Identifying three-dimensional structures of ...

RESEARCH RESOURCE STRUCTURAL BIOLOGY

Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases Qifang Xu,1 Kimberly L. Malecka,1 Lauren Fink,1 E. Joseph Jordan,2 Erin Duffy,1* Samuel Kolander,1† Jeffrey R. Peterson,1 Roland L. Dunbrack Jr.1‡

INTRODUCTION

Protein kinases play important roles in many cellular signaling pathways, such as cell cycle regulation and apoptosis (1). Problems in kinase regulation can lead to diverse illnesses ranging from cancer (2) to obesity (3). Activity of most kinases is partially regulated by the phosphorylation status and position of the activation loop, which begins with the highly conserved DFG (Asp-Phe-Gly) motif and ends with a sequence usually similar to APE (Ala-Pro-Glu) (4). In many kinases, the nonphosphorylated activation loop occupies a position that interferes with substrate binding. When phosphorylated, usually by trans-autophosphorylation (meaning, by a second instance of the same kinase), the activation loop becomes repositioned, providing access to the active site for substrates and rearranging several residues required for catalysis (5). Many kinases contain additional sites outside the activation loop that are also trans-autophosphorylated (6). Several kinase structures have been reported in which a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer is present in the active site of another monomer of the same protein in the crystal (7–15). In these structures, the position of the phosphorylation site and adjacent residues resembles those of substrates in structures of substrate peptides bound to kinases (16–18). Phosphorylation sites reported in autophosphorylation complexes in crystals include a tyrosine in the juxtamembrane region that is N-terminal to the kinase domain of the receptor tyrosine kinase c-KIT [Protein Data Bank (PDB: 1PKG)] (7), a tyrosine in the “kinase insert 1 Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111, USA. 2Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA. *Present address: Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA. †Present address: Albert Einstein College of Medicine, Bronx, NY 10461, USA. ‡Corresponding author. E-mail: [email protected]

region” of fibroblast growth factor receptor 1 (FGFR1) [PDB: 3GQI (8)] and of FGFR3 [PDB: 4K33 (14)], a tyrosine in the C-terminal tail of FGFR2 [PDB: 3CLY (9)] and of epidermal growth factor receptor (EGFR) [PDB: 4I21 (19)], and a tyrosine in the activation loop of insulin-like growth factor 1 receptor (IGF1R) [PDB: 3D94 (10)]. In all of these, the tyrosine side chain of the substrate kinase is hydrogen-bonded to the catalytic Asp side chain of the active site HRD (His-Arg-Asp) motif of the enzyme kinase [the site in FGFR1 (PDB: 3GQI) has been mutated to Phe but is correctly positioned if it were Tyr]. Furthermore, each of these residues is an experimentally verified autophosphorylation site in these kinases. For serine/threonine kinases, autophosphorylation complexes of the activation loop Thr residues of p21-activated kinase (PAK1) [PDB: 3Q4Z (11)] and interleukin-1 receptor–associated kinase 4 (IRAK4) [PDB: 4U97 and 4U9A (15)] have been described, as have autophosphorylation complexes of the C-terminal regulatory regions of human [PDB: 2WEL (12)] and Caenorhabditis elegans [PDB: 3KK8 and 3KK9 (13)] calcium/calmodulin-dependent kinase II (CaMKII). Because of the importance of understanding kinase activation processes and kinase-substrate recognition, we sought to identify undetected autophosphorylation complexes in crystals of kinases in the PDB using a structural bioinformatics approach. Using the symmetry information for each crystal provided by the PDB, we constructed all distinct interfaces between monomers in 3525 kinase crystals in the PDB (as of 24 October 2015) and measured the distance between the Asp oxygen atoms of the HRD motif in one monomer and the hydroxyl groups on Ser, Thr, and Tyr of the other monomer, and vice versa. This approach properly identified the 10 previously described autophosphorylation complexes listed above and identified five more that were not described as such in the relevant papers. The newly identified autophosphorylation complexes include (i) the activation loop Tyr of the human

www.SCIENCESIGNALING.org

1 December 2015

Vol 8 Issue 405 rs13

1

Downloaded from http://stke.sciencemag.org/ on December 1, 2015

Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation complexes in x-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which five complexes had not previously been described in the publications describing the crystal structures. These five complexes consist of tyrosine residues in the N-terminal juxtamembrane regions of colony-stimulating factor 1 receptor (CSF1R, Tyr561) and ephrin receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore, we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets.

RESEARCH RESOURCE

RESULTS

Potential autophosphorylation complexes in crystals of protein kinases As of 24 October 2015, the PDB contained 3525 crystal structures of proteins that contain a Ser or Thr kinase domain or a Tyr kinase domain as identified with the associated hidden Markov models (HMMs) provided by Pfam (30) (Pfam models “Pkinase” and “Pkinase_Tyr,” respectively). These PDB entries contain structural information on 237 human kinases, 25 mouse kinases, and 103 kinases from other species. To identify possible autophosphorylation complexes in each crystal, we needed to build coordinates for a portion of the crystal large enough to contain at least one example of all distinct protein-protein interactions that make

up the crystal, using methods described previously (31, 32). We started our analysis with the asymmetric unit (ASU), the smallest part of the crystal structure to which symmetry rules can be applied to produce coordinates for the entire crystal (33). We used information on the symmetry of each crystal provided by the PDB to build a unit cell of the crystal, a cuboid object (or parallelepiped) consisting of multiple copies of the ASU. Once we built a compact unit cell for a crystal, we built a 3 × 3 × 3 collection of unit cells of the crystal by copying each unit cell and translating it in the ±x, ±y, and ±z directions as needed. This was done to guarantee that we have all possible interactions of the original ASU with all other neighboring ASUs in the crystal. Each protein in the assembly of 27 unit cells is given an identifier in a standard format, such as “A:2_555.” The letter A is the chain identifier of the original ASU, which might consist of chains A, B, and C, for instance, if there are three such chains in the ASU. The first number after the colon is the identifier of the symmetry operator (a rule to copy, rotate, and translate the coordinates of the ASU) used to orient the ASUs in the unit cell (“2” in this example). The number 555 indicates the unit cell at the center of our 3 × 3 × 3 box; neighboring unit cells of the center cell in the x, y, and z directions are designated by adding or subtracting 1 from these numbers. As another example, “B:3_655” indicates author chain B, symmetry operator 3 for the space group of the crystal form, and a neighboring unit cell one step in the positive x direction. After building a 3 × 3 × 3 collection of unit cells for each crystal, we determined the list of distinct protein-protein interfaces in the crystal for further analysis. Formation of a hydrogen bond between the substrate hydroxyl group of the Ser, Thr, or Tyr side chain and the catalytic aspartic acid residue side chain of the active site HRD motif is a critical step in the mechanism of phosphorylation by protein kinases (34). Therefore, we calculated the distances between hydroxyl oxygen atoms of Thr, Ser, or Tyr of each monomer with the Asp carboxylate atoms (Od1 and Od2) of the HRD motif for each of the other monomer in each of the distinct homodimers in each crystal. Because some kinase structures in the PDB contain phosphorylated amino acids and phosphomimetic mutations of phosphorylation sites to Asp or Glu, we also measured the distances of the appropriate oxygen atoms of these side chains and the active site Asp. Additionally, substrate residue side chains often form hydrogen bonds with the side chain of arginine or lysine residues located two or four amino acids after the HRD motif (HRD+2 and HRD+4) (35), and where this residue was present, we calculated potential hydrogen bond distances between hydrogen bond acceptor oxygen atoms in the substrate side chain and hydrogen bond donor nitrogens in the Arg or Lys side chain of HRD+2 or HRD+4 (usually only one of these is present in the sequence). With this procedure, we identified 15 potential unique autophosphorylation complexes in 26 PDB entries. These structures and the autophosphorylation sites contained in them are listed in Table 1, which provides details on each autophosphorylation complex including the chains involved and their symmetry relationship, the relevant substrate and enzyme residues, the substrate sequence about the phosphorylation site, the surface area of interaction of the two kinases, and the HRD-phosphosite hydrogen bond distance. Some crystals contain more than one unique instance of the autophosphorylation complex, and these are indicated in Table 1. Often, the complexes are very similar, but in some cases, they may have distinct structural features, such as the locations and extent of domain-domain interactions between the kinases. The mean and median hydrogen bond lengths (oxygento-oxygen atom distances) are 3.16 and 2.91 Å, respectively (picking only the shortest distance for each phosphosite, if there are multiple structures), which are typical of hydroxyl/carboxylate hydrogen bonds. The mean and median interface surface areas are 913 and 848 Å2, respectively, indicating extensive interactions between the kinases beyond the residues

