similar studies on the θC1B also detected differential phospholipid sensitivities of different residues for PDBu binding.40 In conventional PKC, the Ca2+ binding in C2 domain many fold increases its affinity for the PS rich anionic membrane. Presumably, Ca2+ acts as a bridge between the C2 domain and the anionic PS in membrane. This has been confirmed by the elucidation of the PKCαC2 domain crystal structure in presence of PS consisting of the abridged Ca2+ ions.75 In novel PKCs, the Ca2+ nonresponsive C2 domains require the coordination of the C1 domain for the membrane anchoring and possibly modulate the spatial distribution of novel PKCs through C1−C2 and other protein−protein interactions and binding to the receptor for activated C-kinase (RACKs).229 The C2 pocket lining in the conventional PKCs aligns several aspartic acid residues that coordinate 2−3 Ca2+ ions possibly through opposite charge interaction230 whereas in novel C2 domain these key residues are missing, which lacks the ability for Ca2+ binding.231 In PKCα, PS plays an important role in disrupting the intramolecular tethering between C1A and C2 domains (electrostatic interactions between Asp-55 and Arg-252, and between Arg-42 and Glu-282), which stabilizes the closed conformation of PKCα in its inactive state.76,227 Single point mutation in any of the above four residues increases PKCα’s ability for membrane anchoring and reduces PS dependency.232 A further mutational study of the cationic residue Arg-77 in PKCαC1A into alanine demonstrates that it interacts nonspecifically with anionic phospholipids prior to the penetration of the hydrophobic residues in the membrane.232 However, an inaccessible C1A is not common to all conventional PKCs; for example, C1A and C1B of PKCγ are conformationally flexible to penetrate the membrane and bind to DAG without the aid of PS.77 Like the typical PKC C1 domains, the C1 domain of MRCKα and MRCKβ also show PS dependence for their binding to phorbol esters.99

but one is DAG/phorbol-ester-sensitive, and the other is not. For example, whereas the C1 domain of chimerin α1 and ROCK2 have similar pocket volume, the former binds to phorbol ester with 0.2 nM affinity, and the latter does not bind to DAG/phorbol ester at all. PKCδC1B and PKCθC1A have similar binding site volume, although the former binds to phorbol ester with 0.58 nM affinity, and the latter shows lower affinity with >200 nM. PKCαC1B and Munc13.1 C1 show similar affinity for PDBu although the pocket volume of the former is double the size of the latter. Similar observations were also made with the indolactam and benzolactam compounds.42 This means that the binding site geometry may not be the only determinant for binding of a ligand for a particular isoform. What are the key determinants for the responsiveness of a C1 domain toward phorbol ester/DAG? The current understanding is that PKC C1 domains anchor to the membranes as a ternary system including the C1 domain, the activator, and the anionic phospholipids present in the membrane, which bind to the positively charged residues present in the rim of the upper third of the activator binding cleft.74,76,226−228 Alteration of any component in this ternary system therefore could affect the ligand binding affinity. For example, in the atypical PKCζ, several arginine residues lining the activator binding pocket contribute significantly to its unresponsiveness toward the activator.233 It has been suggested that these arginine residues reduce access of ligands to the binding cleft and change the electrostatic profile of the C1 domain surface without altering the basic structure of the binding cleft. Mutation into arginines of the first four corresponding residues in the PKCδC1B domain completely abolishes its high binding affinity for PDBu in vitro, and with only marginal remaining activity for TPA in vivo.233 Both in vitro and in vivo studies have demonstrated that the loss of affinity to activators is cumulative with the sequential introduction of the arginine residues along the rim of activator binding cavity. In silico modeling revealed that these arginine residues reduce access of ligands to the binding cleft due to the change of electrostatic potential of the C1 domain surface toward positive although the basic structure of the binding cleft is still maintained. Finally, mutation of the four arginine residues of the atypical PKC C1 domains to the corresponding residues in the PKCδC1B domain made them DAG/phorbol-ester-responsive.233 In the atypical Vav1C1, the presence of E-9, E-10, T-11, T24, and Y-26 along the rim of the binding cleft reduces the overall lipophilicity of the rim, impairing membrane association, and thereby preventing the formation of the ternary complex.234 In Munc13.1 occlusion of the Trp-587 (W-22 in C1) in the activator binding pocket reduces its affinity for the activators.53 A sequence comparison of all DAG-binding PKC C1 domains with the activator binding residues of PKCδC1B (PDB ID: 1PTQ)45 reveals a general activator binding motif X8X9X10P11T12F13 for residues 8−13, and X20X21X22G23X24 for residues 20−24 and Q27 (Q28 for C1A of PKCε and PKCη), where X8 is an aliphatic or aromatic hydrophobic residue), X9 is a nonconsensus residue, X10 is a polar uncharged glutamine/ serine or a hydrophobic valine, X20 is an aromatic or aliphatic hydrophobic residue, X21 is an aliphatic hydrophobic residue, X22 is either tryptophan or tyrosine, and X24 is a hydrophobic residue. The inability of RAF1 C1 and KSR C1 to bind phorbol ester or DAG can be explained by the absence of some of these consensus residues in their sequences. Both of these C1

