Phospho-priming Confers Functionally Relevant Specificities for

Subscriber access provided by - Access paid by the | UCSB Libraries

Article

Phospho-priming Confers Functionally Relevant Specificities for Rad53 Kinase Autophosphorylation Eric Sheng-Wen Chen, Jui-Hung Weng, Yu-Hou Chen, Shun-Chang Wang, Xiao-Xia Liu, Wei-Cheng Huang, Tsutomu Matsui, Yoshiaki Kawano, JiahnHaur Liao, Liang-Hin Lim, Kai-Fa Huang, Wen-Jin Wu, and Ming-Daw Tsai Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00689 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 5, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Phospho-priming Confers Functionally Relevant Specificities for Rad53 Kinase Autophosphorylation Eric Sheng-Wen Chen@,†,‡,†, Jui-Hung Weng@,§, ‡, Yu-Hou Chen@,‡ , Shun-Chang Wang@, XiaoXia Liu@,¶, Wei-Cheng Huang@, Tsutomu Matsui#, Yoshiaki Kawano||, Jiahn-Haur Liao@, LiangHin Lim@,†, Kai-Fa Huang@, Wen-Jin Wu@, and Ming-Daw Tsai@,† ,* @

Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan;

†

Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan;

§

Institute of Biochemical Sciences, Department of Chemistry, National Tsing Hua University,

Hsinchu 300, Taiwan; #

Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford

University, Menlo Park, California 94025, USA. ||

RIKEN SPring-8 Center.1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.

KEYWORDS: Ser/Thr kinases; phospho-priming; Autophosphorylation; Kinase activation.

ACS Paragon Plus Environment

1

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 38

ABSTRACT

The vast majority of in vitro structural and functional studies on the activation mechanism of protein kinases use the kinase domain alone. Well demonstrated effects of regulatory domains or allosteric factors are scarce for serine/threonine kinases. Here we use a site-specifically phosphorylated SCD1-FHA1-kinase three-domain construct of the serine/threonine kinase Rad53 to show the effect of phospho-priming, an in vivo regulatory mechanism, on the autophosphorylation intermediate and specificity. Unphosphorylated Rad53 is a flexible monomer in solution but is captured in an asymmetric enzyme:substrate complex in crystal with the two FHA domains separated apart from each other. Phospho-priming induces formation of a stable dimer via intermolecular pT-FHA binding in solution. Importantly, autophosphorylation of unprimed and phospho-primed Rad53 produced predominantly inactive pS350-Rad53 and active pT354-Rad53, respectively. The latter mechanism was also demonstrated in vivo. Our results show that, while Rad53 can display active conformations under various conditions, simulation of in vivo regulatory conditions confers functionally relevant autophosphorylation.


2

Page 3 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

INTRODUCTION

Protein kinases have been widely shown to serve as crucial molecular switches in cellular signaling networks. Most protein kinases, including Ser/Thr kinases that are the focus of this study, are expressed in the inactive form, and their activation often requires autophosphorylation, or phosphorylation by an upstream kinase, at the activation segment1, 2. The mechanism of autophosphorylation has been investigated extensively for many kinases and the structure of the dimeric intermediate of autophosphorylation has been reported for a number of kinases in recent years3-12. These dimeric intermediates include face-to-face5,

7, 8

; face-to-face with activation

segment exchange4, 9; head-to-tail dimers of EGFR (autophosphorylation of the C-terminal segment)3; and back-to-back dimers of B-RAF6, 13. However, the majority of past studies were performed for the kinase domain alone, in the absence of regulatory domains or regulatory factors. Recent reports of two receptor tyrosine kinases show that the structural properties of multi-domain constructs that include regulatory domains differ from those of the corresponding kinase domain alone constructs14-18. For the kinases that are activated by a priming phosphorylation, the effect of phospho-priming on the auto-activation of the kinase has not been reported to-date. The Chk2-like kinases are part of the cell cycle checkpoint kinase cascades associated with DNA replication stress or DNA damage19-22. They are characterized by the presence of an SQ/TQ cluster domain (SCD), a phosphothreonine (pT)-specific forkhead-associated (FHA) domain, and a serine/threonine kinase domain (Figure 1A). Yeast ortholog Rad53, a Chk2-like kinase, contains an additional second SCD-FHA module at the C-terminus23, 24. Moreover, Chk2-like kinases belong to RD kinases where the catalytic loop contains a conserved arginine immediately preceding the conserved catalytic aspartate, and the coordinated conformation with activation segment can control the kinase activity1. As shown in Figure 1B, auto-activation of Chk2-like


3

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 38

kinases has been suggested to depend on the priming phosphorylation at the N-terminal SCD by ATM/ATR-like kinases (step 1), leading to dimerization by binding with the N-terminal FHA domain of a second kinase molecule (step 2)24-28. The resulting dimerization in turn promotes trans autophosphorylation at the activation segment of the kinase domain (step 3), in order to maintain the kinase at the active conformation29, 30. However, the structural and biochemical bases for this model remain to be established since the priming phosphorylation was not included in the recent structural analyses of Chk2 or Rad5331-33. Another complexity in understanding the structural basis of kinase auto-activation in vivo is the existence of multiple phosphorylation sites and multiple pathways. For example, the four phosphorylatable TQ motifs (T5, T8, T12, and T15) in the N-terminal SCD1 of Rad53 have been shown to work redundantly in supporting Rad53-dependent checkpoint survival34, 35. In addition, our recent study has identified more than 20 autophosphorylation sites for endogenous Rad53 in vivo, including the three conserved Ser/Thr residues at the activation segment (S350, T354, and T358), in response to methyl methanesulfonate (MMS)-induced S phase DNA damage36. It has also

been

shown that

in

addition

to

the pT-FHA binding-induced dimerization,

autophosphorylation of Rad53 in vivo can also be mediated by the scaffold protein Rad936, 37. In this study, we focus on the pT-FHA binding-dependent auto-activation mechanism of Rad53. We prepared site-specifically phospho-primed, three-domain Rad532-466 (Figure 1C) to examine the dimeric structural intermediate and the autophosphorylation specificity. We asked how phospho-priming can regulate the dimerization of a multi-domain kinase, and whether dimerization in turn controls the specificity and mechanism of the trans autophosphorylation of the kinase. We observed an asymmetric transient dimer for unprimed Rad532-466 and a stable


4

Page 5 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

symmetric dimer for phospho-primed Rad532-466, and showed that they differ in the site specificity of autophosphorylation.

MATERIALS AND METHODS

Materials. ATP, 4-mercaptophenylacetic acid (MPAA), Tris (2-carboxyethyl) phosphine (TCEP), and Isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased from Sigma-Aldrich, and the crosslinker bis(sulfosuccinimidyl) glutarate (BS2G) from Thermo Scientific Pierce. The other chemical reagents for protein purification and chemical ligation were purchased from Merck and Acros. Phos-tagTM Acrylamide AAL-107 was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Unphosphorylated Rad53 and Dun1 proteins were prepared as described in Supporting Information (SI). Site-specifically phosphorylated Rad53 proteins were prepared from native chemical ligation as described in Figure S1 and Table S1. The identities of all protein products were confirmed by mass spectrometry analyses following in-gel digestion. Crystallization and structural determination. The Rad53SFK or SeMet-Rad53SFK was crystallized by the sitting drop vapor-diffusion method by which 0.5 μL of the protein solution (12 mg/mL in the purification buffer) and 0.5 μL of crystallization solution (100 mM cacodylate buffer at pH 6.5, 0.4 M NaCl, 1.5 M (NH4)2SO4 were equilibrated with 70 μL reservoir at 293K. Additional 20% glycerol was added to the crystallization buffer as a cryoprotectant before removing the crystals from crystallization drops. For soaking experiments, 1 mM AMPPNP was added into crystallization drops 1 day before collecting crystals. Diffraction intensities were integrated and scaled with programs DENZO and SCALEPACK in the HKL2000 package38. Initial phasing and model building were carried out using PHENIX-AutoSol39. Model extension and rebuilding was performed by ARP/wARP40 using native data. Final models were built after


5

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 38

several runs of refinement in REFMAC541 and manual re-building in Coot42. The final models were analyzed with PROCHECK43. Structural presentations were generated using the PyMOL Molecular Graphics System (Version 1.7.4 Schrödinger, LLC). SAXS measurements, analyses, and modeling. SAXS data were collected at Synchrotron BL23A SWAXS station. Data were analyzed by ATSAS 2.6.144. EOM structure pool was generated by inputting structural fragments of SCD, FHA, and Kinase domain. Rigid body modeling was performed by CORAL. The restraints were based on chemical cross linking experiment using cross linker BS2G [bis(sulfosuccinimidyl)glutarate, Thermo Scientific]. More details are described in SI Method. ADP-based phosphatase-coupled kinase assay. The specific autophosphorylation were measured by Universal Kinase Activity Kit (R&D Systems) that utilizes the nucleoside diphosphatase, CD39L2/ENTPD6, to release the β-phosphate from ADP. Various dilutions of kinase samples from 5 μM to 0.078 μM were subjected to auto-kinase reaction for 10 min at 30 °C in 25 mM HEPES buffer at pH 7.0, 0.15 M NaCl, 10 mM MgCl2, 10 mM CaCl2, 0.2 mM ATP, and 0.1 mg coupling phosphatase. The reaction was terminated using Malachite Green Reagents that also react with the released β-phosphate, resulting in a maximal optical absorbance at 620 nm, which serves as the readout of ADP levels to monitor the kinetics of auto-kinase reaction. In vitro autophosphorylation, phosphate-affinity SDS-PAGE, and MS analysis. Unprimed and phospho-primed Rad53SFK (5 μM) were mixed with different concentrations of ATP (5 μM, 20 μM or 200 μM) in 50 mM HEPES buffer at pH 7.2, 0.1 M NaCl, 10 mM MgCl 2 at 30 °C. Aliquots of reaction mixture were collected and mixed with 2x SDS-PAGE loading sample buffer to stop the reaction, and then separated with 7.5% SDS-PAGE containing 10 μM Phos-tagTM Acrylamide AAL-107 and visualized by Coomassie Brilliant Blue R-250 staining. The