www.SCIENCESIGNALING.org

1 December 2015

Vol 8 Issue 405 rs13

2

Downloaded from http://stke.sciencemag.org/ on December 1, 2015

nonreceptor tyrosine kinase LCK [PDB: 2PL0 (20)], which is similar to the IGF1R structure (10); (ii) a second tyrosine autophosphorylation site (Tyr1166) in the activation loop of human IGF1R [PDB: 3LVP (21)]; (iii) a Tyr in the N-terminal juxtamembrane region of human colony-stimulating factor 1 receptor (CSF1R) [PDB: 3LCD (22)] that is homologous to the tyrosine observed in the c-KIT complex (7); (iv) a Tyr in the N-terminal juxtamembrane region of ephrin receptor A2 (EPHA2) [PDB: 4PDO (23)] that represents a phosphorylation site near but distinct from that in the c-KIT and CSF1R complexes; and (v) a Ser in the C-terminal tail of human CDC2/CDC28-like kinase 2 (CLK2) [PDB: 3NR9 (24)]. We also identified several additional structures of autophosphorylation complexes of PAK1 including PDB: 4O0R, 4O0T (25); 4P90 (26); 4ZY4, 4ZY5, 4ZY6 (27); and 4ZLO, 4ZJI, and 4ZJJ (28). Two of these structures, PDB: 4ZY4 and 4ZY5, contain an autophosphorylation complex with a fully ordered activation loop in the substrate kinase, whereas the others, including PDB: 3Q4Z (11), are missing the coordinates of 8 to 11 residues within the activation loop. Comparison of all the newly identified sites to the structures of previously published peptide substrate–kinase complexes in the PDB indicated that these sites are consistent with catalysis of phosphorylation. However, comparison of one of the previously described autophosphorylation complexes, EGFR [PDB: 4I21 (19)], was not consistent with recently published structures of EGFR in complexes with peptide substrates. Because of the central importance of activation loop autophosphorylation in the regulation of tyrosine kinases, we performed site-directed mutagenesis of the autophosphorylation complex interface of LCK that we identified in PDB: 2PL0, changing several residues in the G helix that contacts the activation loop of the opposing monomer, and analyzed the phosphorylation of these when expressed in cells. A mutation in Pro447 in LCK has previously been identified as activating and is associated with T cell leukemia (29). Whereas some mutations in the interface region impaired autophosphorylation, mutation of Pro447 to Gly, Ala, or Leu maintained or increased autophosphorylation activity. By comparing all autophosphorylation complexes with each other and with the structures of peptide-bound kinases, we discovered common structural elements in subsets of the complexes, providing insights regarding the substrate specificity of kinases in general and autophosphorylation sites in particular, and the importance of domain-domain interactions in the phosphorylation of some substrates in the context of full-sized proteins as distinct from peptide substrates. Furthermore, because several of the autophosphorylation sites are conserved in other kinases, the relevance of these complexes likely extends to other clinically relevant drug targets. We show that this is particularly true of the IGF1R, LCK, and PAK1 activation loop sites, which are found at homologous positions within the activation loop in the sequences of a large number of kinases.

RESEARCH RESOURCE Table 1. New and previously documented autophosphorylation complexes in the PDB.All kinases in the autophosphorylation complexes listed are human kinases except ceCaMKII from C. elegans. Residues in the sequence that are disordered in the structure are in lowercase. The phosphorylation site is the middle of the 13 residues of each sequence shown in red. Distance (Dist) is in Å. The HRD column provides the residue number of the catalytic site aspartic acid and the OH column indicates phosphorylated residue number (Y, T, or S). The enzyme and substrate columns provide the ASU chain ID given by the authors. When the enzyme or substrate kinase does not come No.

Kinase KIT

2 3

CSF1R EPHA2

4 5 6 7

IGF1R LCK IGF1R PAK1 PAK1 PAK1 PAK1 PAK1 PAK1 PAK1 PAK1 PAK1 PAK1 PAK1 PAK1 IRAK4 IRAK4 FGFR1 FGFR3 FGFR2 CLK2

8 9 10 11 12 13 14 15

ceCaMKII ceCaMKII CaMKII EGFR

New

Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Y Y

PDB

Location

Enzyme

Substrate

Sequence

Dist

HRD

OH

ASA

BA

1PKG

Juxtamem

Y561 Y594

Act. loop Act. loop Act. loop Act. loop Act. loop Act. loop Act. loop Act. loop Act. loop Act. loop Act. loop Act. loop Act. loop Act. loop Act. loop Act. loop Act. loop Kinase ins. Kinase ins. C-term tail N-term tail

D1135 D364 D1135 N389 N389 N389 N389 D389 N389 N389 N389 D389 D389 D389 D389 D311 D311 D623 D617 D626 D290

Y1165 Y394 Y1166 T423 T423E T423E T423E pT423 T423 T423 T423 T423 T423 T423 T423 T345 T345 Y583F Y577 Y769 S142

3KK8 3KK9 2WEL 4I21

C-term C-term C-term C-term

2.82 3.29 2.62 3.15 3.21 2.51 2.91 3.14 2.84 2.62 2.61 3.19 2.54 2.99 2.92 2.83 2.83 2.93 2.85 3.00 4.83 3.78 3.00 3.35 2.82 5.36 5.40 4.19 4.17 2.71 2.47 2.81

D778 D739

3D94 2PL0 3LVP 3Q4Z 4O0R 4O0T 4P90 4ZLO 4ZY4 4ZY5 4ZY6 4ZJI 4ZJI 4ZJJ 4ZJJ 4U97 4U9A 3GQI 4K33 3CLY 3NR9

eingNNXVXIDPT eingnNXVXIDPT syeGNSYTFIDPT yvDPHTYEDPNQA yvDPHTYEDPNQA DIYETDYYRKGGK LIEDNEYTAREGA IYETDYYRKGGKG eQSKRSTMVGTPY EQSKRSEMVGTPY EQSKRSEMVGTPY eqSKRSEMVGTPY eqSKRSTMVGTPY EQSKRSTMVGTPY EQSKRSTMVGTPY eqSKRSTMVGTPY eqSKRSTMVGTPY eQSKRSTMVGTPY EQSKRSTMVGTPY EQSKRSTMVGTPY faqtVMTSRIVGT faqtVMTSRIVGT RPPGLEFSFNPSH RPPGLDYSFDTSE LTTNEEYLDLSQP sSRRAKSVEDDAE sSRRAKSVEDDAE AIHRQDTVDCL* AIHRQDTVDCL* mMHRQETVDCLKK VVDADEYLIPQqg vvdadEYLIPqqg

pY568

Juxtamem Juxtamem

B A A A:2_764 B:2_654 A A:2_655 B B B B B:2_646 A:1_556 B B B D C D C B B A:4_456 A A C A:4_645 A A A B A

D792

3LCD 4PDO

A B:2_655 A:4_555 A B A:4_555 A C A A A A B A A A A B A B A A A A:3_645 A:3_655 B:5_555 A A:5_555 A:1_655 A:3_644 A B:1_655

N134 N134 D136 D837

pT284 pT284 T287 Y1016

939 826 843 819 816 997 878 848 875 819 874 813 761 863 904 870 1230 1214 1169 1190 1187 1154 824 843 897 1501 1554 1310 700 668 792 558

PISA PISA — — — Auth., PISA PISA — Auth., PISA — PISA — Auth., PISA PISA PISA PISA PISA PISA PISA PISA Auth., PISA Auth., PISA — — Auth. Auth., PISA PISA Auth. Auth. — — —

tail tail tail tail

adjacent to the phosphorylation site in the sequence. For each structure, we mapped the interaction areas separately to the surfaces of the enzyme and substrate kinases (fig. S1). Table 1 also lists the symmetry operators required to determine the position of the enzyme and substrate kinase in each autophosphorylation complex. We found that 26 PDB entries contain 33 autophosphorylation complexes; 18 of these 33 are between kinases in different copies of the ASU and 13 are between kinase monomers in different unit cells, demonstrating the importance of considering the full symmetry of each crystal in identifying potential autophosphorylation complexes. We expect that, in autophosphorylation complexes, the enzyme kinase should be in a conformation consistent with known active kinase structures in the PDB. The substrate kinases may be in an active or inactive conformation, and the state observed in the crystal may be informative in determining under what conditions trans-autophosphorylation might take place. For these reasons, in Table 2, we list three features that distinguish active from inactive kinases for both enzymes and substrates listed in Table 1: (i) the position of

the phenylalanine ring of the DFG motif; (ii) the presence or absence of a salt bridge between a Lys residue of the N-terminal b sheet and a Glu of the C helix; (iii) the existence (or lack thereof) of van der Waals interactions of residue side chains of the “regulatory spine” (36), which comprises the His side chain of the HRD motif, the Phe of the DFG motif, and two additional hydrophobic residues in the N-terminal domain. The presence of an intact regulatory spine is associated with active enzyme structures (36). Most of the enzyme kinases in the autophosphorylation complexes are consistent with the active conformation (Table 2): the DFG motif is labeled “DFGin,” meaning that the Phe ring is under the C helix and the activation loop is positioned such that a substrate may bind in the enzyme active site; the distance of the Nz atom of the Lys side chain and Oe1 or Oe2 atoms of the Glu side chain are within hydrogen-bonding distance (Arg, Met302->Leu) in complex with compound 823. 10.2210/pdb3dj6/pdb (2009). C.-H. Lee, J. H. Chung, The hCds1 (Chk2)-FHA domain is essential for a chain of phosphorylation events on hCds1 that is induced by ionizing radiation. J. Biol. Chem. 276, 30537–30541 (2001).

