Phosphorylation of the Human Full-Length Protein Kinase Cι

20 May 2008 - length PKCι was expressed in Sf9 insect cells, purified, and digested with trypsin and endoproteinase. Asp-N, and its phosphorylation a...
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Phosphorylation of the Human Full-Length Protein Kinase Cι Boris Macek,‡ Christian Benda,§ Anja Jestel,§ Klaus Maskos,§ Matthias Mann,‡ and Albrecht Messerschmidt*,‡ Abteilung Proteomics and Signaltransduktion, Max-Planck-Institut fu ¨ r Biochemie, Am Klopferspitz 18, 82152 Martinsried, Germany, and Proteros Biostructures GmbH, Am Klopferspitz 19, 82152 Martinsried, Germany Received January 22, 2008

Atypical protein kinases C, including protein kinase Cι (PKCι), play critical roles in signaling pathways that control cell growth, differentiation and survival. This qualifies them as attractive targets for development of novel therapeutics for the treatment of various human diseases. In this study, the fulllength PKCι was expressed in Sf9 insect cells, purified, and digested with trypsin and endoproteinase Asp-N, and its phosphorylation analyzed by liquid chromatography-high accuracy mass spectrometry. This strategy mapped 97% of the PKCι protein sequence and revealed seven new Ser/Thr phosphorylation sites, in addition to the two previously known, pThr403 in the activation loop and pThr555 in the turn motif of the kinase domain. Most of the newly identified phosphorylation sites had low estimated occupancies (below 2%). Two phosphorylation sites were located in domain connecting amino acid sequence stretches (pSer217 and pSer237/pSer238) and may contribute to an improved stability and solubility of the protein. The most interesting new phosphorylation site was detected in a wellaccessible loop of the PB1 domain (pSer35/pSer37) and may be involved in the interactions of the PB1 domain with different partners in the relevant signaling pathways. Keywords: Protein kinase Cι • PB1 domain • mass spectrometry • phosphorylation • protein solubility

Introduction Protein kinases C constitute a family of Ser/Thr kinases of the AGC group of protein kinases1 and are subdivided into conventional (PKCR, PKCβI, PKCβII, PKCγ), novel (PKCδ, PKCε, PKCθ, PKCη), and atypical (PKCζ, PKCι/λ, PKCµ) isoforms, depending on their cofactor requirements.2 They contain a C-terminal kinase domain and N-terminal regulatory domains depending on the subfamily.3 Conventional PKCs have a functional C1 domain consisting of a tandem repeat of two zinc-finger CA1 and CA2 subdomains, which bind phosphatidylserine and diacylglycerol/phorbol esters and a C2 domain, that serves as a Ca2+-regulated phospholipd-binding module. Novel PKCs exhibit a functional C1 domain and a novel C2 domain, which binds neither Ca2+ nor membrane phospholipids. Atypical PKCs have a PB1 and an atypical C1 domain consisting of one zinc finger repeat, which only accepts phosphatidylserine. PB1 domains are scaffold modules that adopt a ubiquitin-like β-grasp fold and interact with each other in a front-to-back mode to form heterodimers or homooligomers.4 The different PB1 domain adaptors provide specificity for PB1 kinases like atypical PKCs, MEKK2/MEKK3 or MEK5R. PKCs have been shown to play an essential role in a wide range of cellular functions including mitogenic signaling, cytoskeleton rearrangement, glucose metabolism, differentia* To whom correspondence should be addressed. E-mail:messersc@ biochem.mpg.de. ‡ Max-Planck-Institut fu ¨ r Biochemie. § Proteros Biostructures GmbH.