6

Page 7 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

electrophoretic mobility of autophosphorylated Rad53 sample would be retarded by the acrylamide-pendant Phos-tagTM relative to the corresponding unphosphorylated Rad53 samples45, 46

. For MS analysis, the discernible shifted bands resulting from the ATP to protein ratio of 1 were

performed by in-gel trypsin digestion, followed by LC-MS/MS analysis. In addition, the aliquots of reaction mixtures from the ATP to protein ratio of 4 or 40 were separated by standard 7.5% SDS-PAGE (without Phos-tagTM) and visualized by Coomassie Brilliant Blue R-250 staining. The resulting Rad53 bands, including phosphorylated and unphosphorylated Rad53 from each aliquot, were subjected to in-gel trypsin digestion and LC-MS/MS analysis. The ES% at a given phosphorylation site was determined by a semi-quantitative label-free method47. Kinase activities of autophosphorylated Rad53 toward biological substrate Dun1. 5 µM Rad53SFK recombinant protein or its mutant was pre-activated by autophosphorylation with 200 µM ATP for 5 min. 0.1 µM activated Rad53 was used to phosphorylate 4.9 µM Dun1 D328A with 50 µM ATP in the same condition as autophosphorylation of Rad53, and analyzed in SDS-PAGE containing 10 µM Phos-tagTM. All autophosphorylation analyses by electrophoretic mobility shifts and by MS were performed in three independent reactions and analyses. The kinase reaction using Dun1 as substrate was also performed three times. All error bars represent standard deviation. In vivo autophosphorylation analyses. Conditions of yeast cells culture and lysate preparation are described in SI Method. Genotypes of yeast strains used in this experiment are described in Table S2. The in vivo phosphorylation was analyzed by a quantitative spike-in SILAC method as described in our recent report, including the detailed procedures of sample preparation36. Four independent biological experiments were performed, two at 45 min and two at 90 min. The results are similar and thus only the 45 min data are shown. All error bars represent standard deviation.


7

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 38

NMR experiments of for Rad53F and pT8-Rad53SF. NMR experiments were performed at 293 K on a Bruker Avance 800 MHz NMR spectrometer equipped with a cryogenic probe. All NMR samples (approximately 300 L) were contained in 5 mm O.D. Shigemi tubes. The sample concentrations were 0.55 mM and 0.33 mM for Rad53F and pT8-Rad53SF, respectively. The NMR buffers contained 8% D2O, 0.5 mM EDTA, 5 mM DTT-d10, 10 mM sodium phosphate at pH 6.5. Experimental parameters: pulse sequence: fhsqcf3gpph (with delay for binomial water suppression d19 set to 72 s), spectral width: 14.463 ppm (centered at 4.702 ppm) for 1H, 66.000 ppm (centered at 107.000 ppm) for 15N. 16 scans with 256 FIDs (total 1 hr and 18 min), and 128 scans with 154 FIDs (total 12 hr 13 min) were collected for Rad53F and pT8-Rad53SF, respectively. Data processing: 60-degree shifted sine bell window function; zero-filling once in both dimensions, and forwarded complex linear prediction with 32 coefficient points were utilized to enhance digital resolution in the 15N dimension. All spectra were processed using the Topspin software (Bruker). For sequential resonance assignments of unligated Rad53F, 3D triple resonance experiments of HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH and HNCO were collected using non-uniform sampling scheme and processed with the compressed sensing scheme under Topspin 3.5. The resonance assignments were made using the program CARA (http://www.nmr.ch). Due to fast relaxation caused by dimerization, partial resonance assignments were obtained for pT8-Rad53SF by comparing to those of Rad53F and were confirmed by a 3D HNCA data set. Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis. The procedures for SV-AUC are based on the previously reported method48 with minor modifications. The samples were dissolved in 50 mM HEPES buffer at pH 7.5, 150 mM NaCl, 2 mM EDTA, and 5 mM β-mercaptoethanol and analyzed using a Beckman-Coulter XL-A analytical ultracentrifuge. The samples were loaded into 12-mm standard double-sector Epon charcoal-filled centrepieces


8

Page 9 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

that were mounted in an An-60 Ti rotor for ultracentrifugation at 42,000 rpm at 20 °C and monitoring

at

280

nm.

The

raw

data

was

analyzed

using

SEDFIT

software

(http://www.analyticalultracentrifugation.com/default.htm). The plots of c(s, fr) and c(s, M) were generated using MATLAB 7.0 software (MathWork, Inc.). The highest concentration Rad53SF and the lowest concentration Rad53SFK samples were performed with three different batches of samples, whereas additional concentrations were performed with only one batch.

RESULTS

Phospho-priming induces dimerization of Rad53SF and Rad53SFK via pT-FHA binding. The native chemical ligation method49, 50 or the related expressed protein ligation method51 has been used to prepare site-specifically phosphorylated kinases, leading to valuable mechanistic insights for several kinases51-54. We used this approach to prepare Rad53SF (residues 2-164, consisting of SCD1 and FHA1 domains) and Rad53SFK (residues 2-466, consisting of SCD1, FHA1, and kinase domains), respectively, with specific phosphorylation at T5, T8, T12, or T15 (Figure 1C). As shown in Figure S1, ligation was performed between a C-terminal peptide (residues 2-18, with phosphorylation at one of the four threonine residues) and a truncated Rad53 construct (from residue 19, with Leu19 replaced by Cys). We then examined the effects of pT-FHA binding on the dimerization of Rad53SF and Rad53SFK by sedimentation velocity analytical ultracentrifugation (SV-AUC) analyses. Plots of c(s) analysis for Rad53SF and Rad53SFK are summarized in Figure 1 D and E, respectively, whereas complete c(s, fr) and c(s, M) distribution plots in a range of concentrations are shown in Figure S2 to S5. The results indicate that unphosphorylated Rad53SF and Rad53SFK are predominantly monomeric, while the phospho-primed constructs form dimers with different dimerization propensities. The four isomers of the two-domain pT-Rad53SF varies


9

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

AHs_Chk2

S

F

K

543 aa

Mm_Chk2

S

F

K

546 aa

Xl_Cds1 S Sc_Rad53 S

F F

K

K

S

D

F

504 aa 821 aa

Page 10 of 38

B

C S

1

P P

S

F 2

F

P

K

Rad53SF

S

F

3

Rad53SFK S

F

K

Rad53FKSF

F

K

P

K

E

F

2-164 T5QPT8QQST12QAT15Q 2-466 S

F

19-732

R35 R83

R70

G113 G133

15N

S85 1H

Figure 1. Protein Constructs and Their Oligomerization States. (A) Domain architectures for Chk2-like kinases in different organisms (Hs: Homo sapiens, Mm: Mus musculus, Xl: Xenopus laevis, Sc: Saccharomyces cerevisiae). S, F, and K designate SCD, FHA, and kinase domains, respectively. (B) Current model for the autophosphorylation of Chk2-like kinases. Steps 1-3 are explained in the text. (C) Specific Rad53 constructs used in this work. (D) Plots of c(s) analysis for unphosphorylated Rad53SF and its four phosphorylation isomers and the corresponding R70A mutant, at 107.5 μM. (E) Same as D for the three-domain Rad53SFK, at 5.8 μM. Complete analyses with concentration dependence are shown in Fig. S2 to S5. All 18 experiments in D and E were performed with three independent batches of samples except pT15-R70A due to its instability. (F) NMR Data Support the pT8-Rad53SF Dimer. Overlaid 2D 1H,15N-HSQC spectra of the pT8Rad53SF (blue) and unligated Rad53F (green) show peak broadening upon pT8-SCD1 ligation. Expansion of the G113 and G133 cross peaks are shown to demonstrate the line broadening effect. The signals of R35, R70 and R83 side chain NH and S85 backbone NH for pT8-Rad53SF are either shifted or broadened beyond detection.

from monomer to dimer depending on the position of pT and the concentration, whereas all four isomers of the three-domain pT-Rad53SFK exist mainly as dimers, except that the pT12 isomer precipitates and the pT15 isomer oligomerizes at higher concentrations (Figure S4). The dimerization is the result of pT-FHA binding, as only monomer was observed when the crucial pT-binding residue Arg70 in FHA1 was substituted by alanine. The enhanced dimerization propensity of pT-Rad53SFK relative to pT-Rad53SF is likely caused by the dimerization propensity of the kinase domain31, 33.


10

Page 11 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Since the pT-Rad53SF construct is relatively small, it is possible to use NMR to provide structural support for pT-FHA binding and dimerization, by examining the effect of phospho-priming on the chemical shifts and line widths. We have previously used NMR to solve the structures of the FHA1 domain alone55 and in complex with a pT-SCD1 peptide35. As shown in Figure 1F, substantial peak broadening in 2D 1H,15N-HSQC NMR spectra of the pT8-FHA1 dimer was observed. In addition, signal perturbation for residues in the FHA1 domain supports interaction between the pT8-SCD1 and the FHA1 domain. Thus NMR data also support that the dimerization of pT8Rad53SF is a result of intermolecular pT-FHA binding. Overall, the results show that phospho-priming induces dimerization via pT-FHA binding for both Rad53SF and Rad53SFK, and that the dimerization propensities are not identical between the four pT isomers of each construct.