www.SCIENCESIGNALING.org

1 December 2015

Vol 8 Issue 405 rs13

22

Downloaded from http://stke.sciencemag.org/ on December 1, 2015

60. S. Pronk, S. Páll, R. Schulz, P. Larsson, P. Bjelkmar, R. Apostolov, M. R. Shirts, J. C. Smith, P. M. Kasson, D. van der Spoel, B. Hess, E. Lindahl, GROMACS 4.5: A high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29, 845–854 (2013). 61. X. Zhu, J. L. Kim, J. R. Newcomb, P. E. Rose, D. R. Stover, L. M. Toledo, H. Zhao, K. A. Morgenstern, Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors. Structure 7, 651–661 (1999). 62. C. M. Dumaual, B. A. Steere, C. D. Walls, M. Wang, Z.-Y. Zhang, S. K. Randall, Integrated analysis of global mRNA and protein expression data in HEK293 cells overexpressing PRL-1. PLOS One 8, e72977 (2013). 63. J. M. Bland, D. G. Altman, Transformations, means, and confidence intervals. BMJ 312, 1079 (1996). 64. D. A. Keedy, I. Georgiev, E. B. Triplett, B. R. Donald, D. C. Richardson, J. S. Richardson, The role of local backrub motions in evolved and designed mutations. PLOS Comput. Biol. 8, e1002629 (2012). 65. A. Kaushansky, A. Gordus, B. Chang, J. Rush, G. MacBeath, A quantitative study of the recruitment potential of all intracellulartyrosine residues on EGFR, FGFR1 and IGF1R. Mol. Biosyst. 4, 643–653 (2008). 66. S. C. Mayer, A. L. Banker, F. Boschelli, L. Di, M. Johnson, C. H. Kenny, G. Krishnamurthy, K. Kutterer, F. Moy, S. Petusky, M. Ravi, D. Tkach, H.-R. Tsou, W. Xu, Lead identification to generate isoquinolinedione inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment. Bioorg. Med. Chem. Lett. 18, 3641–3645 (2008). 67. L. M. Miller, S. C. Mayer, D. M. Berger, D. H. Boschelli, F. Boschelli, L. Di, X. Du, M. Dutia, M. B. Floyd, M. Johnson, C. H. Kenny, G. Krishnamurthy, F. Moy, S. Petusky, D. Tkach, N. Torres, B. Wu, W. Xu, Lead identification to generate 3-cyanoquinoline inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment. Bioorg. Med. Chem. Lett. 19, 62–66 (2009). 68. G. J. Kleywegt, M. R. Harris, J. Zou, T. C. Taylor, A. Wählby, T. A. Jones, The Uppsala Electron-Density Server. Acta Crystallogr. D Biol. Crystallogr. 60, 2240–2249 (2004). 69. J. Wu, Y. D. Tseng, C.-F. Xu, T. A. Neubert, M. F. White, S. R. Hubbard, Structural and biochemical characterization of the KRLB region in insulin receptor substrate-2. Nat. Struct. Mol. Biol. 15, 251–258 (2008). 70. S. R. Hubbard, Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog. EMBO J. 16, 5572–5581 (1997). 71. M. A. Lemmon, J. Schlessinger, Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010). 72. K. Neet, T. Hunter, Vertebrate non-receptor protein-tyrosine kinase families. Genes Cells 1, 147–169 (1996). 73. C. Chen, B. H. Ha, A. F. Thévenin, H. J. Lou, R. Zhang, K. Y. Yip, J. R. Peterson, M. Gerstein, P. M. Kim, P. Filippakopoulos, S. Knapp, T. J. Boggon, B. E. Turk, Identification of a major determinant for serine-threonine kinase phosphoacceptor specificity. Mol. Cell 53, 140–147 (2014). 74. G. Zhu, K. Fujii, Y. Liu, V. Codrea, J. Herrero, S. Shaw, A single pair of acidic residues in the kinase major groove mediates strong substrate preference for P-2 or P-5 arginine in the AGC, CAMK, and STE kinase families. J. Biol. Chem. 280, 36372–36379 (2005). 75. Z. Wang, J. Liu, A. Sudom, M. Ayres, S. Li, H. Wesche, J. P. Powers, N. P. C. Walker, Crystal structures of IRAK-4 kinase in complex with inhibitors: A serine/threonine kinase with tyrosine as a gatekeeper. Structure 14, 1835–1844 (2006). 76. J. H. Bae, T. J. Boggon, F. Tomé, V. Mandiyan, I. Lax, J. Schlessinger, Asymmetric receptor contact is required for tyrosine autophosphorylation of fibroblast growth factor receptor in living cells. Proc. Natl. Acad. Sci. U.S.A. 107, 2866–2871 (2010). 77. K. C. Hart, S. C. Robertson, D. J. Donoghue, Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation. Mol. Biol. Cell 12, 931–942 (2001). 78. M. Ceridono, F. Belleudi, S. Ceccarelli, M. R. Torrisi, Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis. Biochem. Biophys. Res. Commun. 327, 523–532 (2005). 79. J. Wang, S. Steinbacher, M. Augustin, P. Schreiner, D. Epstein, M. J. Mulvihill, A. P. Crew, The crystal structure of a constitutively active mutant RON kinase suggests an intramolecular autophosphorylation hypothesis. Biochemistry 49, 7972–7974 (2010). 80. N. Schiering, S. Knapp, M. Marconi, M. M. Flocco, J. Cui, R. Perego, L. Rusconi, C. Cristiani, Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a. Proc. Natl. Acad. Sci. U.S.A. 100, 12654–12659 (2003). 81. O. Nayler, F. Schnorrer, S. Stamm, A. Ullrich, The cellular localization of the murine serine/arginine-rich protein kinase CLK2 is regulated by serine 141 autophosphorylation. J. Biol. Chem. 273, 34341–34348 (1998). 82. M. Soundararajan, A. K. Roos, P. Savitsky, P. Filippakopoulos, A. N. Kettenbach, J. V. Olsen, S. A. Gerber, J. Eswaran, S. Knapp, J. M. Elkins, Structures of Down syndrome kinases, DYRKs, reveal mechanisms of kinase activation and substrate recognition. Structure 21, 986–996 (2013). 83. O. Fedorov, K. Huber, A. Eisenreich, P. Filippakopoulos, O. King, A. N. Bullock, D. Szklarczyk, L. J. Jensen, D. Fabbro, J. Trappe, U. Rauch, F. Bracher, S. Knapp,

RESEARCH RESOURCE

122.

123.

124. 125. 126. 127.

128.

tion, Taxonomy and Sequences resource. Nucleic Acids Res. 41, D483-D489 (2013). B. Zhao, A. Smallwood, J. Yang, K. Koretke, K. Nurse, A. Calamari, R. B. Kirkpatrick, Z. Lai, Modulation of kinase-inhibitor interactions by auxiliary protein binding: Crystallography studies on Aurora A interactions with VX-680 and with TPX2. Protein Sci. 17, 1791–1797 (2008). A. Cook, E. D. Lowe, E. D. Chrysina, V. T. Skamnaki, N. G. Oikonomakos, L. N. Johnson, Structural studies on phospho-CDK2/cyclin A bound to nitrate, a transition state analogue: Implications for the protein kinase mechanism. Biochemistry 41, 7301–7311 (2002). D. L. Theobald, D. S. Wuttke, THESEUS: Maximum likelihood superpositioning and analysis of macromolecular structures. Bioinformatics 22, 2171–2172 (2006). W. L. DeLano, The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, CA, 2002). S. J. Hubbard, J. M. Thornton, NACCESS (Department of Biochemistry and Molecular Biology, University College London, London, 1993). W. Wang, B. A. Malcolm, Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis. Biotechniques 26, 680–682 (1999). G. G. Chiang, B. M. Sefton, Specific dephosphorylation of the Lck tyrosine protein kinase at Tyr-394 by the SHP-1 protein-tyrosine phosphatase. J. Biol. Chem. 276, 23173–23178 (2001).

Acknowledgments: We thank J. Wu for useful discussions and V. Modi for preparing the models of the asymmetric LCK and IGF1R dimers. Funding: This work was funded by NIH grants R01 GM084453 (R.L.D.) and R01 GM083025 (J.R.P.). Author contributions: R.L.D. conceived the project. R.L.D. and J.R.P. supervised the project. Q.X. and R.L.D. wrote the paper. Q.X., R.L.D., E.D., E.J.J., and S.K. performed calculations and analysis. K.L.M. and L.F. performed experiments on LCK and analyzed the experimental data. Competing interests: R.L.D. is a member of the Scientific Advisory Board of Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB). Data and materials availability: The coordinates of the structures described in this paper are available in the Supplementary Materials.

Submitted 12 January 2015 Accepted 11 November 2015 Final Publication 1 December 2015 10.1126/scisignal.aaa6711 Citation: Q. Xu, K. L. Malecka, L. Fink, E. J. Jordan, E. Duffy, S. Kolander, J. R. Peterson, R. L. Dunbrack Jr., Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases. Sci. Signal. 8, rs13 (2015).