7. STRUCTURAL BASIS OF LIGAND BINDING The above discussion on the structural features of the C1 domain and their ligand binding affinities for diverse ligands raises the pertinent question: whether structure influences the binding affinity, and if so, to what extent? A recent modeling study on the structural determinants for DAG/phorbol ester binding to the C1 domains revealed that the value of volume/surface area of the ligand binding pocket had poor correlation with the value of ligand binding affinity.42 The binding pocket volume/surface area of a typical C1 domain could be lower, higher, or similar to the atypical one, 12125

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domains consist of two β-sheets and an α-helix, but four residues at the consensus position 22−25 in the activatorbinding region are different from that of typical C1 domains. Furthermore, the Q-27 has been replaced by N (RAF1 C1) and F (KSR C1), respectively, which abolishes the activator binding affinity.34 This structural motif discussed above is based on just one activator-bound structure. Availability of more activatorbound C1 structures would help validating this ligand binding motif. To summarize, the responsiveness of a C1 domain toward DAG/phorbol ester largely depends on the residues lining the activator binding site, pharmacophore of the ligand, phosphatidylserine sensitivity of the membrane binding residues, and the overall structure and orientation of the residues. Needless to say, identification of the determinants for a C1 domain’s responsiveness is an active area of current research.

According to the crystal structure of the phorbol-13-acetatebound PKCδC1B, phorbol ester binds to the hydrophilic cleft of the C1 domain forming hydrogen bonds (Figure 5A), and caps the hydrophobic surface of the protein. With the continuous hydrophic surface, the protein−ligand complex then inserts into membranes following the stabilization of the activated protein−ligand−membrane complex. Molecules that bind to the C1 domain, but not necessarily stabilize the interaction with the membrane, could only partially activate PKC. This concept has been tested by several groups by using ligand with a chain with variable lipophilicity. In one such study, Bertolini et al.240 designed several molecules in which phorbol ester was substituted with polar functional groups in the chain, and these compounds inhibited PKC in millimolar concentrations.240 The Shibasaki group241 developed a compound in which the C12 position of phorbol ester was modified with a tetra(ethylene glycol) moiety. This compound inhibited PKCα in contrast to the activation showed by TPA. The same group242,243 also modified the phorbol ester scaffold with a perfluorinated alkyl group and a polyether hydrophilic chain that showed significant inhibition of PKCα as compared to PKCδ. Following the same principle, phorbol and ingenol, having no ester group bind to the C1 domain and inhibit PKC, while their esters activate PKC. Phorbol esters with higher hydrophobicity activated/translocated PKC more than that of the less hydrophobic one64,67,244 and also influence cellular response.245 The other consequence of altering the liphophilicity of the ligand is to drive the kinase to a new location where it does not find its natural substrate, showing altered biological response. For example, while 12-deoxyphorbol 13-tetradecanoate translocated PKCδ first to the plasma membrane, then to internal membranes and to the nuclear membrane, 12-deoxyphorbol 13phenylacetate translocated PKCδ predominantly to the nuclear membrane and little to the plasma membrane.67 Because the localization of PKC will dictate to which substrates it will phosphorylate, these different patterns of localization should correlate with different patterns of response. This could be the reason why 12-deoxyphorbol 13-tetradecanoate is a tumor promoter and 12-deoxyphorbol 13-phenylacetate is an inhibitor of tumor promotion. In contrast to the mechanism described above, PKC inhibition by calphostin C follows a different mechanism that involves light.170−172 To find out which type of biological response is associated with a particular chemical modification, a library of molecules can be synthesized on the basis of a particular chemical scaffold246 and tested for different biological activities in a high throughput manner. This proof of concept was successfully tested for the DAG-lactones which contained a signature “chemical zip” which can be decoded only by one particular biological response among many.247