Global and kinase domain structures of the unprimed Rad53SFK. Further structural and functional analyses of this work focused on the more biologically relevant three-domain Rad53SFK. First, the crystal structure of unprimed Rad53SFK was solved at 2.8 Å. The refinement statistics are shown in Table 1. Each asymmetric unit (ASU) consists of two unique protein chains A and B. Figure 2A shows the overall structure of the two chains, in which the FHA1 phosphothreonine recognition loops (between β strand pairs β3’-β4’, β4’-β5’, and β6’-β7’, colored magenta in chain B) face outward to be available for ligand binding. The complete sequence and secondary structures are shown in Figure S6. For comparison, we summarize the basic structural properties of face-to-face dimeric structures suggested to be autophosphorylation intermediates of Ser/Thr kinases in Table 2. Other than Rad53SFK, the only multi-domain structure in Table 2 is the two-domain construct of Chk2 (FHA-


11

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 38

kinase domains, designated as Chk2FK)32. It is interesting that there are notable differences between the two structures: The kinase domains are more tilted in Rad53SFK than in Chk2FK; the FHA Table 1. Data Collection and Refinement Statistics for Structural Determination of Rad531-466.

NSRRC 13B 30-3.3 (3.42-3.3)* P3221

Apo form † 5XZW NSRRC 15A 30-2.8 (2.9-2.8)* P3221

AMPPNP complex ‡ 5XZV SPring-8 BL41XU 30-3.1 (3.2-3.1)* P3221

117.7, 117.5, 141.3 0.97888 17466 0.085 21.2 (2.0) 99.9 (99.6) 6.3 (5.8)

115.8, 115.8, 141.3 0.90000 25178 0.054 55.5 (5.2) 98.6 (100) 9.1 (9.4)

116.7, 116,7, 140.8 1.00000 17877 0.060 51.3 (4.4) 100 (100) 10.9 (9.4)

30-2.8 23184 / 1994 0.260 / 0.281

30-3.1 16975 / 902 0.250 / 0.268

0.008 1.400

0.012 1.568

5599 / 70 49 / 40.0

5444 / 53.5

SeMet Rad531-466 PDB ID Data collection Resolution (Å) Space group Cell dimensions a, b, c (Å) Wavelength (Å) No. unique reflections Rmerge I / σI Completeness (%) Redundancy Refinement Resolution (Å) Reflections (working/test) Rwork / Rfree R.m.s deviations Bond lengths (Å) Bond angles () No. atoms / B-factors Protein Water Ligand Ramachandran plot (%) Most favored Additional allowed regions Generously allowed regions Disallowed regions

54 / 67.9 87.9 12.1

89.7 9.9

0

0.5

0

0

*Values in parentheses are for the highest resolution shell. †In chain A, the electron density is not discernible for part of the SCD1 domain (residue 1-14), part of the loops of FHA1 domain (79-86 and 135-137), the linker region between the FHA1 and the kinase domains (165-190), part of the activation segment (344-356), and the linker between helices αEF and αF (368-382). In addition, the residues 409, 410, 426-428, and 447-449 of chain A have no discernible electron density for model building either. In chain B, the electron density is not discernible for the SCD1 domain (1-31), the linker region between the FHA1 and the kinase domains (158-188), part of the activation segment (343-353), and the linker between helices αEF and αF (369-383). ‡Residues with non-discernible electron density: chain A, 1-15, 79-84, 135-137, 165-189, 345358, 368-382, 409-410, 425-429, 447-449 and 454-455; chain B, 1-31, 135-139, 158-188, 234237, 343-353, 369-382, the gamma and beta phosphate of AMPPNP.


12

Page 13 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

A

C

L(β4′- β5′) R70 L(β3′- β4′)

D (a)

Chain A

5’

7’

N189

3’

αC

L259

4’

L(β6′- β7′)

FHA1

K227

L248

E244 F340

6’

D339 Q157

αA

H317

N-lobe

D319

D319

(b)

Chain B

αC D’319

αC

L259 L248

K227

L342

E244

F340 H317

C-lobe T354

Chain A Chain B (assembled R-spine) (dissembled R-spine)

B

D347 (CHK2) D319 (Rad53)

D319

E F

FHA 90o Kinase

Rad53SFK

Chk2FK

E

β8

(a)

β7

Chain A αF

β6

(b)

L342

β7

β8

K321 D319 3.0 3.6 8.1

D319

R318

Chain B

K321 10.4

T358 αF αEF

R454

β8

(c)

β6

10.8 T358

β9

β7 R318

PKA αF

D166

β6 2.9 R165

3.0 2.8

7.8

T354

2.2 E365 2.4 αEF

K168

T201

2.8 10.1 4.3

R280 3.0

pT197

E208

Figure 2. Structural Properties of Unphosphorylated Rad53SFK. (A) Structure of the chain A: chain B dimer. The RD containing catalytic loop (residues 317-324) and the activation segment from DFG to APE motifs (339-365) are colored in brown and yellow in chains A and B, respectively (the dotted red line represents the missing part of the activation segment in chain B). Side chains for R70 of chain B and D319 of chain A are shown in sticks, while those for R-spine residues in spheres. The residues with undiscernible electron densities are specified in Table 1. (B) Cartoon comparison for the relative orientations of the FHA domain and the kinase domain between Rad53SFK and Chk2FK. (C) Detailed structures for the assembly and disassembly of R-spine in chain A and chain B, respectively. (D) Partial structures showing binding of AMPPNP to Rad53SFK chain A (a), and superposition of this structure with the kinase domain of Chk2 bound with MgADP (PDB 2CN5, green) (b). The 2Fo-Fc electron density map around the AMPPNP, contoured at 0.9σ level, is shown in blue (a). (E) Partial structures in the catalytic loop and activation segment of Rad53SFK chain A (a), chain B (b), and PKA (c, PDB 1ATP). (F) Structurebased sequence alignment from catalytic loop to helix αEF of RD serine/threonine kinases (Sc_Rad53: PDB 5CAF, Hs_Chk2: 2CN5, Hs_Aurora A: 1OL5, Mm_PKA: 1ATP, Hs_PKB: 1OL6, Hs_CDK2: 1QMZ, Rn_MAKP1: 2ERK, Hs_IRAK4: 2OID).


13

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 38

domains of Chk2FK interact with each other and both point upward while those of Rad53SFK are separated and point to different direction (cartoon illustration in Figure 2B and structural comparison in Figure S7). Furthermore, Rad53SFK is unusual in that only chain A, not chain B, displays an “assembled regulatory spine (R-spine)” characteristic of an active kinase56, 57 (Figure 2C). In relation to this property, the side chain of E244 forms salt bridge with K227 in chain A, and the αC helix moves inward in chain A relative to chain B to form the canonical closed conformation. These properties for chain A are conserved functional features for phosphotransfer from bound ATP to a protein substrate1, 57.

Table 2: Face-to-face Dimers of Possible Autophosphorylation Intermediates of Ser/Thr Kinases Allosteric regulator

Activation segment

R-spine†

PDB code & Ref

None

None

Nonexchanged

Assembled: disassembled

5XZW. This work

SCD+FHA +kinase

AMPPNP

None

Nonexchanged


5XZV. This work

3 / Rad53

SCD+FHA +kinase

None

pT5

Assumed

4 / Rad53

Kinase*

None

None

Not available

Assembled: assembled

4PDP & 4PDS (AMPPNP)33

5 / Chk2

Kinase

ADP

None

Exchanged


2CN531

6 / Chk2

FHA+ Kinase*

None

None

Not available

Disassembled: disassembled

3I6U32

7 / Aurora A

Kinase

AMPPCP

TPX

Exchanged


4C3P9

8 / IRAK4

Kinase*

Inhibitor

None

Nonexchanged


4U978

9 / SLK

Kinase (K25T)

Inhibitor

None

Exchanged


2J514

10 / LOK

Kinase

Inhibitor

None

Exchanged


2J7T4

11 / Mst2

Kinase*

None

None

Nonexchanged


4LG4 & 4LGD (RASSF5)7

12 / PAK

Kinase*

AMPPNP

None

Nonexchanged


3Q4Z58

Number Name

/

Domains

Substrate Inhibitor

1 / Rad53

SCD+FHA +kinase

2 / Rad53

/

This work (SAXS model)

*

A kinase-dead mutant was used. Assembled R-spine is characteristic of an active kinase56, 57

†


14

Page 15 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

The structure of the apo-dimer crystal soaked with AMPPNP was also solved (at 3.1 Å, Table 1). The nucleotide was found in the active protomer (chain A) as shown in Figure 2Da. The conformations of both protomers were not perturbed and remained with assembled/disassembled R-spines. The position and conformation of the bound AMPPNP are very similar to that of ADP bound to the kinase domain alone of Chk231 (Figure 2Db). Taken together, the results suggest that the unphosphorylated Rad53 can sample the active conformation to allow functional binding of ATP for autophosphorylation reaction, which may provide the molecular basis for the autophosphorylation of unprimed Rad53 in vitro and in bacteria in the absence of DNA damage37, 59.

Structural analyses of the roles of the three Ser/Thr residues at the activation segment. The respective roles of residues S350, T354, and T358 at the activation segment were not entirely clear, since autophosphorylation has been shown to occur at all three residues in vitro (see later sections) and in response to S phase DNA damage in vivo, albeit at a very low level for pT35836. In addition, T387 of Chk2 (corresponding to T358 of Rad53) was considered a crucial phosphorylation site exerting its function through phosphorylation29. We thus assessed the role of each residue by comparing the conformations of the catalytic loop and the activation segment of chain A (Figure 2Ea) and chain B (Figure 2Eb) with that of phosphorylated, fully activated protein kinase A (PKA) (Figure 2Ec)1, 60. Since T354 is conformationally analogous to T197 in PKA, it should be the prototypical activation loop phospho-residue which, upon phosphorylation, mediates the electrostatic interaction with the conserved R318 of the catalytic loop. Similar to the conserved T201 of the P+1 loop in PKA, T358 in chain A of Rad53 forms an H-bond network with D319 and K321 of the catalytic loop, highlighting the phosphorylation independent role of T358 in


15

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 38

maintaining the active conformation. These analyses led to the structure-based sequence alignment of the catalytic cleft shown in Figure 2F where the residues highlighted in yellow (T354/Rad53; T383/Chk2) are the prototypical activation loop phospho-residue, while those highlighted in green (T358/Rad53; T387/Chk2) are the conserved threonine residues in the P+1 loop that play a critical function in the unphosphorylated state. Even though the electron density of S350 is non-discernible possibly due to its flexibility, the above analyses suggest that S350 is not highly conserved among kinases, and that its phosphorylation may not be as relevant to functions as pT354 and pT358.