www.SCIENCESIGNALING.org

1 December 2015

Vol 8 Issue 405 rs13

23

Downloaded from http://stke.sciencemag.org/ on December 1, 2015

106. P. R. Graves, K. M. Winkfield, T. A. J. Haystead, Regulation of zipper-interacting protein kinase activity in vitro and in vivo by multisite phosphorylation. J. Biol. Chem. 280, 9363–9374 (2005). 107. L. Hagerty, D. H. Weitzel, J. Chambers, C. N. Fortner, M. H. Brush, D. Loiselle, H. Hosoya, T. A. J. Haystead, ROCK1 phosphorylates and activates zipper-interacting protein kinase. J. Biol. Chem. 282, 4884–4893 (2007). 108. H. Chen, J. Ma, W. Li, A. V. Eliseenkova, C. Xu, T. A. Neubert, W. T. Miller, M. Mohammadi, A molecular brake in the kinase hinge region regulates the activity of receptor tyrosine kinases. Mol. Cell 27, 717–730 (2007). 109. Z. Kalender Atak, K. De Keersmaecker, V. Gianfelici, E. Geerdens, R. Vandepoel, D. Pauwels, M. Porcu, I. Lahortiga, V. Brys, W. G. Dirks, H. Quentmeier, J. Cloos, H. Cuppens, A. Uyttebroeck, P. Vandenberghe, J. Cools, S. Aerts, High accuracy mutation detection in leukemia on a selected panel of cancer genes. PLOS One 7, e38463 (2012). 110. J. J. Ellis, B. Kobe, Predicting protein kinase specificity: Predikin update and performance in the DREAM4 challenge. PLOS One 6, e21169 (2011). 111. R. I. Brinkworth, R. A. Breinl, B. Kobe, Structural basis and prediction of substrate specificity in protein serine/threonine kinases. Proc. Natl. Acad. Sci. U.S.A. 100, 74–79 (2003). 112. N. Kumar, D. Mohanty, MODPROPEP: A program for knowledge-based modeling of protein–peptide complexes. Nucleic Acids Res. 35, W549-W555 (2007). 113. N. Kumar, D. Mohanty, Identification of substrates for Ser/Thr kinases using residuebased statistical pair potentials. Bioinformatics 26, 189–197 (2010). 114. J. A. Endicott, M. E. M. Noble, L. N. Johnson, The structural basis for control of eukaryotic protein kinases. Annu. Rev. Biochem. 81, 587–613 (2012). 115. M. Lei, M. A. Robinson, S. C. Harrison, The active conformation of the PAK1 kinase domain. Structure 13, 769–778 (2005). 116. C. L. Corless, C. M. Barnett, M. C. Heinrich, Gastrointestinal stromal tumours: Origin and molecular oncology. Nat. Rev. Cancer 11, 865–878 (2011). 117. S. Hirota, K. Isozaki, Y. Moriyama, K. Hashimoto, T. Nishida, S. Ishiguro, K. Kawano, M. Hanada, A. Kurata, M. Takeda, G. Muhammad Tunio, Y. Matsuzawa, Y. Kanakura, Y. Shinomura, Y. Kitamura, Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 279, 577–580 (1998). 118. Y. Liu, N. S. Gray, Rational design of inhibitors that bind to inactive kinase conformations. Nat. Chem. Biol. 2, 358–364 (2006). 119. Z. H. Foda, M. A. Seeliger, Kinase inhibitors: An allosteric add-on. Nat. Chem. Biol. 10, 796–797 (2014). 120. Q. Xu, R. L. Dunbrack Jr., Assignment of protein sequences to existing domain and family classification systems: Pfam and the PDB. Bioinformatics 28, 2763–2772 (2012). 121. S. Velankar, J. M. Dana, J. Jacobsen, G. van Ginkel, P. J. Gane, J. Luo, T. J. Oldfield, C. O’Donovan, M.-J. Martin, G. J. Kleywegt, SIFTS: Structure Integration with Func-

Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases Qifang Xu, Kimberly L. Malecka, Lauren Fink, E. Joseph Jordan, Erin Duffy, Samuel Kolander, Jeffrey R. Peterson and Roland L. Dunbrack Jr. (December 1, 2015) Science Signaling 8 (405), rs13. [doi: 10.1126/scisignal.aaa6711]

The following resources related to this article are available online at http://stke.sciencemag.org. This information is current as of December 1, 2015.

Article Tools

Related Content

References Permissions

"Supplementary Materials" http://stke.sciencemag.org/content/suppl/2015/11/25/8.405.rs13.DC1 The editors suggest related resources on Science's sites: http://stke.sciencemag.org/content/sigtrans/3/111/ra17.full http://stke.sciencemag.org/content/sigtrans/7/350/ra105.full http://stke.sciencemag.org/content/sigtrans/5/245/ra74.full http://stke.sciencemag.org/content/sigtrans/4/195/re2.full http://stke.sciencemag.org/content/sigtrans/7/354/ra114.full This article cites 121 articles, 56 of which you can access for free at: http://stke.sciencemag.org/content/8/405/rs13#BIBL Obtain information about reproducing this article: http://www.sciencemag.org/about/permissions.dtl

Science Signaling (ISSN 1937-9145) is published weekly, except the last December, by the American Association for the Advancement of Science, 1200 New York Avenue, NW, Washington, DC 20005. Copyright 2015 by the American Association for the Advancement of Science; all rights reserved.

Downloaded from http://stke.sciencemag.org/ on December 1, 2015

Supplemental Materials

Visit the online version of this article to access the personalization and article tools: http://stke.sciencemag.org/content/8/405/rs13

www.sciencesignaling.org/cgi/content/full/8/405/rs13/DC1

Supplementary Materials for Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases Qifang Xu, Kimberly L. Malecka, Lauren Fink, E. Joseph Jordan, Erin Duffy, Samuel Kolander, Jeffrey R. Peterson, Roland L. Dunbrack Jr.* *Corresponding author. E-mail: [email protected] Published 1 December 2015, Sci. Signal. 8, rs13 (2015) DOI: 10.1126/scisignal.aaa6711

This PDF file includes: Fig. S1. Surfaces of autophosphorylation complexes and the individual enzyme and substrate kinases. Fig. S2. Sequence alignment of the activation loops of tyrosine kinases. Fig. S3. Sequence alignment of the activation loops of kinases of the IRAK family. Fig. S4. Sequence alignment of the activation loops of all human serine/threonine kinases  with  Ser  or  Thr  at  the  −12  position  of  the  activation  loop. Fig. S5. Sequence alignment of the kinase insert loops of FGFR kinases. Fig. S6 Sequence alignment of C-terminal extensions of FGFR2 and related tyrosine receptor kinases. Fig. S7. Sequence alignment of the N-terminal regions of CLK family members. Fig. S8. Sequence alignment of the C-terminal tails of CaMKII family members. Fig. S9. Sequence alignment of the C-terminal tails of EGFR, ErbB2, ErbB3, and ErbB4. Table S1. LCK autophosphorylation experimental data. Table S2. Fifty-eight domain-swapping PDB dimers in 17 distinct crystal forms from ProtCID. Legends for data S1 and S2 Other Supplementary Material for this manuscript includes the following: (available at www.sciencesignaling.org/cgi/content/full/8/405/rs13/DC1) Data file S1 (.pdb format). Coordinates of kinase autophosphorylation complexes.

Data file S2 (.pdb format). Models of asymmetric autophosphorylatin complexes of IGF1R and LCK.

Continued 1

Fig. S1. Surfaces of autophosphorylation complexes and the individual enzyme and substrate kinases. In each image, the enzyme kinases are colored in blue, substrate kinases are colored in orange. The interface areas are colored in magenta, with distance cutoff 5 Å. Enzymes and substrates are rotated and translated to show the interfacial surfaces, located on the right side of each figure. Coordinates from the first interface listed in Table 1 were used. For PAK1, structures from PDB: 4ZY4 and 4ZJI are also included.

2

Gene AATK ABL1 ABL2 BMX BTK FER FES FGR FYN HCK ITK LCK LMTK2 LYN PTK6 RYK SRC SRMS TEC TXK YES1

Uniprot LMTK1_HUMAN ABL1_HUMAN ABL2_HUMAN BMX_HUMAN BTK_HUMAN FER_HUMAN FES_HUMAN FGR_HUMAN FYN_HUMAN HCK_HUMAN ITK_HUMAN LCK_HUMAN LMTK2_HUMAN LYN_HUMAN PTK6_HUMAN RYK_HUMAN SRC_HUMAN SRMS_HUMAN TEC_HUMAN TXK_HUMAN YES_HUMAN

Activatioon loop sequence DYGLAHCKYREDYFV--------------TADQLWVPLRWIAPE DFGLSRLMTGDTYTA---------------HAGAKFPIKWTAPE DFGLSRLMTGDTYTA---------------HAGAKFPIKWTAPE DFGMTRYVLDDQYVS---------------SVGTKFPVKWSAPE DFGLSRYVLDDEYTS---------------SVGSKFPVRWSPPE DFGMSRQEDGGVYSS---------------SGLKQIPIKWTAPE DFGMSREEADGVYAA--------------SGGLRQVPVKWTAPE DFGLARLIKDDEYNP---------------CQGSKFPIKWTAPE DFGLARLIEDNEYTA---------------RQGAKFPIKWTAPE DFGLARVIEDNEYTA---------------REGAKFPIKWTAPE DFGMTRFVLDDQYTS---------------STGTKFPVKWASPE DFGLARLIEDNEYTA---------------REGAKFPIKWTAPE DYGIGFSRYKEDYIE--------------TDDKKVFPLRWTAPE DFGLARVIEDNEYTA---------------REGAKFPIKWTAPE DFGLARLIKEDVYLS----------------HDHNIPYKWTAPE DNALSRDLFPMDYHC--------------LGDNENRPVRWMALE DFGLARLIEDNEYTA---------------RQGAKFPIKWTAPE DFGLARLLKDDIYSP---------------SSSSKIPVKWTAPE DFGMARYVLDDQYTS---------------SSGAKFPVKWCPPE DFGMTRYVLDDEYVS---------------SFGAKFPIKWSPPE DFGLARLIEDNEYTA---------------RQGAKFPIKWTAPE

CSF1R EPHA10 FLT1 FLT3 FLT4 JAK1 JAK2 JAK3 KDR KIT PDGFRA PDGFRB RET SYK TYK2 ZAP70

CSF1R_HUMAN EPHAA_HUMAN VGFR1_HUMAN FLT3_HUMAN VGFR3_HUMAN JAK1_HUMAN JAK2_HUMAN JAK3_HUMAN VGFR2_HUMAN KIT_HUMAN PGFRA_HUMAN PGFRB_HUMAN RET_HUMAN KSYK_HUMAN TYK2_HUMAN ZAP70_HUMAN