2928 Journal of Proteome Research 2008, 7, 2928–2935 Published on Web 05/20/2008

tion and regulation of cell survival and apoptosis.5–9 Many of these cellular functions are related to human diseases. Inhibitors of PKCs are currently in clinical trials for various types of cancer, and a PKCβ inhibitor is in trials for diabetes-related retinopathy.10 Atypical PKCs play important roles in controlling cell growth and survival most likely through their regulation of critical signaling pathways including those that activate the AP-1 and NF-κB transcription factors (see ref 11 and references therein). PKCι has been proposed to be an attractive target to develop novel therapeutics against colon cancer12 and chronic myelogenous leukemia.13 The crystal structures of the catalytic domains or full-length proteins of various members of the AGC family have been determined (cAMP-dependent protein kinase/ PKA,14 PKB/AKT,15,16 PKCβII,17 PKCθ,18 PKCι,19 PDK1,20 Aurora21 and Grk222). Furthermore, the X-ray structures of the Cys2 activator (C1B) domain of murine PKCδ,23 the C2 domain of rat PKCβ24 and PKCδ25 and of the PB1 domain (in complex with Par6R) of human PKCι26 have been reported. Like all protein kinases, their catalytic domain is composed of an N-terminal lobe consisting mainly of a β-sheet and a predominantly R-helical C-terminal lobe.27,28 The ATP-binding site is located between the two lobes. There are further structural features common to all protein kinases: (i) the glycine-rich loop between β-strands 1 and 2sthis is a part of the ATP-binding site and its primary function is to position the γ-phosphate of ATP for phosphoryl transfer, (ii) the magnesium positioning loop, (iii) the activation loop, and (iv) the peptide positioning loop.29 Beside this structural relation, AGC-protein kinases share numerous functional similarities such as activation in response to second messengers and, importantly, dependence 10.1021/pr800052z CCC: $40.75

 2008 American Chemical Society

Phosphorylation of the human PKCι on phosphorylation for activity. Members of the family are phosphorylated on a conserved threonine residue within their activation segment. Protein kinases A, B, and C share a C-terminal turn motif and a hydrophobic motif (HM, immediately at the C-terminus).30 Phosphorylation at the activation loop triggers the rapid phosphorylation of a Thr or Ser in a Pro-rich domain of the turn motif. Protein kinases B and C exhibit a Ser/Thr phosphorylation site in the hydrophobic motif.3 Exceptions are the atypical PKCs, where this residue is a Glu, which may mimic a phosphorylation site. The sequence of PKA terminates with a Phe just before this phosphorylation site in PKB and PKC. All PKBs and PKCs have this phenylalanine. The crystal structures of PKA,14 PKB/AKT,16 PKCθ,18 PKCι19 and PKCβ II17 show that this phenylalanine inserts into a hydrophobic pocket on the back side of the active site situated in the N-lobe. Thus, the interaction between the hydrophobic motif and the hydrophobic pocket of the catalytic core stabilizes its structure. The development of specific inhibitors directed at the catalytic core of AGC kinases for the treatment of various human diseases has recently been reviewed.31 The high degree of homology in the catalytic core ATP-binding site is a serious issue in this context. A precise knowledge of the spatial structure of the kinase domain of the relevant protein kinase target will be of great benefit for the development of highly specific inhibitors as disease therapies. In this context, we have recently cloned the catalytic domain of human PKCι, expressed and purified the protein and determined its crystal structure in complex with the inhibitor bis(indolyl)maleimide 1 (BIM1).19 The key outcome of this structure determination was the observation of the well-defined phosphorylation site (pThr555) in the turn motif, which is different to the one in the turn motif of PKA. The same architecture of the turn motif phosphorylation site (pThr641) has now been observed in the crystal structure of PKCβ II.17 To obtain insight into the regulatory aspects of PKCι from a structural point of view, we have recombinantly produced fulllength PKCι with the aim to solve its crystal structure. As the phosphorylation state of a protein is pivotal not only for its function, but also for its ability to be crystallized, we have determined it by LC-MS/MS spectrometry. We report seven novel phosphorylation sites on the protein and discuss putative functional implications.

Materials and Methods Cloning, Expression, and Purification of Human PKCι. Fulllength PKCι was cloned for overexpression in insect cell line Sf9 as previously described.19 In brief, the sequence encoding human PKCι (1-587) was cloned from cDNA derived from human testis, verified by DNA sequencing (identical sequence to TrEMBL entry Q8WW06, differences from Swiss-Prot P41743: Leu476Met, His499Leu and Pro551Arg) and cloned into pFastBacHT A (Invitrogen, Carlsbad, CA) via the BamHI and XbaI restriction sites, resulting in an N-terminal (His)6-fusion with TEV protease recognition site. Recombinant viruses for infection were obtained by standard procedures recommended by the manufacturer (Invitrogen, Carlsbad, CA). For protein expression, 2 × 106 Sf9-cells/mL were infected with a multiplicity of infection of approximately 5 and grown in aerated plastic bags for 65-72 h at 28 °C. Cells from 10 L of insect cell culture were harvested, resuspended in 50 mL of buffer A (20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 20 mM imidazole, 5% (v/v) glycerol, 1%