Phospho-primed Rad53SFK forms a stable dimer different from unprimed Rad53SFK monomer or its transient dimer. Small-angle X-ray scattering (SAXS) data were obtained for unprimed Rad53SFK monomer and pT5- and pT8-Rad53SFK dimers in solution (Figure 3A), while pT12- and pT15-Rad53SFK were unsuitable for SAXS analysis due to precipitation and oligomerization, respectively, at high concentration as mentioned earlier (Figure S4). The samples were free of aggregation based on the Guinier analysis at low scattering angles (q) (Figure S8). The corresponding pair-distance distribution functions P(r) and Kratky plots are shown in Figure 3 B and C, respectively, and the data are summarized in Table 3. Overall, the data support that unprimed Rad53SFK is a highly flexible monomer whereas pT5- and pT8-Rad53SFK are more compact dimers, based on the following properties of unprimed Rad53SFK relative to pT-Rad53SFK: the larger value of radius of gyration (Rg), the failure to converge to the baseline in the Kratky plot, the smaller Porod volume, and the wider distributions of Rg and Dmax selected by the ensemble optimization method (EOM) analysis61 (Figure 3D). Since the data for pT5- and pT8-Rad53SFK dimers are very similar, and since more supporting biochemical results for the pT5 one have been demonstrated24, 35, we focused on pT5-Rad53SFK in


16

Page 17 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Table 3. Summary of SAXS data. Protein construct

Rad53SFK

pT5Rad53SFK

pT8Rad53SFK

Q region (Å-1)

0.05-0.35

0.05-0.35

0.05-0.35

Rg (Å) from Guinier

37.7 ± 0.25

32.9 ± 0.14

34.6 ± 0.25

Rg (Å) from P(r)

38.31 ± 0.12 32.40 ± 0.11

34.30 ± 0.19

Dmax (Å) from P(r)

131

96

102

Porod volume (Å3) estimate from 83543 P(r)

150844

174176

Dry volume (Å3) calculated from 65938 sequence

131875

131875

Calculated tetrameric molecular 51934 weight from sequence (Da)

103708

103708

Software employed Primary data reduction

NSRRC 23A SWAXS package

Data processing

ATSAS 2.6.1

SAXS profile computation

CORAL

Three-dimensional representations

PyMOL

generating possible models for the dimeric structure from SAXS results. At first we tried to fit the crystal structure to the scattering data. However, the scattering curves computed from the resulting structural model did not conform well to the experimental data of pT5-Rad53SFK, based on a Chi value of 9.6 (Figure 3Ea and 3F). We then tried to obtain a more plausible model of the dimeric pT5-Rad53SFK solution structure by adding additional information: (i) The two kinase domains were modeled with the dimeric crystal structure of the segment-exchanged kinase domain of Chk231. (ii) The two FHA domains


17

Biochemistry

were constrained by reciprocal binding between pT5 and the FHA domain, based on the solution structure of the complex between pT5-SCD1 peptide and the FHA1 domain35. (iii) We performed chemical cross-linking experiments with BS2G and identified several intramolecular cross links

unprimed 20 pT5 1612 pT8 0.0001 P(r)

12 88 4 00

0

0.1 0.2 q (Å-1)

D

10X -1 0.1

-2 0.01

pT5-Rad53SFK

-4 0.0001

0

0.001 -3

0.1

0.2

q (Å-1)

00 0.06 0.12 0.18 0.060.12 0.12 0.18 0.06 0.18 -1) (Å-1 qq (Å )

E

10X

Data 0.1-1 Fit 0.01-2

c 2= 1.16

0.001-3

FHA

Data Fit

90o

c 2 = 1.17

0.0001 0.0001 -4 -4 0 0.08 0.16 0.08 0.16 0 0.08 0.16 q (Å-1) q (Å-1 ) q (Å-1) (b) -1 x10-1 x10-2 x10 0.22 0.088 Pool 0.22 Pool Pool Sel. Sel. Sel. 0.11 0.044 0.11

Kinase

Rg distribution Frequency

0

(c) Dmax distribution Frequency

0.088

00

40 (Å)

x10-2

25

125 (Å)

50 (Å)

75

x10-1

Pool Sel.

25

0.16 1.6 0.08 0.8

50 (Å)

FHA

75

x10-1

90o

Pool Sel.

00 00 200 75 150 225 300 75 150 225 300 (Å) (Å)

75 Å

(b)

00

0.16 Pool 1.6 Sel. 0.8 0.08

0.044 00 50

60

60 Å

Model from crystal. Chi = 9.6

70 Å

00 20

0.18 0.18

pT8-Rad53SFK (a)

10X

Data 0.1-1 Fit

c 2 = 1.09

0.08 0.08 0.13 0.13

1.00E-04 1.00E-04 -4-4

0.0E+000 50 100 150 r (Å)

model a CORAL model b -3 Crystal -3 pT5pT5

1.00E-03 1.00E-03 -3-3

4.0E-054

0.01 -2

-3 0.001

pT5 1.00E-02 pT8 1.00E-02 -2-2 0.0001

8.0E-058

0

Unprimed

Ensemble Fit Intensity (d. u.)

(a)

unprimed pT5 12 1.2E-04 pT8 0.0001 Q2 * I

Intensity (d. u.)

0.1-1 0.01-2 0.001-3 0.0001-4

X

10X 10 1.00E-01-1 unprimed 1.00E-01-1

x10-5

x10-5

10X

F

C

u.) (d.u.) Intensity Intensity (d.

B

A

80 Å

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 38

Kinase 75 Å

50 Å

Model from Chk2 dimer. Chi = 1.1

Figure 3. SAXS Analyses of Unprimed Rad53SFK, pT5-Rad53SFK, and pT8-Rad53SFK. (A) SAXS scattering intensities. (B) Pair-distance distribution functions derived from SAXS. (C) Kratky analysis. (D) Analysis of SAXS data by EOM: (a) averaged theoretical scattering intensities from an ensemble of conformations (red) and the experimental curves (blue); (b) the Rg frequency distribution function (red) from an initial pool of 50000 structures (blue); and (c) the Dmax frequency distribution function (red) from an initial pool of 50000 structures (blue). (E) Structural models for pT5-Rad53SFK by CORAL: (a) the model based on two monomers from the crystal of Rad53SFK; (b) the model based on the segment-exchanged dimer structure of the Chk2 kinase domain31 and additional restraints described in the text. (F) Fitting of the theoretical scattering curves calculated from the two models in (E) to the experimental data of pT5Rad53SFK. The graph indicates that the model from Chk2 dimer (b) fits better to the experimental data.


18

Page 19 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

between loop β2’-β3’ of the FHA domain and the loop connecting the FHA domain and kinase domain (Table S3), which were included as additional constraints. (iv) The FHA domain was allowed to move relative to the kinase domain. The resulting model agrees well with experimental data with Chi = 1.1 (Figure 3Eb and 3F). In this model, the FHA domain and the kinase domain in each protomer are closer to each other, and the dimer becomes more compact as indicated by the spatial dimensions in Figure 3Eb. The result suggests that phospho-priming not only induces dimerization, but also controls the structure of the dimer.

Phospho-priming facilitates Rad53 autophosphorylation and controls its specificity at T354. As phospho-priming triggers dimerization of Rad53 with a specific conformation, we anticipated that it will facilitate autophosphorylation at very low concentrations. To demonstrate this effect, we measured the specific activity of the autophosphorylation of unprimed Rad53SFK and pT8-Rad53SFK at varying protein concentrations by an ADP-based phosphatase-coupled kinase universal assay62. As shown in Figure 4A, the specific activity of unprimed Rad53SFK was enhanced by increasing concentration as expected. On the other hand, the specific activity of pT8Rad53SFK remained constant from 0.08 M (where the unprimed was inactive) to 5 M, indicating that its specific activity of autophosphorylation is concentration-independent above the concentrations of pT-FHA binding-mediated dimerization. We next compared site specificities between unprimed Rad53SFK monomers and pT-Rad53SFK dimers based on phosphorylation-dependent electrophoretic mobility shifts (Figure 4B) and labelfree semi-quantitative MS analysis47. Only pT8-Rad53SFK is shown in Figure 4B, while the other three isomers pT5-, pT12-, and pT15-Rad53SFK are shown in Figure S9A as they behaved almost identically to pT8-Rad53SFK. The protein concentration was fixed at 5 M, where the


19

Biochemistry

concentration-dependent autophosphorylation activity of unprimed Rad53SFK has plateaued (Figure 4A) and all four isomers of pT-Rad53SFK exist mainly as dimers (Figure 1E and S4). Moreover, we varied the availability of ATP and the reaction time in order to compare the autophosphorylation patterns at initial and progressive stages. As shown in Figure 4B, pT8-Rad53SFK autophosphorylation produced a major band (band 3) promptly and throughout all conditions. On the other hand, unprimed Rad53SFK gave two light

20 0 -20 0.0

40 40 20 20 00

Unprimed Rad53SFK ATP/kinase = 4:1

E

F(a)

40

1 5 10 30 (min)