DFGLARDIMNDSNYI--------------VKGNARLPVKWMAPE GFGRGPRDRSEAVYT---------------TMSGRSPALWAAPE DFGLARDIYKNPDYV--------------RKGDTRLPLKWMAPE DFGLARDIMSDSNYV--------------VRGNARLPVKWMAPE DFGLARDIYKDPDYV--------------RKGSARLPLKWMAPE DFGLTKAIETDKEYY-------------TVKDDRDSPVFWYAPE DFGLTKVLPQDKEYY-------------KVKEPGESPIFWYAPE DFGLAKLLPLDKDYY-------------VVREPGQSPIFWYAPE DFGLARDIYKDPDYV--------------RKGDARLPLKWMAPE DFGLARDIKNDSNYV--------------VKGNARLPVKWMAPE DFGLARDIMHDSNYV--------------SKGSTFLPVKWMAPE DFGLARDIMRDSNYI--------------SKGSTFLPLKWMAPE DFGLSRDVYEEDSYV--------------KRSQGRIPVKWMAIE DFGLSKALRADENYY-------------KAQTHGKWPVKWYAPE DFGLAKAVPEGHEYY-------------RVREDGDSPVFWYAPE DFGLSKALGADDSYY-------------TARSAGKWPLKWYAPE

ALK AXL DDR1 DDR2 FGFR1 FGFR2 FGFR3 FGFR4 IGF1R INSR INSRR LMTK3 LTK MERTK MET MST1R MUSK NTRK1 NTRK2 NTRK3 PTK2 PTK2B PTK7 ROR1 ROR2 ROS1 TYRO3

ALK_HUMAN UFO_HUMAN DDR1_HUMAN DDR2_HUMAN FGFR1_HUMAN FGFR2_HUMAN FGFR3_HUMAN FGFR4_HUMAN IGF1R_HUMAN INSR_HUMAN INSRR_HUMAN LMTK3_HUMAN LTK_HUMAN MERTK_HUMAN MET_HUMAN RON_HUMAN MUSK_HUMAN NTRK1_HUMAN NTRK2_HUMAN NTRK3_HUMAN FAK1_HUMAN FAK2_HUMAN PTK7_HUMAN ROR1_HUMAN ROR2_HUMAN ROS1_HUMAN TYRO3_HUMAN

DFGMARDIYRASYYR--------------KGGCAMLPVKWMPPE DFGLSKKIYNGDYYR--------------QGRIAKMPVKWIAIE DFGMSRNLYAGDYYR--------------VQGRAVLPIRWMAWE DFGMSRNLYSGDYYR--------------IQGRAVLPIRWMSWE DFGLARDIHHIDYYK--------------KTTNGRLPVKWMAPE DFGLARDINNIDYYK--------------KTTNGRLPVKWMAPE DFGLARDVHNLDYYK--------------KTTNGRLPVKWMAPE DFGLARGVHHIDYYK--------------KTSNGRLPVKWMAPE DFGMTRDIYETDYYR--------------KGGKGLLPVRWMSPE DFGMTRDIYETDYYR--------------KGGKGLLPVRWMAPE DFGMTRDVYETDYYR--------------KGGKGLLPVRWMAPE DYGLAHSNYKEDYYL--------------TPERLWIPLRWAAPE DFGMARDIYRASYYR--------------RGDRALLPVKWMPPE DFGLSKKIYSGDYYR--------------QGRIAKMPVKWIAIE DFGLARDMYDKEYYS------------VHNKTGAKLPVKWMALE DFGLARDILDREYYS------------VQQHRHARLPVKWMALE DFGLSRNIYSADYYK--------------ANENDAIPIRWMPPE DFGMSRDIYSTDYYR--------------VGGRTMLPIRWMPPE DFGMSRDVYSTDYYR--------------VGGHTMLPIRWMPPE DFGMSRDVYSTDYYRLFNPSGNDFCIWCEVGGHTMLPIRWMPPE DFGLSRYMEDSTYYK---------------ASKGKLPIKWMAPE DFGLSRYIEDEDYYK---------------ASVTRLPIKWMSPE ALGLSKDVYNSEYYH---------------FRQAWVPLRWMSPE DLGLSREIYSADYYR--------------VQSKSLLPIRWMPPE DLGLFREVYAADYYK--------------LLGNSLLPIRWMAPE DFGLARDIYKNDYYR--------------KRGEGLLPVRWMAPE DFGLSRKIYSGDYYR--------------QGCASKLPVKWLALE

Y13 Y13-annotation Y283 unannotated Y393 auto Y439 auto Y566 auto Y551 LYN,SYK Y714 auto Y713 auto Y412 auto Y420 auto Y411 auto Y512 LCK Y394 auto Y295 unannotated Y397 auto Y342 auto Y492 pseudokinase Y419 auto Y380 auto Y519 auto Y420 auto Y426 auto

Y1282 Y702 Y796 Y740 Y653 Y656 Y647 Y642 Y1165 Y1189 Y1145 Y296 Y676 Y753 Y1234 Y1238 Y755 Y680 Y706 Y709 Y576 Y579 Y960 Y645 Y645 Y2114 Y685

auto unannotated auto auto auto auto auto auto auto auto auto unannotated auto auto auto auto auto auto auto auto RET SRC pseudokinase pseudokinase pseudokinase unannotated auto

Y14 Y14-annotation

Y809 Y801 Y1053 Y842 Y1068 Y1034 Y1007 Y980 Y1059 Y823 Y849 Y857 Y905 Y525 Y1054 Y492

auto pseudokinase auto auto auto auto auto auto auto auto auto auto auto auto auto unspecified

Y1283 Y703 Y797 Y741 Y654 Y657 Y648 Y643 Y1166 Y1190 Y1146 Y297 Y677 Y754 Y1235 Y1239 Y756 Y681 Y707 Y710 Y577 Y580 Y961 Y646 Y646 Y2115 Y686

unspecified auto auto auto auto auto auto auto auto auto auto unannotated unannotated auto auto auto unannotated auto auto auto RET SRC pseudokinase pseudokinase pseudokinase unannotated auto

Fig. S2. Sequence alignment of the activation loops of tyrosine kinases. Phosphorylation sites at DFG+13 and DFG+14 are shown in red, bold type. Other probable phosphorylation sites of the activation loop are shown in blue; those annotated as such are shown in blue bold type. The residue numbers for the +13 and +14 sites are given after the sequence. The annotation were parsed from UniProt text files. "auto" indicates that the site is annotated as autophosphorylated in Uniprot. "unspecified" means it is annotated as phosphorylated but the kinase is not given. For the others, a specific kinase is given by gene name. "unannotated" means there is no annotation for that site in UniProt. Some kinases are likely pseudokinases and are marked as such.

3

Uniprot IRAK1_HUMAN IRAK2_HUMAN IRAK3_HUMAN IRAK4_HUMAN

Activation Loop Sequence DFGLARFSRFAGSSPSQSSMVARTQTVRGTLAYLPEE HPMAHLCPVNKRSKYTMM----KTHLLRTSAAYLPED DFAMAHFRSHLEHQSCTIN---MTSSSSKHLWYMPEE DFGLARASEKFAQTV-------MTSRIVGTTAYMAPE

Site T381 T372 T331 T345

Annotation None None None Phosphorylated

Fig. S3. Sequence alignment of the activation loops of kinases of the IRAK family. The sequences are not highly conserved, but 387 all IRAKs have Thr (bold red) at -14 from the APE site (E = -1). Two additional phosphorylation sites in IRAK4 and Thr of IRAK1 are shown in bold blue, and are annotated as phosphorylation sites in UniProt.

Family AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC

Gene AKT1 AKT2 AKT3 LATS1 LATS2 PDPK1 PDPK2P PKN1 PKN2 PKN3 PRKACA PRKACB PRKACG PRKCA PRKCB PRKCD PRKCE PRKCG PRKCH PRKCI PRKCQ PRKCZ PRKG1 PRKG2 PRKX PRKY ROCK1 ROCK2 RPS6KA1 RPS6KA1 RPS6KA2 RPS6KA2 RPS6KA3 RPS6KA3 RPS6KA4 RPS6KA4 RPS6KA5 RPS6KA5 RPS6KA6 RPS6KA6 RPS6KB1 RPS6KB2 SGK1 SGK2 SGK3 STK38 STK38L

UniProt AKT1_HUMAN AKT2_HUMAN AKT3_HUMAN LATS1_HUMAN LATS2_HUMAN PDPK1_HUMAN PDPK2_HUMAN PKN1_HUMAN PKN2_HUMAN PKN3_HUMAN KAPCA_HUMAN KAPCB_HUMAN KAPCG_HUMAN KPCA_HUMAN KPCB_HUMAN KPCD_HUMAN KPCE_HUMAN KPCG_HUMAN KPCL_HUMAN KPCI_HUMAN KPCT_HUMAN KPCZ_HUMAN KGP1_HUMAN KGP2_HUMAN PRKX_HUMAN PRKY_HUMAN ROCK1_HUMAN ROCK2_HUMAN KS6A1_HUMAN KS6A1_HUMAN KS6A2_HUMAN KS6A2_HUMAN KS6A3_HUMAN KS6A3_HUMAN KS6A4_HUMAN KS6A4_HUMAN KS6A5_HUMAN KS6A5_HUMAN KS6A6_HUMAN KS6A6_HUMAN KS6B1_HUMAN KS6B2_HUMAN SGK1_HUMAN SGK2_HUMAN SGK3_HUMAN STK38_HUMAN ST38L_HUMAN