research articles (v/v) Triton X-100, 5 mM 2-mercaptoethanol, and protease inhibitor cocktail (Roche-Diagnostics, Penzberg, Germany)) and homogenized with an Ultraturrax homogenizer (IKA Werke GmbH, Staufen, Germany). The homogenate was clarified by centrifugation (51000g, 30 min) and applied to a Ni2+ charged Streamline IMAC-column (GE Healthcare, Freiburg, Germany) equilibrated in buffer A. After washing thoroughly with buffer A without detergent, the recombinant protein was eluted in buffer B (20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 0.5 M imidazole, 5% (v/v) glycerol, and 5 mM 2-mercaptoethanol). Fractions containing PKCι were pooled, concentrated, supplemented with TEV protease and dialyzed overnight against buffer A without detergent. After reapplying to a Ni-chelate column, the target protein, containing the amino acid residues GAMDP from the TEV cleavage site upstream of the N-terminal methionine of human PKCι, was detected in the flow-through. It was dialyzed against buffer C (25 mM sodium citrate, pH 6.5, 50 mM NaCl, 5% (v/v) glycerol, 0.5 mM EDTA, and 2 mM DTT) and loaded on a MonoQ column (GE Healthcare, Freiburg, Germany). Elution of PKCι was done by applying a gradient of buffer C containing 500 mM NaCl. Two well-separated elution peaks at approximately 50 (peak1) and 250 (peak2) mM salt, both containing PKCι as judged from SDS-PAGE were obtained. The fractions of interest were pooled, dialyzed (20 mM Tris 7.5, 150 mM NaCl, and 2 mM DTT) and concentrated (10-15 mg/mL). The final yield of recombinant PKCι was 15 mg of at least 99% homogeneous protein from 10 L of initial cell culture. For mass spectrometry analysis, the buffer was changed by dialysis against 20 mM ammonium bicarbonate. Activity Test. The kinase activity assay of PKCι full-length protein was performed according to the Kinase-Glo Plus Luminescent Kinase Assay (Promega, Madison, WI). The assay is based on measuring kinase activity by quantifying the amount of ATP remaining in solution following a kinase reaction. The assay is performed in a single well of a 384-well plate (plate reader: TECAN Infinite F200, TECAN, Switzerland) by adding a volume of Kinase-GloPlus Reagent equal to the volume of a completed kinase reaction and measuring luminescence. The luminescence signal is correlated with the amount of ATP present and is inversely correlated to the amount of kinase activity. The luminescence signal comes from the mono-oxygenation of luciferin. The luciferase reaction produces 1 photon of light per turnover. The PKCι was diluted in 10 µL of kinase reaction buffer (20 mM HEPES/NaOH, pH 7.4, 0.05% (v/v) Triton X-100, 0.1 mg/mL phosphatidylserine and 0.01 mg/mL diacylgylcerinol (5× stock in 20 mM MOPS, pH 7.2, 25 mM β-glycerophosphate, 1 mM DTT, 1 mM sodium orthovanadate, 1 mM CaCl2)) with 50 µM PKCtide (synthetic peptide with ERMRPRKRQGSVRRRV amino acid sequence) and 10 µM ATP. The specific unit is defined as turnover of 1 nmol ATP/(min/ mg of protein). Liquid Chromatography-Mass Spectrometry Analysis. To enable comprehensive sequence coverage of PKCι, the protein was separately digested with two proteases and digests were separately analyzed using nano LC-MS/MS. In the first analysis, 10 µg of the purified PKCι was dissolved in 6 M urea/2 M thiourea, reduced by 1 mM dithiotreitol for 30 min, alkylated with 5 mM iodoacetamide for 45 min and digested with endoproteinase Lys-C (Waco Chemicals, Richmond, VA) for 3 h at the room temperature (RT). After 4× dilution with 20 mM ammonium bicarbonate, the solution was Journal of Proteome Research • Vol. 7, No. 7, 2008 2929