30 60 20 40 10 20

00

ATP/kinase= 40:1 (1 min) Unp pT5 pT8 pT12 pT15 pT8-R70A

ES (%)

40

60 60

3

*

*

60

D

0 1

5 10 30 60 90 (min)

100 80 80 60 60 40 40 20 20 00

ATP/kinase= 1:1 (30 min)

ES (%)

80

C

ATP/kinase 40:1

p(T179-S185) pT354

pS350 pT358

band 3

80 80 band 1 60 60

40 40

band 2

20 20 00

Unp Unp pT5 pT8 pT12pT15 Enzyme: pre-autophosphorylated Rad53FKSF 5+1 5+5 5+10 5+30 (min)

(b) ES (%)

B

Unprimed Rad53SFK pT8-Rad53SFK ATP/kinase 1:1 4:1 M M 0 1 5 10 30 1 5 10 30 (min) 140 70 120 Unp 55 Unp 70 2 55 100 55 1 80 0 M 70 60 70 70 pT8 55 pT8 55 40 3 55 20 Unp 70 0 D339A 55 -20 * Hyper autophosphorylation 0 1 2 3 4 5 0.5

ES (%)

Specific activity (pmole/μg/min)

A

ES (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 38

100 80 80 60 60 40 40 20 20 00

Substrate: D339A-Rad53SFK 1 5 10 30 (min)

Figure 4. Autophosphorylation Analyses In Vitro. (A) Specific activities of the autophosphorylation of unprimed Rad53SFK and pT8-Rad53SFK at various concentrations. The left panel is the inset of the low concentration region indicated by the blue rectangle. (B) Phosphorylation-dependent electrophoretic mobility shifts for unprimed Rad53SFK and pT8Rad53SFK, at different time points and under different [ATP]/[protein] ratios. The bottom panel shows D339A-Rad53SFK as a negative control. (C) Bar graphs showing the estimated stoichiometry (ES) of the specific autophosphorylation sites of Rad53SFK for the major shifted bands 1 and 2 of unprimed (Unp) and phospho-primed Rad53SFK (band 3, for the four positional isomers pT5, pT8, pT12, and pT15) at 30 min with [ATP]/[protein] = 1. (D) Time dependence of ES% for the total reaction mixture of unprimed Rad53SFK at [ATP]/[protein] = 4. Note that p(T179-S185) is the mixture of mono-phosphorylated species of pT179, pS180, pS181, pT182, pS184 and pS185. (E) The ES% of the total reaction mixtures of indicated Rad53 autophosphorylation after 1 min at [ATP]/[protein] = 40. (F) Same as D, for the enzyme Rad53FKSF after 5 min of pre-autophosphorylation (a), and the substrate D339A-Rad53SFK (b), at [ATP]/[protein] = 40. All experiments were performed in three repeats, and all error bars represent standard deviation.


20

Page 21 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

bands (1 and 2) gradually when [ATP]/[protein] = 1. As the ratio increased, band 1 appeared more rapidly, and extensive “hyper-autophosphorylation” occurred. As a control, mutation of the conserved D339 at the Mg2+ binding loop to alanine abolished the autophosphorylation activity of Rad53SFK (bottom row). Based on MS analyses with [ATP]/[protein] = 1 at 30 min (Figure 4C), bands 1 and 2 are predominantly pS350-Rad53SFK and a mixture of mono-phosphorylated species, respectively, while band 3 is predominantly pT354-Rad53SFK. MS analyses of the total reaction mixtures with [ATP]/[protein] = 4 further showed that pS350 is the preferred autophosphorylation site for unprimed Rad53SFK throughout the reaction time course 1 – 30 min (Figure 4D). With [ATP]/[protein] = 40, the pS350 preference for unprimed and pT354 preference for pT-Rad53SFK are also clear at 1 min (Figure 4E) and 30 min (Figure S9B). The result at 30 min also shows that hyper-autophosphorylation mainly arises from phosphorylation at the linker region T179-S185 between the FHA1 domain and the kinase domain. Such hyper-autophosphorylation could not occur for phospho-primed Rad53SFK likely because it remained mostly dimeric throughout the reaction (Figure S9C). It also explains the lower plateaued activity of pT8-Rad53SFK in Figure 4A. The different specificities of the initial autophosphorylation between unprimed and pT-Rad53SFK constructs could be attributed to the dimerization mediated through FHA-pT binding. To provide support for this point, we show that the R70A mutation altered the autophosphorylation specificity of pT8-Rad53SFK, resulting in a pattern similar to that of unprimed Rad53SFK with higher levels of pS350 over pT354 (Figure 4E). The results taken together demonstrate that although unprimed Rad53SFK can sample active conformations to enable autophosphorylation, the autophosphorylation is concentration dependent and is predominantly specific to pS350. On the other hand, phospho-priming induces formation of


21

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 38

stable dimers, enables concentration-independent autophosphorylation, and confers the autophosphorylation specificity of pT354. It is important to point out that the pT354 versus pS350 specificity was made possible by the use of MS analysis. A recent study on the kinase domain alone of Rad53 used an antibody specific to both pS350 and pT354 but reported the result as pT35433.

Correlation of functional differences with structural differences in solution. Since autophosphorylation occurs in solution, it is reasonable to conclude that the structural model of pT5-Rad53SFK in Figure 3Eb, which is based on solution SAXS data, is the structure responsible for the pT354 specificity. To provide support that the unprimed Rad53SFK indeed undergoes autophosphorylation via an active:inactive E:S complex as that captured in crystals, we reconstituted the complex using a longer “enzyme construct” (Rad53FKSF, residues 19-732, Figure 1C) and a kinase-dead “substrate construct” D339A-Rad53SFK. At first we pre-incubated the intended enzyme Rad53FKSF with MgATP for 5 min and then mixed it with D339A-Rad53SFK (1:4, with [ATP]/[protein] = 40) and monitored the reaction with time. The phosphorylation patterns of both samples were analyzed separately as shown in Figure 4Fa and Fb, respectively. The results indicate that unprimed Rad53FKSF was pre-autophosphorylated in an expected pattern (pS350 is predominant over pT354, and extensive hyper-autophosphorylation), while the intended substrate D339A-Rad53SFK was also phosphorylated with time to reach the same pattern as the enzyme molecule. Taken together, the results support that the autophosphorylation of unprimed Rad53SFK in solution could also go through an active:inactive E:S intermediate. In addition, the results also support that the autophosphorylation events of unprimed Rad53SFK result mainly from trans autophosphorylation.


22

Page 23 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Further MS analyses to dissect the functional roles of the three Ser/Thr residues at the activation segment. We then further used MS analyses to dissect the functional roles of the three Ser/Thr residues at the activation loop by autophosphorylation-dependent mobility shifts in Figure 5A and the corresponding semi-quantitative MS analysis in Figure 5B for S350A, T354A, and T358A mutants. In support of the important role of T358 (Figure 2Ea), the results show that T358A mutation and K321A mutation substantially reduced the autophosphorylation activity of unprimed Rad53SFK (Figure 5Aa and 5Ba). In addition, T358A mutation also substantially slowed down the autophosphorylation of the pT8-Rad53SFK dimer (Figure 5Ab and 5Bb). These results support the pivotal role of T358 in stabilizing the active conformation for Rad53 autophosphorylation. Although T354 has been suggested to be essential for Rad53 phosphorylation of Dun163, the relative roles of S350 and T354 remain to be demonstrated since both sites can be phosphorylated in vitro (as described above) as well as in vivo36. The mobility shifts in Figure 5Aa indicate that S350A is relatively unchanged while T354A is substantially reduced from WT, which was further analyzed by MS. As shown in Figure 5Ba, S350A or T354A mutation still allowed autophosphorylation of the other site within the activation loop, though with a delayed effect for pS350 (in T354A) and an enhanced effect for pT354 (in S350A) relative to WT, supporting that autophosphorylation can occur at both sites, though pS350 is preferred by the unprimed WT Rad53SFK. Interestingly, the hyper-autophosphorylation at p(T179-S185) was nearly abolished in T354A (where pS350 was present) but remained unchanged in S350A (where pT354 was present), suggesting that even though S350 is the predominant site of autophosphorylation for unprimed Rad53SFK, T354 is the most important autophosphorylation site for Rad53SFK activation as predicted from the above structural analysis.


23

Biochemistry

A

B(a)80

ATP/kinase 40:1

(a)

M 0 1 3 5 10 30 60 90 (min)

ES (%)

Unprimed 70 Rad53SFK 55

S350A- 70 Rad53SFK 55 T354A- 70 Rad53SFK 55 T358A- 70 Rad53SFK 55

(b)

pT870 T358A55 Rad53SFK

C Enzyme:

Substrate: D328A-Dun1 M 0 1 5 10 30 (min)

Unprimed 70 Rad53SFK 55 S350A70 Rad53SFK

55

T354A70 Rad53SFK 55

10

1

30 (min)

60 40 20 0 80 60 40 20 0 80 60 40 20 0 80 60 40 20 0

WT

S350A

T354A

T358A p(T179- pS350 pT354 pT358 S185)

K321A- 70 Rad53SFK 55 (b) pT870 Rad53SFK 55

ES (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

20 15 10 5 0 20 15 10 5 0

pT8-Rad53SFK

1

30 (min)

pT8-T358A-Rad53SFK

p(T179S185)

pS350

pT354

Substrate: D328A-Dun1 *

*

M

*

T358A70 Rad53SFK 55

*

*

* K321A70 Rad53SFK 55

0

1

5

10

30 (min)

Page 24 of 38

Figure 5. Effects of Activation Segment Mutations on the Autophosphorylation of Rad53SFK. (A) In vitro autophosphorylation of Rad53SFK and its mutants S350A, T354A, T358A, and K321A (a), and pT-Rad53SFK and its mutant pT8-T358A (b), at [ATP]/[protein] = 40, monitored by phosphorylation-dependent electrophoretic mobility shifts. (B) Bar graphs showing the ES% of indicated trans autophosphorylation at different time points of the corresponding samples in A, except K321A (almost no shifts). (C) Kinase activities of autophosphorylated Rad53 toward biological substrate Dun1. Dun1 kinase dead mutant (D328A) was resolved by SDS-PAGE containing Phos-tagTM. The major band that consists of phosphorylation site at T380 is indicated.