Activation Loop Sequence DFGLCKEGIKDGA-----------------------------------TMKTFCGTPEYLAPE DFGLCKEGISDGA-----------------------------------TMKTFCGTPEYLAPE DFGLCKEGITDAA-----------------------------------TMKTFCGTPEYLAPE DFGLCTGFRWTHDXHQRC------------------------------LAHSLVGTPNYIAPE DFGLCTGFRWTHNXHQRC------------------------------LAHSLVGTPNYIAPE DFGTAKVLSPESKQA---------------------------------RANSFVGTAQYVSPE DFGTAKVLSPESKQA---------------------------------RANSFVGTAQYVSPE DFGLCKEGMGYGD-----------------------------------RTSTFCGTPEFLAPE DFGLCKEGMGYGD-----------------------------------RTSTFCGTPEFLAPE DFGLCKEGIGFGD-----------------------------------RTSTFCGTPEFLAPE DFGFAKRVKG--------------------------------------RTWTLCGTPEYLAPE DFGFAKRVKG--------------------------------------RTWTLCGTPEYLAPE DFGFAKRVKG--------------------------------------RTWTLCGTPEYLAPE DFGMCKEHMMDGV-----------------------------------TTRTFCGTPDYIAPE DFGMCKENIWDGV-----------------------------------TTKTFCGTPDYIAPE DFGMCKENIFGES-----------------------------------RASTFCGTPDYIAPE DFGMCKEGILNGV-----------------------------------TTTTFCGTPDYIAPE DFGMCKENVFPGT-----------------------------------TTRTFCGTPDYIAPE DFGMCKEGICNGV-----------------------------------TTATFCGTPDYIAPE DYGMCKEGLRPGD-----------------------------------TTSTFCGTPNYIAPE DFGMCKENMLGDA-----------------------------------KTNTFCGTPDYIAPE DYGMCKEGLGPGD-----------------------------------TTSTFCGTPNYIAPE DFGFAKKIGFGK------------------------------------KTWTFCGTPEYVAPE DFGFAKKIGSGQ------------------------------------KTWTFCGTPEYVAPE DFGFAKKLVD--------------------------------------RTWTLCGTPEYLAPE DFGFAKKLVD--------------------------------------RTWTLCGTPEYLAPE DFGTCMKMNKEGMV----------------------------------RCDTAVGTPDYISPE DFGTCMKMDETGMV----------------------------------HCDTAVGTPDYISPE DFGLSKEAIDHEK-----------------------------------KAYSFCGTVEYMAPE DFGLSKEAIDHEK-----------------------------------KAYSFCGTVEYMAPE DFGLSKEAIDHDK-----------------------------------RAYSFCGTIEYMAPE DFGLSKEAIDHDK-----------------------------------RAYSFCGTIEYMAPE DFGLSKESIDHEK-----------------------------------KAYSFCGTVEYMAPE DFGLSKESIDHEK-----------------------------------KAYSFCGTVEYMAPE DFGLSKEFLTEEKE----------------------------------RTFSFCGTIEYMAPE DFGLSKEFLTEEKE----------------------------------RTFSFCGTIEYMAPE DFGLSKEFVADETE----------------------------------RAYSFCGTIEYMAPD DFGLSKEFVADETE----------------------------------RAYSFCGTIEYMAPD DFGLSKESVDQEK-----------------------------------KAYSFCGTVEYMAPE DFGLSKESVDQEK-----------------------------------KAYSFCGTVEYMAPE DFGLCKESIHDGT-----------------------------------VTHTFCGTIEYMAPE DFGLCKESIHEGA-----------------------------------VTHTFCGTIEYMAPE DFGLCKENIEHNS-----------------------------------TTSTFCGTPEYLAPE DFGLCKEGVEPED-----------------------------------TTSTFCGTPEYLAPE DFGLCKEGIAISD-----------------------------------TTTTFCGTPEYLAPE DFGLCTGLKKAHRTEFYRNLNHSLPSDFTFQNMNSKRKAETWKRNRRQLAFSTVGTPDYIAPE DFGLCTGLKKAHRTEFYRNLTHNPPSDFSFQNMNSKRKAETWKKNRRQLAYSTVGTPDYIAPE

Site T308 T309 T305 S909 S872 S241 S214 T774 T816 T718 T198 T198 T198 T497 T500 T507 T566 T514 T513 T412 T538 T410 T517 T609 T203 T203 T233 T249 S221 S573 S570 S218 S227 S577 S568 S196 S581 S212 S232 S581 T252 T228 T256 T253 T320 S281 S282

Annotation IKKE PDPK1 PDPK1 STK3/MST2 unannotated auto unannotated PDPK1 PDPK1 unspecified PDPK1 unspecified auto PDPK1 PDPK1 auto PDPK1 PDPK1 PDPK1 PDPK1 PDPK1 PDPK1 unannotated unannotated unannotated unannotated unannotated unannotated PDPK1 unspecified unannotated PDPK1 PDPK1 unannotated MAPK1 auto MAPK1 auto unspecified unspecified PDPK1 unannotated PDPK1 PDPK1 PDPK1 auto auto

CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK

BRSK1 BRSK2 CAMK1 CAMK1D CAMK1G CAMK4 CHEK2 DCLK1 DCLK2 DCLK3 HUNK MAPKAPK2 MAPKAPK3 MAPKAPK5 MARK1 MARK2 MARK3 MARK4

BRSK1_HUMAN BRSK2_HUMAN KCC1A_HUMAN KCC1D_HUMAN KCC1G_HUMAN KCC4_HUMAN CHK2_HUMAN DCLK1_HUMAN DCLK2_HUMAN DCLK3_HUMAN HUNK_HUMAN MAPK2_HUMAN MAPK3_HUMAN MAPK5_HUMAN MARK1_HUMAN MARK2_HUMAN MARK3_HUMAN MARK4_HUMAN

DFGMASLQVGDS------------------------------------LLETSCGSPHYACPE DFGMASLQVGDS------------------------------------LLETSCGSPHYACPE DFGLSKMEDPGS------------------------------------VLSTACGTPGYVAPE DFGLSKMEGKGD------------------------------------VMSTACGTPGYVAPE DFGLSKMEQNG-------------------------------------IMSTACGTPGYVAPE DFGLSKIVEHQV------------------------------------LMKTVCGTPGYCAPE DFGHSKILGETS------------------------------------LMRTLCGTPTYLAPE DFGLATIVDG--------------------------------------PLYTVCGTPTYVAPE DFGLATVVEG--------------------------------------PLYTVCGTPTYVAPE DFGLAKHVVR--------------------------------------PIFTVCGTPTYVAPE DFGLSNCAGILGYSD---------------------------------PFSTQCGSPAYAAPE DFGFAKETTSHN------------------------------------SLTTPCYTPYYVAPE DFGFAKETTQN-------------------------------------ALQTPCYTPYYVAPE DFGFAKIDQG--------------------------------------DLMTPQFTPYYVAPQ DFGFSNEFTVGN------------------------------------KLDTFCGSPPYAAPE DFGFSNEFTFGN------------------------------------KLDTFCGSPPYAAPE DFGFSNEFTVGG------------------------------------KLDTFCGSPPYAAPE DFGFSNEFTLGS------------------------------------KLDTFCGSPPYAAPE

T189 T174 T177 T180 T178 T200 T383 T546 T550 T512 T222 T222 T201 T182 T215 T208 T211 T214

LKB1 LKB1 CaMKK1 CaMKK1 unannotated CaMKK1 auto unannotated unannotated unannotated unannotated MAPK14 MAPK14 MAPK11 LKB1 LKB1 LKB1 LKB1

4

CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK CAMK

MELK MKNK1 MKNK2 NIM1K NUAK1 NUAK2 OBSCN PASK PNCK PRKAA1 PRKAA2 PRKD1 PRKD2 PRKD3 PSKH1 PSKH2 SIK1 SIK2 SIK3 SNRK STK11 TSSK1B TSSK2 TSSK3 TSSK4 TSSK6

MELK_HUMAN MKNK1_HUMAN MKNK2_HUMAN NIM1_HUMAN NUAK1_HUMAN NUAK2_HUMAN OBSCN_HUMAN PASK_HUMAN KCC1B_HUMAN AAPK1_HUMAN AAPK2_HUMAN KPCD1_HUMAN KPCD2_HUMAN KPCD3_HUMAN KPSH1_HUMAN KPSH2_HUMAN SIK1_HUMAN SIK2_HUMAN SIK3_HUMAN SNRK_HUMAN STK11_HUMAN TSSK1_HUMAN TSSK2_HUMAN TSSK3_HUMAN TSSK4_HUMAN TSSK6_HUMAN

DFGLCAKPKGNKDY----------------------------------HLQTCCGSLAYAAPE DFDLGSGMKLNNSCTPITTP----------------------------ELTTPCGSAEYMAPE DFDLGSGIKLNGDCSPISTP----------------------------ELLTPCGSAEYMAPE DFGFSTVSKKGE------------------------------------MLNTFCGSPPYAAPE DFGLSNLYQKDK------------------------------------FLQTFCGSPLYASPE DFGLSNLYHQGK------------------------------------FLQTFCGSPLYASPE DFGFAQNITPAE------------------------------------LQFSQYGSPEFVSPE DFGSAAYLERGK------------------------------------LFYTFCGTIEYCAPE DFGLSKIQAGN-------------------------------------MLGTACGTPGYVAPE DFGLSNMMSDGE------------------------------------FLRTSCGSPNYAAPE DFGLSNMMSDGE------------------------------------FLRTSCGSPNYAAPE DFGFARIIGEKS------------------------------------FRRSVVGTPAYLAPE DFGFARIIGEKS------------------------------------FRRSVVGTPAYLAPE DFGFARIIGEKS------------------------------------FRRSVVGTPAYLAPE DFGLASARKKGDDC----------------------------------LMKTTCGTPEYIAPE DFGLAYSGKKSGDW----------------------------------TMKTLCGTPEYIAPE DFGFGNFYKSGE------------------------------------PLSTWCGSPPYAAPE DFGFGNFFKSGE------------------------------------LLATWCGSPPYAAPE DFGFSNLFTPGQ------------------------------------LLKTWCGSPPYAAPE DFGFSNKFQPGK------------------------------------KLTTSCGSLAYSAPE DLGVAEALHPFAADD---------------------------------TCRTSQGSPAFQPPE DFSFSKRCLRDDSGRMA-------------------------------LSKTFCGSPAYAAPE DFGFSKRCLRDSNGRII-------------------------------LSKTFCGSAAYAAPE DFGFAKVLPKSHRE----------------------------------LSQTFCGSTAYAAPE DFGFAKMVPSNQPVGCSPSYRQVNCFSH--------------------LSQTYCGSFAYACPE DFGFGRQAHGYPD-----------------------------------LSTTYCGSAAYASPE