research articles further incubated with trypsin (Promega, Madison, WI) for 16 h at RT. The enzyme-to-protein ratio was in both cases 1/100. In the second analysis, 10 µg of PKCι was dissolved in 20 mM ammonium bicarbonate and digested with endoproteinase Asp-N (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) for 16 h at RT. The enzyme-to-protein ratio was 1/50. Prior to MS measurements, both samples were purified using C18 stage-tips, eluted into a microwell-plate using the HPLC solvent B (see below), partially dried in a vacuum centrifuge and resuspended in loading solution containing 2% acetonitrile and 1% trifluoroacetic acid to the final volume of 10 µL. The trypsin and endoproteinase Asp-N digests were analyzed separately on a 1100 nano-HPLC system (Agilent Technologies, Santa Clara, CA) coupled with a LTQ-FT mass spectrometer (Thermo Fisher Scientific, Waltham, MA) using a nanoelectrospray interface (Proxeon Biosystems, Odense, Denmark). Peptides were loaded onto an in-house made 75 µM reverse-phase HPLC column filled with 3 µM Reprosil C18 beads (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) in the HPLC solvent “A” (0.5% acetic acid in Milli-Q water), and separated using the following gradient of HPLC solvent “B” (80% acetonitrile, 0.5% acetic acid in Milli-Q water): 0 min (2%B), 22 min (2%B), 25 min (10%B), 105 min (30%B), 122 min (50%B), 129 min (60%B), 130 min (80%B), 133 min (80%B), 137 min (2%B). The flow rate during sample loading was 0.5 µL/min and during elution, 0.25 µL/min. The LTQ-FT mass spectrometer (Thermo Fisher, Waltham, MA) was operated in the positive ion mode. For the analysis of tryptic peptides, survey scans were recorded in the FT-ICR analyzer and followed by data-dependent collision-induced dissociation (CID) of the five most-intense ions in the linear ion trap (LTQ). Additional neutral loss-dependent MS3 scans were triggered for all precursor ions showing a loss corresponding to the mass of phosphoric acid during CID. Since fewer peptides were expected in the endoproteinase Asp-N digest, the survey scan in the FT-ICR analyzer was followed by selected ion monitoring (SIM) scans of the three most intense ions in the FT ICR analyzer and their simultaneous CID fragmentation in the LTQ. The neutral loss-dependent MS3 scan was activated in the same fashion as in the analysis of tryptic peptides. Raw spectra were processed using the in-house developed open-source software DTASuperCharge (http://msquant.sourceforge.net) and searched using Mascot search engine (Matrix Science, London, England) against a specialized database containing the sequences of the expressed PKCι and 44 commonly observed protein contaminants. The search parameters were as follows: trypsin or endoproteinase Asp-N specificity, maximum 3 missed cleavages, initial precursor ion mass tolerance 25 ppm, fragment ion mass tolerance 0.5 Da. Phosphopeptides were validated using the in-house developed open-source software MSQuant (http://msquant.sourceforge.net), and automatically scored for post-translational modifications (PTM-scored) as described previously.32 Only peptides measured with absolute mass deviation of 10 ppm or better were accepted. Phosphorylation sites were determined from the PTM scores and confirmed manually. Since no specific enrichment of phosphopeptides was performed prior to LC-MS analysis, the approximate occupancies or stoichiometries of detected phosphorylation sites were estimated from the ratios of maximum peak intensities of detected phosphopeptides and corresponding unmodified peptides (see main text). 2930

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Figure 1. Coomassie stained SDS-PAGE of purified PKCι. Lane M, selected molecular mass markers; lane 1, eluate 1 of ion exchange chromatography; lane 2, eluate 2 of ion exchange chromatography.

Motif Scan of PKCι Primary Structure. The amino acid sequence of human PKCι was scanned for possible phosphorylation motifs serving as substrates for other putative protein kinases with program Scansite 2.033 using all available protein kinase motifs and medium stringency level.