To provide further support for that pT354- but not pS350-Rad53SFK possesses kinase activity, we showed that pre-autophosphorylated Rad53SFK (consisting of predominantly pS350 and some pT354) and mutant S350A (consisting of pT354 but not pS350) phosphorylated full-length recombinant Dun1 (a biological substrate of Rad53) to give a specific band (with a * in the figure) initially (Figure 5C), which was shown by MS to be the activation loop-phosphorylated pT380Dun1. On the other hand, T354A and T358A (both consist of pS350 but not pT354) as well as K321A (no autophosphorylation) were unable to phosphorylate Dun1.

In vivo autophosphorylation analyses. Finally, we performed in vivo analyses to provide further support for the in vitro results and interpretation. Although we have previously reported the in vivo autophosphorylation analyses of Rad53 upon methyl methanesulfonate (MMS)-induced DNA damage36, here we used hydroxyurea (HU) as a genomic insult to induce the Rad9-


24

Page 25 of 38

independent DNA replication checkpoint, in order to minimize complication by the Rad9dependent (pT-FHA binding-independent) mechanism. By use of the same quantitative MS approach, “spike-in SILAC”64, two types of phosphopeptide analyses could be performed (Figure S10A): the relative levels of phosphorylation (the WT data were set to 100%) using the normalized heavy phosphopeptide as the reference, and the percent site occupancy of phosphorylation based on depletion of the corresponding unphosphorylated peptide. As a control, we first showed by the depletion analysis that the Mec1/Tel1-targeted SQ/TQ phosphorylation after HU treatment was not significantly affected by the mutation of R70A (Figure 6A), indicating that the rad53-R70A strain did not impair the signaling relay from Mec1/Tel1 to Rad53 noticeably. We then examined how the R70A mutation affected autophosphorylation at the activation segment. Unlike pS350 and pT354, the relative level of pT358 was not upregulated by the HU-induced DNA replication checkpoint responses in S phase (Figure S10B), supporting the conclusion from our in vitro structural and functional analysis that the important functional form

A

B pSCD1 pS24 pSCD2 pT543 0 -20 -40

-60 -80

-100

pS350

Relative level (%)

Relative depletion (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

WT+HU (150mM; 45min) R70A+HU

pT354

100 50 0

C Unprimed Rad53 Rad53 enzyme : Rad53 substrate

Hyper-auto-P, downstream signaling

S350 pT354

OH +

pS350 T354

OPO3

OPO3 OH

Reciprocal pT-FHA binding P

Kinase activity

Figure 6. Autophosphorylation Analyses In Vivo. (A) Bar graphs showing the estimated phospho-stoichiometries of selected Mec1/Tel1-target sites based on the relative depletion ratios of the corresponding unphosphorylated peptides in the indicated yeast strains 45 min after HU treatment. (B) Bar graphs showing the relative phosphorylation levels of the phosphorylation sites within the activation segment in the indicated strains after S phase HU treatment for 45 min. (C) A proposed model based on the main results of this work. Thicker arrows indicate the predominant autophosphorylation mechanisms.

P

Phospho-priming at SCD1


25

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 38

of T358 is the unphosphorylated form. Furthermore, as shown in Figure 6B, the relative levels of pT354 vs. pS350 was reduced by ca. 50% at 45 min after HU treatment, supporting the in vitro finding that the intermolecular binding of phospho-SCD1 with FHA1 could enable the autophosphorylation specifically at T354. DISCUSSION Using semi-synthetic, site-specifically phosphorylated proteins, we have demonstrated that the N-terminal priming-phosphorylation of Rad53SFK can allosterically regulate its solution conformation and the site-specificity of trans autophosphorylation within the activation loop. Unphosphorylated Rad53SFK is monomeric in solution and undergoes relatively nonspecific autophosphorylation, likely via a unique transient E:S complex that can bind ATP analog at the active protomer. Phospho-primed Rad53SFK forms a stable dimer that leads to functionally relevant autophosphorylation at pT354. In addition, we have also dissected the roles of the multiple phosphorylatable sites at the activation segment, and identified hyper-autophosphorylation at the linker between FHA and kinase domains. Figure 6C shows a proposed model for the differences between unprimed (used in most in vitro studies) and phospho-primed (mimicking in vivo regulation) autophosphorylation mechanisms of Rad53 based on these results.

Possible differences between single- and multiple-domain constructs. While emphasizing the functional importance of phospho-priming, it is important to point out that, even in the absence of phospho-priming, mono- and multi-domain constructs of a kinase can also show different properties. For example, even though the kinase domain alone of Rad53 has a high propensity for dimerization33, the multi-domain Rad53SFK is predominantly monomeric in solution (Figure 1E


26

Page 27 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

and S2). A possible explanation is that intramolecular dynamic movements between domains (which are not constrained in the absence of phospho-priming) may destabilize dimerization through the kinase domain. In addition, the structure of Rad53 kinase in the previous study33 and that of Rad53SFK in this work also differ in the R-spine assembly (both assembled for kinase domain alone, and assembled:disassembled for Rad53SFK) (Table 2). For human Chk2, the kinase domain alone and the two-domain (FHA + kinase, no phospho-priming) constructs also gave somewhat different dimer interfaces as well as different R-spine assembly (both assembled and both disassembled, respectively)31, 32.

Possible mechanisms and pathways for the autophosphorylation of kinases. Even though we have emphasized the unique features of the structure of unprimed Rad53SFK, the most significant aspect of our results is the effect of phospho-priming on both the structure and the autophosphorylation specificity of Rad53SFK. Based on our results of Rad53 and previous reports, we summarize the following important issues on the autophosphorylation mechanism of Ser/Thr kinases here: (i) The kinase domain alone of some Ser/Thr kinases is able to sample active conformations and being captured in dimeric structures under higher concentration used for crystallization, as shown in Table 2. These dimeric structures have been suggested as autophosphorylation intermediates. However, whether they lead to functionally relevant autophosphorylation should be examined for each case by MS analysis. (ii) In the presence of allosteric regulation such as phospho-priming of Rad53, the kinase conformation could then be restricted to the functionally relevant ones. The flexibility of activation loop should also be constrained upon phospho-priming, since the autophosphorylation pattern of activation loop is significantly altered in phospho-primed dimers (Figure 4). This constraining of the activation loop


27

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 38

may also play regulatory roles to confer functionally relevant conformations and position the sidechain of T354 for trans autophosphorylation. To fulfill these regulatory roles, swap of activation loop could be a potential intermediate in the compact phospho-dimer, as this mechanism has been demonstrated in the allosteric effects of TPX2 on Aurora A dimer, which not only enables the assembly of R-spine in the enzyme protomer but also rearranges the hydroxyl group of the prototypical activation loop phospho-residue (T288, highlight in yellow in Figure 2F) towards the γ-phosphate of AMPPCP in the substrate promoter9. Based on these analyses, we have included the swap of activation loop in our SAXS structural model of the pT5-Rad53SFK dimer (Figure 3Eb), though it remains to be validated by atomic resolution structures.

Multiple auto-activation mechanisms in vivo. Adding to the complexity of a specific mechanism, there are multiple autophosphorylation mechanisms in vivo. For Ser/Thr kinases, a recent report demonstrated that Aurora A kinase could be activated by two mechanisms – autophosphorylation in the early stage of mitosis, and allosteric activation by TPX2 in the spindle microtubules9. Rad53 can also be activated by multiple mechanisms in vivo36, 37 and possibly in vitro also. The present study uncovered molecular details for one each of the possible in vivo and in vitro mechanisms of Rad53 autophosphorylation and identified their differences. The work also lends support for the concept of “conformational landscape”65-69. In this concept, kinases are highly conformationally flexible and they can be activated or auto-activated in multiple ways, and likewise can bind to multiple activators and inhibitors.

Conclusion. In vitro studies designed for elucidating the molecular basis of in vivo functions are often limited by the difficulty in simulating in vivo regulatory mechanisms. By analyzing both


28

Page 29 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

unprimed and phospho-primed Rad53SFK, we have uncovered new insight, as well as identified the differences, in its auto-activation mechanisms in the absence and presence of allosteric regulation. Our results provide experimental evidence supporting that phospho-priming in vivo induces the kinase to the biologically relevant conformation and leads to more effective autophosphorylation and more accurate site specificity. In addition, our results support that the recruiting process in cell signaling could also define the conformation, specificity, and mechanism.


29

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 38

ASSOCIATED CONTENT The following files are available free of charge. Supplemental material and method, figures and tables (PDF) Data deposition: The atomic coordinates have been deposited in the Protein Data Bank (PDB ID for apo Rad53SFK and Rad53SFK in complex with AMPPNP are 5XZW and 5XZV, respectively).

AUTHOR INFORMATION Corresponding Author *Ming-Daw Tsai. E-mail: [email protected]

ORCID for M.-D. T.: 0000-0003-1374-0414 ORCID for W.-J.W.: 0000-0002-7253-5384 ORCID for J.-H. W.: 0000-0002-0432-3515 ORCID for W.-C. H.: 0000-0003-4437-3530

Present Addresses E.S.-W. Chen: Institute of Stem Cell and Translational Cancer Research, Chang Gung Memorial

†

Hospital at Linkou, Taoyuan 333, Taiwan. X.X. Liu: Department of Biophysics, University of Texas Southwestern Medical Center, Dallas,

¶

Texas, USA. Author Contributions These authors contributed equally.