T167 T255 T249 T229 T211 T208 S622 T161 T171 T183 T172 S742 S710 S735 T256 T221 T182 T175 T163 T173 T212 T174 T174 T168 T197 T170

auto unspecified unspecified auto LKB1 LKB1 unannotated auto unannotated LKB1 LKB1 auto unspecified auto unannotated unannotated LKB1 LKB1 LKB1 LKB1 unannotated unspecified unannotated unannotated unannotated unannotated

CK1 CK1 CK1

CSNK1G1 CSNK1G2 CSNK1G3

KC1G1_HUMAN KC1G2_HUMAN KC1G3_HUMAN

DFGLAKEYIDPETKKHIPYR----------------------------EHKSLTGTARYMSIN S210 unannotated DFGLAKEYIDPETKKHIPYR----------------------------EHKSLTGTARYMSIN S211 unannotated DFGLAKEYIDPETKKHIPYR----------------------------EHKSLTGTARYMSIN S208 unannotated

CMGC CMGC CMGC CMGC CMGC CMGC CMGC CMGC CMGC CMGC CMGC CMGC CMGC CMGC

CDK15 CDK6 CLK1 CLK2 CLK3 CLK4 DYRK2 DYRK3 DYRK4 GSK3A GSK3B HIPK1 HIPK2 HIPK3

CDK15_HUMAN CDK6_HUMAN CLK1_HUMAN CLK2_HUMAN CLK3_HUMAN CLK4_HUMAN DYRK2_HUMAN DYRK3_HUMAN DYRK4_HUMAN GSK3A_HUMAN GSK3B_HUMAN HIPK1_HUMAN HIPK2_HUMAN HIPK3_HUMAN

DFGLARAKSIPSQ-----------------------------------TYSSEVVTLWYRPPD DFGLARIYSFQM------------------------------------ALTSVVVTLWYRAPE DFGSATYDDE--------------------------------------HHSTLVSTRHYRAPE DFGSATFDHE--------------------------------------HHSTIVSTRHYRAPE DFGSATFDHE--------------------------------------HHTTIVATRHYRPPE DFGSATYDDE--------------------------------------HHSTLVSTRHYRAPE DFGSSCYEHQ--------------------------------------RVYTYIQSRFYRAPE DFGSSCFEYQ--------------------------------------KLYTYIQSRFYRAPE DFGSSCYEHQ--------------------------------------KVYTYIQSRFYRSPE DFGSAKQLVRGE------------------------------------PNVSYICSRYYRAPE DFGSAKQLVRGE------------------------------------PNVSYICSRYYRAPE DFGSASHVSKA-------------------------------------VCSTYLQSRYYRAPE DFGSASHVSKA-------------------------------------VCSTYLQSRYYRAPE DFGSASHVSKT-------------------------------------VCSTYLQSRYYRAPE

S258 S178 T338 T340 T481 T336 T381 T368 T263 S278 S215 T351 T360 T358

unannotated unannotated unannotated unannotated unannotated unannotated MAP3K10 unannotated unannotated unannotated unannotated unannotated unannotated unannotated

NEK NEK NEK NEK NEK NEK NEK NEK NEK NEK NEK

NEK1 NEK10 NEK11 NEK2 NEK3 NEK4 NEK5 NEK6 NEK7 NEK8 NEK9

NEK1_HUMAN NEK10_HUMAN NEK11_HUMAN NEK2_HUMAN NEK3_HUMAN NEK4_HUMAN NEK5_HUMAN NEK6_HUMAN NEK7_HUMAN NEK8_HUMAN NEK9_HUMAN

DFGIARVLNSTVE-----------------------------------LARTCIGTPYYLSPE DFGLAKQKQENS------------------------------------KLTSVVGTILYSCPE DFGVSRLLMGSCD-----------------------------------LATTLTGTPHYMSPE DFGLARILNHDTS-----------------------------------FAKTFVGTPYYMSPE DFGSARLLSNPMA-----------------------------------FACTYVGTPYYVPPE DLGIARVLENHCD-----------------------------------MASTLIGTPYYMSPE DFGIARVLNNSME-----------------------------------LARTCIGTPYYLSPE DLGLGRFFSSETT-----------------------------------AAHSLVGTPYYMSPE DLGLGRFFSSKTT-----------------------------------AAHSLVGTPYYMSPE DFGISKILSSKS------------------------------------KAYTVVGTPCYISPE DYGLAKKLNSEYS-----------------------------------MAETLVGTPYYMSPE

T162 S688 T191 T175 T161 T165 T163 S206 S195 T162 T210

auto unannotated unannotated auto auto auto unannotated NEK9 NEK9 auto auto

OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER OTHER

AURKA AURKB AURKC CAMKK1 CHUK DSTYK EIF2AK1 GAK IKBKB IKBKE PDIK1L PIK3R4 PLK1 PLK2 PLK3 PLK4 RPS6KC1 SGK494 STK32A STK32B STK35 STK36 TBK1 TTK ULK1 ULK2 WNK1 WNK2 WNK3 WNK4

AURKA_HUMAN AURKB_HUMAN AURKC_HUMAN KKCC1_HUMAN IKKA_HUMAN DUSTY_HUMAN E2AK1_HUMAN GAK_HUMAN IKKB_HUMAN IKKE_HUMAN PDK1L_HUMAN PI3R4_HUMAN PLK1_HUMAN PLK2_HUMAN PLK3_HUMAN PLK4_HUMAN KS6C1_HUMAN SG494_HUMAN ST32A_HUMAN ST32B_HUMAN STK35_HUMAN STK36_HUMAN TBK1_HUMAN TTK_HUMAN ULK1_HUMAN ULK2_HUMAN WNK1_HUMAN WNK2_HUMAN WNK3_HUMAN WNK4_HUMAN

DFGWSVHAPSS-------------------------------------RRTTLCGTLDYLPPE DFGWSVHAPSL-------------------------------------RRKTMCGTLDYLPPE DFGWSVHTPSL-------------------------------------RRKTMCGTLDYLPPE DFGVSNQFEGNDA-----------------------------------QLSSTAGTPAFMAPE DLGYAKDVDQGS------------------------------------LCTSFVGTLQYLAPE DLGFCKPEAM--------------------------------------MSGSIVGTPIHMAPE DFGLACTDILQKNTDWTNRNGKRTP-----------------------THTSRVGTCLYASPE DFGSATTISHYPDYSWSAQRRALVE-----------------------EEITRNTTPMYRTPE DLGYAKELDQGS------------------------------------LCTSFVGTLQYLAPE DFGAARELDDDE------------------------------------KFVSVYGTEEYLHPD DFGLSKVCSASGQNPEEPVSVNKC------------------------FLSTACGTDFYMAPE DFASFKPTYLPEDNPADFNY----------------------------FFDTSRRRTCYIAPE DFGLATKVEYDGE-----------------------------------RKKTLCGTPNYIAPE DFGLAARLEPLEH-----------------------------------RRRTICGTPNYLSPE DFGLAARLEPPEQ-----------------------------------RKKTICGTPNYVAPE DFGLATQLKMPHE-----------------------------------KHYTLCGTPNYISPE YFSRWSEVED--------------------------------------SCDSDAIERMYCAPE DFGLSRHVPQGA------------------------------------QAYTICGTLQYMGER DFNIAAMLPRET------------------------------------QITTMAGTKPYMAPE DFNIATVVKGAE------------------------------------RASSMAGTKPYMAPE DFGLSKVCAGLAPRGKEGNQDNKNVNVNKY------------------WLSSACGSDFYMAPE DFGFARAMSTNTM-----------------------------------VLTSIKGTPLYMSPE DFGAARELEDDE------------------------------------QFVSLYGTEEYLHPD DFGIANQMQPDTTSV---------------------------------VKDSQVGTVNYMPPE DFGFARYLQSNM------------------------------------MAATLCGSPMYMAPE DFGFARYLHSNM------------------------------------MAATLCGSPMYMAPE DLGLATLKRAS-------------------------------------FAKSVIGTPEFMAPE DLGLATLKRAS-------------------------------------FAKSVIGTPEFMAPE DLGLATLMRTS-------------------------------------FAKSVIGTPEFMAPE DLGLATLKRAS-------------------------------------FAKSVIGTPEFMAPE

T288 T232 T198 S309 S180 S808 S489 T219 S181 S172 T217 T189 T210 T239 T219 T170 S960 T262 T179 S179 S414 S159 S172 S682 T180 T173 S382 S356 S308 S335

unspecified auto PKA unannotated SGK1 unannotated unannotated unannotated TBK1 auto unannotated unannotated AURKA unspecified unannotated unannotated unannotated unannotated unannotated unannotated unannotated unannotated auto unannotated unannotated unannotated auto auto auto auto