Results Expression Construct and Activity Test. The heterologously expressed construct of PKCι contains at the N-terminus the amino acid residues GAMDP from the TEV cleavage site followed by the whole amino acid sequence (1-587) of PKCι from human testis (TrEMBL Q8WW06). In the last step of the purification, the protein elutes in two peaks from the ion exchange column of identical molecular mass (67 950 Da). The purity of both eluates was tested on an SDS-PAGE (Figure 1), which showed a broad band for each at about 65 kDa and some very weak bands probably from proteolytic fragments of the protein. The purity of the protein can be assessed from this SDS-PAGE to be better than 95%. The specific activity of the PKCι construct was determined with a luminescence assay (see Materials and Methods) and determined to be 3 U/mg both at 22 and 30 °C. The enzyme was about 300 times less active than the full-length PKCι enzyme (also with N-terminal His6-tag) offered from Millipore (Temecula, CA), which had been expressed in Sf21 cells. The purity of both enzyme samples is better than 90% and the values for the specific activity of the Millipore enzyme was taken from the respective product specification sheet. However, these values are difficult to compare since the assay for the Millipore enzyme was carried out at a final concentration of 100 µM ATP in contrast to 10 µM ATP in this study. This means that according to the apparent Km value for ATP the specific activity would be greater by 1.5 order of magnitude. But there still remains a 6-fold reduced activity compared to the Millipore enzyme, which may be due to the different types of SF cells, which were used for the expression of the enzyme.

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Phosphorylation of the human PKCι

Figure 2. Combined amino acid sequence coverage and identified phosphorylation sites on PKCι. The part of the sequence printed in bold letters was detected by either trypsin or endoproteinase Asp-N. Determined phosphorylation sites are marked in dark gray color; potential phosphorylation sites are marked in light gray color.

Phosphorylation Pattern of Full-Length PKCι. We used liquid chromatography-high accuracy mass spectrometry to map phosphorylation sites of human PKCι expressed in the Sf9 insect cell line. The protein was reduced, carbamydomethylated and digested with either endoproteinase Lys-C/trypsin or endoproteinase Asp-N. Peptides were separated on a nanoHPLC column and detected in the LTQ-FT mass spectrometer. Resulting spectra were searched against a custom-made database containing PKCι sequence and validated using MSQuant software. Only peptides measured with accuracy better than 10 ppm were accepted, and all phosphopeptide spectra were manually validated. The combined approach using trypsin and Asp-N resulted in 97.1% sequence coverage of the full-length human PKCι (Figure 2). In the trypsin digest analysis, 54 PKCι peptides were detected, covering 85% of the protein sequence. The information on detected tryptic peptides is listed in Supplementary Table 1. Six phosphopeptides, SPFDIVGSpSDNPDQNTEDYLFQVILEK, VQLpTPDDDDIVR, EGLRPGDTTSpTFCGTPNYIAPEILR, KIDQSEFEGFEYINPLLMpSAEECV, IDQSEFEGFEYINPLLMpSAEECV, and Ap[SS]SLGLQDFDLLR, were detected, revealing phosphorylation events at the PKCι residues Thr403, Ser450, Thr555, Ser582 and Ser237/238 (this phosphorylation site could not be unambiguously identified, indicated by square brackets in the peptide sequence). The spectra of detected phosphopeptides are presented in Figure 3 and Supplementary Figure 1. In the endoproteinase Asp-N digest, 36 peptides were detected, covering 68% of the protein sequence. Detected peptides are listed in the Supplementary Table 2. Six singly phosphorylated peptides, SQFTNERVQLpTPDD, DIVGSpSDNPDQNTE, DTTSpTFCGTPNYIAPEILRGEDYGFSV, DTTSTFCGpTPNYIAPEILRGE, DHAQTVIPYNPp[SS]HESL, and DIMITHFEPp[SIS]FEGLCNEVR, and two doubly phosphorylated peptides, DYGMCKEGLRPGDp[TTST]FCGpTPNYIAPEILRGEandDHAQTVIPYNPp[SS]HEpSL, were detected, revealing phosphorylation events at PKCι residues Ser217, Thr403, Thr407, Ser450, Thr555, Ser35/37 and Ser213/214 (the last two phosphorylation sites could not be unambiguously identified). Combined, the two analyses detected phosphorylation at the following residues: Ser35/37, Ser213/214, Ser217, Ser237/238, Thr403, Thr407, Ser450, Thr555 and Ser582. Since only Thr403 and Thr555 were previously described as phosphorylation sites, this analysis led to detection of seven new phosphorylation events on the protein. In addition to determination of phosphorylation sites, of key importance for this study was to estimate their stoichiometry. We performed no phospho-specific enrichment, and this