‡


30

Page 31 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Funding Sources This work was supported primarily by grants to MDT from National Health Research Institute (NHRI-EX100-10002NI), Academia Sinica (AS-104-TP-B05), and the Taiwan Protein Project of Academia Sinica, funded by Ministry of Science and Technology [MOST105-0210-01-12-01 and MOST106-0210-01-15-04]. Notes All authors declare no conflicting financial interests. ACKNOWLEDGMENT We thank the National Synchrotron Radiation Research Center (NSRRC) for access to beamlines BL13C, BL13B, and BL15A and BL23A. The synchrotron radiation experiments were also performed at the BL12B2, BL44XU, and BL41XU of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2016A2507, 2014A4013, 2013B4008, 2012A4002, 2011B4009, 2011A4006 and 2011A4009). Use of the Stanford Synchrotron Radiation Lightsource (SSRL) was supported by the U.S. Department of Energy DEAC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by grants including P41GM103393. We also thank Academia Sinica for the support of crystallization, mass spectrometry, and biophysical facilities. The analysis of SAXS data and modeling were performed on the Advanced Large-scale Parallel Supercluster (ALPS) with the support of National Center for High-performance Computing. The NMR data were collected at the High Field NMR Center in Academia Sinica. We also thank Dr. Achille Pellicioli for providing monoclonal antibody EL7E1, Dr. Jörg Heierhorst for providing the yeast mutant strains listed in Table S2, Dr. Yoshitaka Bessho for sharing beamline time, and Drs. Kazuya Hasegawa and Tze-Ping Ko for technical assistance.


31

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 38

ABBREVIATIONS Chk2, Checkpoint kinase 2; PKA, protein kinase A; pT, phosphothreonine; FHA, forkheadassociated; Rad53SF, Rad53 residues 2-164, (consisting of SCD1 and FHA1 domains); Rad53SFK, Rad53 residues 2-466, (consisting of SCD1 and FHA1 and kinase domains); SCD, SQ/TQ cluster domain; SV-AUC, sedimentation velocity analytical ultracentrifugation.


32

Page 33 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16.

Nolen, B., Taylor, S., and Ghosh, G. (2004) Regulation of Protein Kinases: Controlling Activity through Activation Segment Conformation, Molecular Cell 15, 661-675. Krupa, A., Preethi, G., and Srinivasan, N. (2004) Structural Modes of Stabilization of Permissive Phosphorylation Sites in Protein Kinases: Distinct Strategies in Ser/Thr and Tyr Kinases, Journal of Molecular Biology 339, 1025-1039. Zhang, X., Gureasko, J., Shen, K., Cole, P. A., and Kuriyan, J. (2006) An Allosteric Mechanism for Activation of the Kinase Domain of Epidermal Growth Factor Receptor, Cell 125, 1137-1149. Pike, A. C. W., Rellos, P., Niesen, F. H., Turnbull, A., Oliver, A. W., Parker, S. A., Turk, B. E., Pearl, L. H., and Knapp, S. (2008) Activation segment dimerization: a mechanism for kinase autophosphorylation of non‐consensus sites, EMBO J 27, 704-714. Wu, J., Li, W., Craddock, B. P., Foreman, K. W., Mulvihill, M. J., Ji, Q. s., Miller, W. T., and Hubbard, S. R. (2008) Small‐molecule inhibition and activation‐loop trans‐ phosphorylation of the IGF1 receptor, Embo J 27, 1985-1994. Rajakulendran, T., Sahmi, M., Lefrancois, M., Sicheri, F., and Therrien, M. (2009) A dimerization-dependent mechanism drives RAF catalytic activation, Nature 461, 542-545. Ni, L., Li, S., Yu, J., Min, J., Brautigam, Chad A., Tomchick, Diana R., Pan, D., and Luo, X. (2013) Structural Basis for Autoactivation of Human Mst2 Kinase and Its Regulation by RASSF5, Structure 21, 1757-1768. Ferrao, R., Zhou, H., Shan, Y., Liu, Q., Li, Q., Shaw, David E., Li, X., and Wu, H. (2014) IRAK4 Dimerization and trans-Autophosphorylation Are Induced by Myddosome Assembly, Molecular Cell 55, 891-903. Zorba, A., Buosi, V., Kutter, S., Kern, N., Pontiggia, F., Cho, Y.-J., and Kern, D. (2014) Molecular mechanism of Aurora A kinase autophosphorylation and its allosteric activation by TPX2, eLIFE 3, e02667. Xu, Q., Malecka, K. L., Fink, L., Jordan, E. J., Duffy, E., Kolander, S., Peterson, J. R., and Dunbrack, R. L., Jr. (2015) Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases, Sci Signal 8, rs13. Beenstock, J., Mooshayef, N., and Engelberg, D. (2016) How Do Protein Kinases Take a Selfie (Autophosphorylate)?, Trends Biochem Sci 41, 938-953. Shah, N. H., Wang, Q., Yan, Q., Karandur, D., Kadlecek, T. A., Fallahee, I. R., Russ, W. P., Ranganathan, R., Weiss, A., and Kuriyan, J. (2016) An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor, Elife 5, e20105. Hu, J., Stites, Edward C., Yu, H., Germino, Elizabeth A., Meharena, Hiruy S., Stork, Philip J. S., Kornev, Alexandr P., Taylor, Susan S., and Shaw, Andrey S. (2013) Allosteric Activation of Functionally Asymmetric RAF Kinase Dimers, Cell 154, 1036-1046. Jura, N., Endres, N. F., Engel, K., Deindl, S., Das, R., Lamers, M. H., Wemmer, D. E., Zhang, X., and Kuriyan, J. (2009) Mechanism for Activation of the EGF Receptor Catalytic Domain by the Juxtamembrane Segment, Cell 137, 1293-1307. Wang, Z., Longo, P. A., Tarrant, M. K., Kim, K., Head, S., Leahy, D. J., and Cole, P. A. (2011) Mechanistic insights into the activation of oncogenic forms of EGF receptor, Nat Struct Mol Biol 18, 1388-1393. Arkhipov, A., Shan, Y., Das, R., Endres, Nicholas F., Eastwood, Michael P., Wemmer, David E., Kuriyan, J., and Shaw, David E. (2012) Architecture and Membrane Interactions


33

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

17.

18. 19. 20. 21. 22.

23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

Page 34 of 38

of the EGF Receptor, Cell 152, 557-569. Endres, Nicholas F., Das, R., Smith, Adam W., Arkhipov, A., Kovacs, E., Huang, Y., Pelton, Jeffrey G., Shan, Y., Shaw, David E., Wemmer, David E., Groves, Jay T., and Kuriyan, J. (2012) Conformational Coupling across the Plasma Membrane in Activation of the EGF Receptor, Cell 152, 543-556. Cabail, M. Z., Li, S., Lemmon, E., Bowen, M. E., Hubbard, S. R., and Miller, W. T. (2015) The insulin and IGF1 receptor kinase domains are functional dimers in the activated state, Nature Communications 6, 6406. Sun, Z., Hsiao, J., Fay, D. S., and Stern, D. F. (1998) Rad53 FHA Domain Associated with Phosphorylated Rad9 in the DNA Damage Checkpoint, Science 281, 272-274. Nyberg, K. A., Michelson, R. J., Putnam, C. W., and Weinert, T. A. (2002) TOWARD MAINTAINING THE GENOME: DNA Damage and Replication Checkpoints, Annual Review of Genetics 36, 617-656. Gottifredi, V., and Prives, C. (2005) The S phase checkpoint: When the crowd meets at the fork, Seminars in Cell & Developmental Biology 16, 355-368. Hoch, N. C., Chen, E. S.-W., Buckland, R., Wang, S.-C., Fazio, A., Hammet, A., Pellicioli, A., Chabes, A., Tsai, M.-D., and Heierhorst, J. (2013) Molecular Basis of the Essential S Phase Function of the Rad53 Checkpoint Kinase, Molecular and Cellular Biology 33, 3202-3213. Bartek, J., Falck, J., and Lukas, J. (2001) Chk2 kinase - a busy messenger, Nat Rev Mol Cell Biol 2, 877-886. Mahajan, A., Yuan, C., Lee, H., Chen, E. S.-W., Wu, P.-Y., and Tsai, M.-D. (2008) Structure and Function of the Phosphothreonine-Specific FHA Domain, Sci. Signal. 1, re12-. Tanaka, K., Boddy, M. N., Chen, X.-B., McGowan, C. H., and Russell, P. (2001) Threonine-11, Phosphorylated by Rad3 and ATM In Vitro, Is Required for Activation of Fission Yeast Checkpoint Kinase Cds1, Mol. Cell. Biol. 21, 3398-3404. Ahn, J.-Y., Li, X., Davis, H. L., and Canman, C. E. (2002) Phosphorylation of Threonine 68 Promotes Oligomerization and Autophosphorylation of the Chk2 Protein Kinase via the Forkhead-associated Domain, Journal of Biological Chemistry 277, 19389-19395. Xu, X., Tsvetkov, L. M., and Stern, D. F. (2002) Chk2 Activation and PhosphorylationDependent Oligomerization, Molecular and Cellular Biology 22, 4419-4432. Li, J., Taylor, I. A., Lloyd, J., Clapperton, J. A., Howell, S., MacMillan, D., and Smerdon, S. J. (2008) Chk2 Oligomerization Studied by Phosphopeptide Ligation, Journal of Biological Chemistry 283, 36019-36030. Lee, C.-H., and Chung, J. H. (2001) The hCds1 (Chk2)-FHA Domain Is Essential for a Chain of Phosphorylation Events on hCds1 That Is Induced by Ionizing Radiation, Journal of Biological Chemistry 276, 30537-30541. Oliver, A. W., Knapp, S., and Pearl, L. H. (2007) Activation segment exchange: a common mechanism of kinase autophosphorylation?, Trends in biochemical sciences 32, 351-356. Oliver, A. W., Paul, A., Boxall, K. J., Barrie, S. E., Aherne, G. W., Garrett, M. D., Mittnacht, S., and Pearl, L. H. (2006) Trans-activation of the DNA-damage signalling protein kinase Chk2 by T-loop exchange, EMBO J 25, 3179-3190. Cai, Z., Chehab, N. H., and Pavletich, N. P. (2009) Structure and Activation Mechanism of the CHK2 DNA Damage Checkpoint Kinase, Molecular Cell 35, 818-829. Wybenga-Groot, L. E., Ho, C. S., Sweeney, F. D., Ceccarelli, D. F., McGlade, C. J.,


34

Page 35 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

34. 35. 36.