8. C1 DOMAIN LIGANDS AS INHIBITORS This section deals with compounds that either inhibit the basal activity of PKC or activate PKC but antagonize the end point of the effect of phorbol esters. Since PKC is a kinase, these PKC modulators are termed as inhibitors in a broad sense. The majority of the ligands described in the preceding sections are PKC activators. In view of the regulating disease states, however, inhibition of a specific PKC isoform is more advantageous than activation. Moreover, in several disease states two different isoforms can have contrasting roles, indicating that one can either activate one isoform or inhibit the other isoform for eliciting same response. The yin and yang of PKCδ and PKCε in tumorigenesis may serve as a typical example of this effect. These two PKC isoforms generally show opposite effects in the regulation of apoptosis, survival, and proliferation.14 While PKCδ is pro-apoptotic and negatively regulates proliferation, PKCε is pro-mutagenic and pro-survival. Other examples of such contrasting responses include PKCδ and PKCε in myocardial infractions,235 PKCγ and PKCε in regulating alcohol actions on GABAA receptor,236 and PKCα and PKCδ in the proliferation and apoptosis in glioma cells.237 PKC inhibitors that target the kinase domain, occupy the ATP-binding region, bind with higher affinity than ATP, and block ATP from binding to the site. These molecules could have similar or bigger structure than that of ATP and may interact with additional sites as compared to ATP. However, these inhibitors have not met much success in terms of selectivity because of the existence of more than 500 kinases in the human genome. The total number of C1 domains is far less than the total number of kinases, and despite high sequence similarity among the C1 domains, several C1-based modulators have been developed. Among them, bryostatin 194 and aplysiatoxin derivatives146,238 are under clinical trial. Like phorbol ester, bryostatin 1 binds to and activates PKC, but paradoxically antagonizes many phorbol ester responses.239 A recently synthesized nontumor promoting aplysiatoxin derivative is shown to behave like brysostatin 1 in inhibiting Epstein− Barr virus early stage antigen (EBV-EA) induced by phorbol ester TPA.146 The major issue for the C1-based modulator design is that, unlike the kinase domain for which rational design of the inhibitor is possible, design of C1-targeted inhibitors are semirational because of the involvement of the lipid membrane, which is difficult to model. Because of the association of the C1-ligand complex to the membrane, it has been found that the lipophilicity of the ligands is an important determinant in the activation and localization of PKCs.

9. SUMMARY AND FUTURE PERSPECTIVES The objective of the present article is to compile the structural and ligand binding features of C1 domains and to point out the caveats in the current knowledge so that readers can get a comprehensive understanding of the subject. From the extensive literature data compiled in this article, the following highlights emerge: (1) There are subtle differences in the volume and surface area of the ligand-binding pocket, despite overall similarity in structures. (2) Topology of the C1 domain in the full-length protein is different for different proteins. (3) 12126