opened up the possibility of estimating the relative occupancies of the phosphorylation sites from the relative signal intensities of phosphorylated vs nonphosphorylated peptides. Recent work by Steen et al.34 and others, performed mostly on synthetic phosphopeptides, indicates that in general, phosphopeptide ionization efficiencies tend to be similar to their unmodified peptides. However, since the ionization efficiencies of these peptides are not identical, it should be kept in mind that these numbers are only estimates. All phosphorylated peptides were also detected in unphosphorylated form, pointing to the fact that phosphorylation was substoichiometric. For most of the detected phosphopeptides, the unmodified counterpart was of much higher abundance (up to 3 orders of magnitude as shown in the Figure 4.), pointing to the low estimated occupancy of newly detected phosphorylation sites. The only notable exceptions were the tryptic peptide EGLRPGDTTSpTFCGTPNYIAPEILR (98% estimated occupancy) and the Asp-N peptide DTTSpTFCGTPNYIAPEILRGEDYGFSV (95% estimated occupancy), which contain the Thr403 phosphorylation site from the activation loop of the PKCι, previously shown to be fully occupied. Information on all identified phosphopeptides, locations and estimated occupancies of phosphorylation sites is summarized in Table 1.

Discussion In this study, we used a state-of-the art proteomics approach, based on nano-LC coupled with a high accuracy FT-ICR mass spectrometer, for in-depth analysis of phosphorylation modifications on the key signaling protein PKCι. Separate analyses of protein digests done by two complementary proteases, trypsin and endoproteinase Asp-N, led to almost complete coverage of the protein sequence (97.1%), leaving only one potential phosphorylation site, Tyr160, uncovered. The expression of human full-length PKCι in the Sf9 insect cell line, commonly used as a robust expression system, delivered an enzyme with a fully occupied phosphosite in the activation loop of the catalytic domain (pThr403) but the turn motif phosphosite (pThr555) with an estimated occupancy of only 5%. It is interesting to note that in the crystal structure of the catalytic domain of PKCι, this pThr was well-defined in the electron density, indicative for occupancy of close to 100%. This residue becomes autophosphorylated once Thr403 has the phosphate group attached. The low estimated occupancy of the phosphosite at Thr555 in the full-length PKCι may be the reason for the reduced enzymatic activity of the analyzed protein. It is known from other PKCs, which all have this turn motif phosphosite, that phosphorylation of this site is crucial in maintaining an active conformation.35 The X-ray structure of the catalytic domain of PKCι19 shows that the phosphorylation of the turn motif stabilizes the kinase domain by fixing the C-terminus at the top of the upper lobe of the kinase core and prevents the flexible C-terminus in covering the substratebinding cavity. We detected a novel phosphorylation site at Thr407. While its occupancy was clearly low, we could not quantify it because the peptide was doubly phosphorylated. A phosphorylation at the homologous residue Thr500 in PKCR has recently been found in a proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry.36 We found two further so far unknown phosphosites in the catalytic subunit, however, with low estimated occupancies (Ser450 ∼ 3% and Ser582 ∼4%). The high occupancy (33%) of Journal of Proteome Research • Vol. 7, No. 7, 2008 2931

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Figure 3. Total ion chromatogram of the PKCι tryptic digest. Intensity differences of up to 3 orders of magnitude between phosphorylated and corresponding unmodified peptides were observed (e.g., for Ap[SS]SLGLQDFDLLR peptide). “RT” ) retention time; “I” ) intensity.

Ser450 determined by the Asp-N digest (see Table 1) is most likely overestimated because this phosphosite precedes the Asp-N cleavage site, which probably changes the cleavage properties of this phosphopeptide. Both serines lie in amino acid sequence stretches that are disordered in the X-ray structure of the catalytic domain of human PKCι19 and were not defined in the electron density of the structure. These partial phosphorylations may contribute to the moderate quality of the crystals of PKCι used in the crystal structure analysis.19 The other phosphosites are located outside the catalytic domain in the PB1 domain (residues Ser35/Ser37), and in a region (residues 185-239) between the atypical C1 and the catalytic domain. The new phosphorylation site at either Ser35 or Ser37 in the PB1 domain has an estimated occupancy of about 18% and could be of functional relevance. In the threedimensional structure of the PB1 domain of human PKCι,26 the side chains of Ser35 and Ser37 are located in the loop connecting β-strand 2 with R-helix 1. Both amino acid residues are well-accessible and sequence alignment of the PB1 domains of PKCι with related atypical PKCs shows that Ser35 is conserved in all vertebrates and that Ser37 is either fully conserved or changed to Thr in insects. Because of the high conservation of these residues and their high accessibility, they are welllocated for interactions with other signaling pathway partners. It remains to be determined if this phosphorylation constitutes a switch between two states of the PB1 domain for interactions with different partners in the relevant signaling pathways. The degree of phosphorylation of Ser217 and Ser237/Ser238 is low; however, the phosphorylation event at Ser237/Ser238 2932