37. 38. 39.

40. 41. 42. 43. 44.

45. 46. 47.

Durocher, D., and Sicheri, F. (2014) Structural basis of Rad53 kinase activation by dimerization and activation segment exchange, Cellular Signalling 26, 1825-1836. Lee, S.-J., Schwartz, M. F., Duong, J. K., and Stern, D. F. (2003) Rad53 Phosphorylation Site Clusters Are Important for Rad53 Regulation and Signaling, Mol. Cell. Biol. 23, 63006314. Lee, H., Yuan, C., Hammet, A., Mahajan, A., Chen, E. S., Wu, M. R., Su, M. I., Heierhorst, J., and Tsai, M. D. (2008) Diphosphothreonine-specific interaction between an SQ/TQ cluster and an FHA domain in the Rad53-Dun1 kinase cascade, Mol Cell 30, 767-778. Chen, E. S.-W., Hoch, N. C., Wang, S.-C., Pellicioli, A., Heierhorst, J., and Tsai, M.-D. (2014) Use of Quantitative Mass Spectrometric Analysis to Elucidate the Mechanisms of Phospho-priming and Auto-activation of the Checkpoint Kinase Rad53 in Vivo, Molecular & Cellular Proteomics 13, 551-565. Gilbert, C. S., Green, C. M., and Lowndes, N. F. (2001) Budding yeast Rad9 is an ATPdependent Rad53 activating machine, Mol Cell 8, 129-136. Otwinowski, Z., and Minor, W. (1997) [20] Processing of X-ray diffraction data collected in oscillation mode, In Methods in Enzymology, pp 307-326, Academic Press. Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L.-W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution, Acta Crystallographica Section D 66, 213-221. Langer, G., Cohen, S. X., Lamzin, V. S., and Perrakis, A. (2008) Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7, Nat. Protocols 3, 1171-1179. Murshudov, G. N., Skubak, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F., and Vagin, A. A. (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallographica Section D 67, 355-367. Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot, Acta Crystallographica Section D 66, 486-501. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) PROCHECK: a program to check the stereochemical quality of protein structures, J. Appl. Cryst. 26, 283-291. Petoukhov, M. V., Franke, D., Shkumatov, A. V., Tria, G., Kikhney, A. G., Gajda, M., Gorba, C., Mertens, H. D. T., Konarev, P. V., and Svergun, D. I. (2012) New developments in the ATSAS program package for small-angle scattering data analysis, Journal of Applied Crystallography 45, 342-350. Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K., and Koike, T. (2006) Phosphatebinding tag, a new tool to visualize phosphorylated proteins, Mol Cell Proteomics 5, 749757. Kinoshita, E., Kinoshita-Kikuta, E., and Koike, T. (2009) Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE, Nat Protoc 4, 1513-1521. Wu, S.-L., Kim, J., Bandle, R. W., Liotta, L., Petricoin, E., and Karger, B. L. (2006) Dynamic Profiling of the Post-translational Modifications and Interaction Partners of Epidermal Growth Factor Receptor Signaling after Stimulation by Epidermal Growth Factor Using Extended Range Proteomic Analysis (ERPA), Molecular & Cellular Proteomics 5, 1610-1627.


35

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

48.

49. 50. 51. 52. 53. 54. 55.

56. 57. 58. 59. 60.

61. 62. 63.

Page 36 of 38

Shen, Y. F., Chen, Y. H., Chu, S. Y., Lin, M. I., Hsu, H. T., Wu, P. Y., Wu, C. J., Liu, H. W., Lin, F. Y., Lin, G., Hsu, P. H., Yang, A. S., Cheng, Y. S., Wu, Y. T., Wong, C. H., and Tsai, M. D. (2011) E339...R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors, Proc Natl Acad Sci U S A 108, 16515-16520. Dawson, P. E., Muir, T. W., Clark-Lewis, I., and Kent, S. B. (1994) Synthesis of proteins by native chemical ligation, Science 266, 776-779. Johnson, E. C. B., and Kent, S. B. H. (2006) Insights into the Mechanism and Catalysis of the Native Chemical Ligation Reaction, Journal of the American Chemical Society 128, 6640-6646. Muir, T. W., Sondhi, D., and Cole, P. A. (1998) Expressed protein ligation: a general method for protein engineering, Proc Natl Acad Sci U S A 95, 6705-6710. Huse, M., Muir, T. W., Xu, L., Chen, Y. G., Kuriyan, J., and Massague, J. (2001) The TGF beta receptor activation process: an inhibitor- to substrate-binding switch, Mol Cell 8, 671682. Pickin, K. A., Chaudhury, S., Dancy, B. C., Gray, J. J., and Cole, P. A. (2008) Analysis of protein kinase autophosphorylation using expressed protein ligation and computational modeling, J Am Chem Soc 130, 5667-5669. Singla, N., Erdjument-Bromage, H., Himanen, J. P., Muir, T. W., and Nikolov, D. B. (2011) A semisynthetic Eph receptor tyrosine kinase provides insight into ligand-induced kinase activation, Chem Biol 18, 361-371. Liao, H., Yuan, C., Su, M.-I., Yongkiettrakul, S., Qin, D., Li, H., Byeon, I.-J. L., Pei, D., and Tsai, M.-D. (2000) Structure of the FHA1 Domain of Yeast Rad53 and Identification of Binding Sites for both FHA1 and its Target Protein Rad9, Journal of Molecular Biology 304, 941-951. Kornev, A. P., Taylor, S. S., and Ten Eyck, L. F. (2008) A helix scaffold for the assembly of active protein kinases, Proceedings of the National Academy of Sciences 105, 1437714382. Taylor, S. S., Shaw, A. S., Kannan, N., and Kornev, A. P. (2015) Integration of signaling in the kinome: Architecture and regulation of the αC Helix, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1854, 1567-1574. Wang, J., Wu, J.-W., and Wang, Z.-X. (2011) Structural Insights into the Autoactivation Mechanism of p21-Activated Protein Kinase, Structure 19, 1752-1761. Ma, J.-L., Lee, S.-J., Duong, J. K., and Stern, D. F. (2006) Activation of the Checkpoint Kinase Rad53 by the Phosphatidyl Inositol Kinase-like Kinase Mec1, J. Biol. Chem. 281, 3954-3963. Zheng, J., Trafny, E. A., Knighton, D. R., Xuong, N., Taylor, S. S., Ten Eyck, L. F., and Sowadski, J. M. (1993) 2.2 A refined crystal structure of the catalytic subunit of cAMPdependent protein kinase complexed with MnATP and a peptide inhibitor, Acta Crystallographica Section D 49, 362-365. Tria, G., Mertens, H. D. T., Kachala, M., and Svergun, D. I. (2015) Advanced ensemble modelling of flexible macromolecules using X-ray solution scattering, IUCrJ 2, 207-217. Wu, Z. L. (2011) Phosphatase-Coupled Universal Kinase Assay and Kinetics for FirstOrder-Rate Coupling Reaction, PLoS ONE 6, e23172. Chen, S.-H., Smolka, M. B., and Zhou, H. (2007) Mechanism of Dun1 Activation by Rad53 Phosphorylation in Saccharomyces cerevisiae, Journal of Biological Chemistry 282, 986995.


36

Page 37 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

64. 65. 66. 67. 68. 69.

Geiger, T., Wisniewski, J. R., Cox, J., Zanivan, S., Kruger, M., Ishihama, Y., and Mann, M. (2011) Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics, Nat. Protocols 6, 147-157. Huse, M., and Kuriyan, J. (2002) The conformational plasticity of protein kinases, Cell 109, 275-282. Agafonov, R. V., Wilson, C., Otten, R., Buosi, V., and Kern, D. (2014) Energetic dissection of Gleevec's selectivity toward human tyrosine kinases, Nat Struct Mol Biol 21, 848-853. Shukla, D., Meng, Y., Roux, B., and Pande, V. S. (2014) Activation pathway of Src kinase reveals intermediate states as targets for drug design, Nat Commun 5, 3397. Kornev, A. P., and Taylor, S. S. (2015) Dynamics-Driven Allostery in Protein Kinases, Trends Biochem Sci 40, 628-647. Wilson, C., Agafonov, R. V., Hoemberger, M., Kutter, S., Zorba, A., Halpin, J., Buosi, V., Otten, R., Waterman, D., Theobald, D. L., and Kern, D. (2015) Using ancient protein kinases to unravel a modern cancer drug’s mechanism, Science 347, 882-886.


37

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 38

For Table of Contents Only

SCD Unprimed Rad53 Rad53 enzyme : Rad53 substrate Hyper-auto-P, downstream signaling P

S350 pT354

OH

OPO3 pS350 T354 OPO3 OH

+

Kinase activity

P

Phospho-priming at SCD1 Reciprocal pT-FHA binding


38

Phospho-priming Confers Functionally Relevant Specificities for

Recommend Documents