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Biographies

C1A and C1B domains have differences in their ligand binding properties. (4) Phorbol-ester- and DAG-induced PKC activation mechanisms are different. (5) The C1 domain can accommodate structurally diverse ligands derived from natural and synthetic sources, and the selectivity requires further improvements. (6) Ligand binding affinity depends on the ligand, binding site residues, structure of domains, and lipid composition. Although a wealth of knowledge has been accumulated since its discovery in the mid-1980s, further studies are required to exploit the full potential of C1 domains as drug targets. The complex pharmacology of C1 domain-containing proteins has made drug discovery efforts extremely challenging. For example, in the case of PKCs the added complexities are that one particular isoform may be involved in different diseases, and several isoforms may be involved in one particular disease state. Again, for a particular disease, two PKC isoforms may show opposing effects. Further complexity arises because in some cases C1 containing PKCs also phosphorylate other C1 containing proteins, such as PKD, RasGRP, and some DAG kinases. While these complications could result in several pharmaceutical companies turning their focus away from C1 domain-based drug discovery, recent FDA approval of ingenol 3-angelate (Picato or PEP005 or ingenol mebutate) provides the proof of principle toward the feasibility of C1 domain based drug discovery. With the ultimate goal of deciphering the atomic mechanism of how ligand−protein−lipid interactions are translated into specific biological responses, future research on C1 domains is expected to evolve around determining the structures of typical C1 domains with various ligands (of which a ligand-bound fulllength PKC would be quite fascinating); determining the role of C1 domains in lesser studied proteins; specifically designing molecules based on either C1A or C1B; identifying determinants in a C1 domain to be either DAG responsive or nonresponsive; and exploring structurally simple natural compound scaffolds to develop activator/inhibitor, among others. The facts that the majority of PKCs are implicated in various types of cancers and that 74% of all marketed cancer drugs are based on natural products,248 a more aggressive search for a natural compound to achieve C1 domain selectivity is highly warranted.

Joydip Das is currently an Associate Professor at the Department of Pharmacological and Pharmaceutical Sciences, University of Houston. He received his Ph.D. from the Indian Institute of Technology, Bombay, on biomolecular interactions in a retinal-binding protein, bacteriorhodopsin. During his post doctoral years at the Medical University of South Carolina and at the Massachusetts Institute of Technology, he extended these studies on visual protein, rhodopsin. At the Massachusetts General Hospital he studied molecular mechanism of anesthesia. His current research goal is to identify presynaptic targets of alcohol in the brain with a long term goal of developing drugs for alcohol addiction.

Ghazi M. Rahman is currently a postdoctoral research fellow in the Department of Experimental Therapeutics at the MD Anderson Cancer Center, Houston, Texas. He received his Masters in pharmacy from the Faculty of Pharmacy, University of Dhaka, Bangladesh. Later, he served as a quality control officer in SK&F Pharmaceuticals, which is now a part of GSK, and also worked as an Assistant Professor in Department of Pharmacy, Northern University Dhaka, Bangladesh. In 2012, he received his Ph.D. from the College of Pharmacy, University of Houston, on structure and function of protein kinase C θ. His current research focuses on the mechanism of the resistance in nonsmall cell lung cancer for cisplatin-type chemotherapeutic agents.

ABBREVIATIONS PKC protein kinase C DAG diacylglycerol PDBu phorbol 12,13-dibutyrate TPA phorbol 12-myristate 13-acetate PKD protein kinase D DGK diacylglycerol kinase KSR kinase suppressor of Ras NFD asparagine phenylalanine aspartic acid

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: 1-713-743-1708. Fax: 1-713743-1884. Notes

The authors declare no competing financial interest. 12127

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NMR RasGRP DPP SPR ITC PEG FRET DiC8 DiC18 PS MRCK

nuclear magnetic resonance Ras guanyl releasing protein 12-deoxyphorbol 13-phenylacetate surface plasmon resonance isothermal calorimetry polyethylene glycol fluorescence resonance energy transfer sn-1,2-dioctanoyl glycerol sn-1,2-dioleoylglycerol phosphatidylserine myotonic dystrophy kinase-related CDC42-binding kinase ROCK Rho-associated coiled-coil-containing protein kinases RASSF5 Ras association (RalGDS/AF-6) domain family member 5 STAC SH3 and cysteine rich domain GABA γ amino butyric acid GST glutathione-S-transferase FDA food and drug administration EMA European medicines agency

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