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is likely to be important for the stability and solubility of the PKCι. In the course of the crystal structure determination of the catalytic domain of PKCι constructs starting at residues 224 and 245 were tested.19 Both groups of constructs could be expressed in insect cells and were found in the soluble as well as in the insoluble fractions. However, the constructs bearing amino acid residues 237 and 238 could be extracted during the Ni-NTA purification step, whereas the purification with the other constructs failed,37 which supports the suggested function. Ser 237 and Ser238 were present in the construct used for the crystal structure determination of human PKCι. However, this N-terminal part was flexible and could not be detected in the electron density. Regardless of their estimated occupancies, all detected phosphorylation sites could be of functional relevance because occupation of phosphosites depends on the stimulation of the respective cells and the studied PKCι sample represents one cellular state only. As new phosphorylation sites were found, it was interesting to look for possible phosphorylating protein kinases. For this purpose, motif scan of the amino acid sequence of PKCι was made with Scansite 2.0. When the highest stringency was used, the scan revealed two hits only, one for the binding of a SH3 domain around the residue Pro101 and another one for binding of PDK1 at the HM around Glu574. The second one can be easily explained since PDK1 is known as activating protein kinase for all PKCs phosphorylating Thr403 in activation loop. The relevance of the first hit must be doubted because there is no interaction of PB1 domains with SH3 domains reported up to now.

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Figure 4. Mass spectra of selected phosphopeptides detected in this study. (a) Phosophopeptide SPFDIVGSpSDNPDQNTEDYLFQVILEK was measured with mass deviation of 1.7 ppm. Fragment (b and y) ions point to Ser450 as modification site; (b) mass spectrum of the phosphopeptide VQLpTPDDDDIVR that includes previously identified phosphorylation at Thr555. The precursor ion was measured with a mass deviation of 1.5 ppm; (c) mass spectrum of the phosphopeptide KIDQSEFEGFEYINPLLMpSAEECV, measured with mass deviation of 2.4 ppm. The fragment ions y7 and y10, with corresponding neutral losses of phosphoric acid, point to Ser582 as modification site. Journal of Proteome Research • Vol. 7, No. 7, 2008 2933

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Table 1. Detected Phosphopeptides and Phosphorylation Sites of PKCι

a

enzyme

phosphopeptide

P-site

estimated occupancy (%)

measured mass

mass deviation (ppm)

Trypsin Trypsin Trypsin Trypsin Trypsin Trypsin Asp-N Asp-N Asp-N Asp-N Asp-N Asp-N Asp-N Asp-N

SPFDIVGSpSDNPDQNTEDYLFQVILEK* VQLpTPDDDDIVR* EGLRPGDTTSpTFCGTPNYIAPEILR* IDQSEFEGFEYINPLLMpSAEECV KIDQSEFEGFEYINPLLMpSAEECV* Ap[SS]SLGLQDFDLLR* DSQFTNERVQLpTPDD DIVGSpSDNPDQNTE DTTSpTFCGTPNYIAPEILRGEDYGFSV DTTSTFCGpTPNYIAPEILRGE* DYGMCKEGLRPGDp[TTST]FCGpTPNYIAPEILRGE DHAQTVIPYNPp[SS]HEpSL* DHAQTVIPYNPp[SS]HESL* DIMITHFEPp[SIS]FEGLCNEVR*

S450 T555 T403 S582 S582 (S237/S238) T555 S450 T403 T407 (T400/T401/S402/T403) T407 (S213/S214) S217 (S213/S214) (S35/ S37)

3