RGC RGC

GUCY2F NPR1

GUC2F_HUMAN ANPRA_HUMAN

DYGFNDILEMLRLS----------------------------------EEESSMEELLWTAPE S705 unannotated DYGLESFRDLDPE-----------------------------------QGHTVYAKKLWTAPE T694 unannotated

5

STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE STE

MAP2K1 MAP2K2 MAP2K5 MAP3K10 MAP3K11 MAP3K12 MAP3K13 MAP3K15 MAP3K19 MAP3K2 MAP3K3 MAP3K4 MAP3K5 MAP3K6 MAP3K9 MAP4K1 MAP4K2 MAP4K3 MAP4K4 MAP4K5 MINK1 MLK4 MYO3A MYO3B NRK OXSR1 PAK1 PAK2 PAK3 PAK4 PAK6 PAK7 SLK STK10 STK24 STK25 STK26 STK3 STK39 STK4 TAOK1 TAOK2 TAOK3 TNIK ZAK

MP2K1_HUMAN MP2K2_HUMAN MP2K5_HUMAN M3K10_HUMAN M3K11_HUMAN M3K12_HUMAN M3K13_HUMAN M3K15_HUMAN M3K19_HUMAN M3K2_HUMAN M3K3_HUMAN M3K4_HUMAN M3K5_HUMAN M3K6_HUMAN M3K9_HUMAN M4K1_HUMAN M4K2_HUMAN M4K3_HUMAN M4K4_HUMAN M4K5_HUMAN MINK1_HUMAN M3KL4_HUMAN MYO3A_HUMAN MYO3B_HUMAN NRK_HUMAN OXSR1_HUMAN PAK1_HUMAN PAK2_HUMAN PAK3_HUMAN PAK4_HUMAN PAK6_HUMAN PAK7_HUMAN SLK_HUMAN STK10_HUMAN STK24_HUMAN STK25_HUMAN STK26_HUMAN STK3_HUMAN STK39_HUMAN STK4_HUMAN TAOK1_HUMAN TAOK2_HUMAN TAOK3_HUMAN TNIK_HUMAN MLTK_HUMAN

DFGVSGQLIDS-------------------------------------MANSFVGTRSYMSPE DFGVSGQLIDS-------------------------------------MANSFVGTRSYMAPE DFGVSTQLVNS-------------------------------------IAKTYVGTNAYMAPE DFGLAREWHKT-------------------------------------TKMSAAGTYAWMAPE DFGLAREWHKT-------------------------------------TQMSAAGTYAWMAPE DFGTSKELSDKS------------------------------------TKMSFAGTVAWMAPE DFGTSKELSDKS------------------------------------TKMSFAGTVAWMAPE DFGTSKRLAGVNP-----------------------------------CTETFTGTLQYMAPE DFGCARRLAWAGLNGTHSD-----------------------------MLKSMHGTPYWMAPE DFGASKRLQTICLSGT--------------------------------GMKSVTGTPYWMSPE DFGASKRLQTICMSGT--------------------------------GMRSVTGTPYWMSPE DFGCSVKLKNNAQTMPG-------------------------------EVNSTLGTAAYMAPE DFGTSKRLAGINP-----------------------------------CTETFTGTLQYMAPE DFGTSKRLAGITP-----------------------------------CTETFTGTLQYMAPE DFGLAREWHRT-------------------------------------TKMSAAGTYAWMAPE DFGISAQIGATLA-----------------------------------RRLSFIGTPYWMAPE DFGVSGELTASVA-----------------------------------KRRSFIGTPYWMAPE DFGVSAQITATIA-----------------------------------KRKSFIGTPYWMAPE DFGVSAQLDRTVG-----------------------------------RRNTFIGTPYWMAPE DFGVAAKITATIA-----------------------------------KRKSFIGTPYWMAPE DFGVSAQLDRTVG-----------------------------------RRNTFIGTPYWMAPE DFGLAREWHRT-------------------------------------TKMSTAGTYAWMAPE DFGVSAQLTSTRH-----------------------------------RRNTSVGTPFWMAPE DFGVSAQLTSTRL-----------------------------------RRNTSVGTPFWMAPE DFGVSAQVSRTNG-----------------------------------RRNSFIGTPYWMAPE DFGVSAFLATGGDITRNK------------------------------VRKTFVGTPCWMAPE DFGFCAQITPEQS-----------------------------------KRSTMVGTPYWMAPE DFGFCAQITPEQS-----------------------------------KRSTMVGTPYWMAPE DFGFCAQITPEQS-----------------------------------KRSTMVGTPYWMAPE DFGFCAQVSKEVP-----------------------------------RRKSLVGTPYWMAPE DFGFCAQISKDVP-----------------------------------KRKSLVGTPYWMAPE DFGFCAQVSKEVP-----------------------------------KRKSLVGTPYWMAPE DFGVSAKNTRTIQ-----------------------------------RRDSFIGTPYWMAPE DFGVSAKNLKTLQ-----------------------------------KRDSFIGTPYWMAPE DFGVAGQLTDTQI-----------------------------------KRNTFVGTPFWMAPE DFGVAGQLTDTQI-----------------------------------KRNTFVGTPFWMAPE DFGVAGQLTDTQI-----------------------------------KRNTFVGTPFWMAPE DFGVAGQLTDTMA-----------------------------------KRNTVIGTPFWMAPE DFGVSAFLATGGDVTRNK------------------------------VRKTFVGTPCWMAPE DFGVAGQLTDTMA-----------------------------------KRNTVIGTPFWMAPE DFGSASMAS---------------------------------------PANSFVGTPYWMAPE DFGSASIMA---------------------------------------PANSFVGTPYWMAPE DFGSASMAS---------------------------------------PANSFVGTPYWMAPE DFGVSAQLDRTVG-----------------------------------RRNTFIGTPYWMAPE DFGASRFHNHT-------------------------------------THMSLVGTFPWMAPE

S222 S226 T315 S262 S281 S269 S312 T808 S226 S520 S526 S501 T838 T806 S308 S171 S170 S170 T187 S174 T187 S303 T184 T190 S211 T185 T423 T402 T436 S474 S560 S602 S189 S191 T190 T174 T178 T180 T231 T183 S181 S181 S177 T187 S165

RAF unspecified unspecified auto auto unannotated unannotated unannotated unannotated unannotated unannotated unannotated auto unspecified auto auto unannotated unannotated unannotated unannotated unannotated auto unannotated unannotated unannotated unannotated auto auto auto auto auto unannotated unspecified unspecified auto auto auto auto unannotated auto unannotated unspecified unannotated unannotated auto

TKL TKL TKL TKL TKL TKL TKL

BMPR1A BMPR1B BMPR2 IRAK1 LIMK1 LIMK2 LRRK2

BMR1A_HUMAN BMR1B_HUMAN BMPR2_HUMAN IRAK1_HUMAN LIMK1_HUMAN LIMK2_HUMAN LRRK2_HUMAN

DLGLAVKFNSDTNEVDV-------------------------------PLNTRVGTKRYMAPE DLGLAVKFISDTNEVDI-------------------------------PPNTRVGTKRYMPPE DFGLSMRLTGNRLVRPGEEDN---------------------------AAISEVGTIRYMAPE DFGLARFSRFAGSSPSQSSMVA--------------------------RTQTVRGTLAYLPEE DFGLARLMVDEKTQPEGLRSLKKPDRK---------------------KRYTVVGNPYWMAPE DFGLSRLIVEERKRAPMEKATTKKRTLRKNDRK---------------KRYTVVGNPYWMAPE DYGIAQYCCRM-------------------------------------GIKTSEGTPGFRAPE

T400 T370 S375 T383 T508 T505 T031

unannotated unannotated unannotated unannotated ROCK1 ROCK1 unannotated

2031

Fig. S4. Sequence alignment of the activation loops of all human serine/threonine kinases with Ser or Thr at the -12 position of the activation loop. Potential and annotated phosphorylation sites in the activation loop are indicated in red. The residue number of the -12 site and any kinases annotated as targeting the site are given after each sequence. If a specific kinase is listed, Uniprot lists that kinase as responsible for phosphorylation at the -12 site; “auto” indicates annotation of autophosphorylation in Uniprot; “unspecified” indicates that Uniprot has an annotation for phosphorylation at the -12 site but does not indicate a specific kinase; “unannotated” indicates that Uniprot does not have an annotation for the -12 site. The PAK1 structures (e.g. PDB: 4ZY4) may be used as hypothetical autophosphorylation complexes for these sites.

Uniprot FGFR1_HUMAN FGFR2_HUMAN FGFR3_HUMAN FGFR4_HUMAN

Kinase insert sequence REYLQARRPPGLEYCYNPSHNPEE REYLRARRPPGMEYSYDINRVPEE REFLRARRPPGLDYSFDTCKPPEE REFLRARRPPGPDLSPDGPRSSEG

Site Y583 Y586 Y577 -

Fig. S5. Sequence alignment of the kinase insert loops of FGFR kinases. Sequence alignment of the kinase insert regions in FGFR1, FGFR2, FGFR3, and FGFR4 containing the autophosphorylation sites in FGFR1 (PDB: 3GQI) and FGFR3 (PDB: 4K33). The homologous autophosphorylation sites are shown in red, bold type. FGFR4 does not contain a known phosphorylation site in this region. Other phosphorylation sites annotated as autophosphorylated in UniProt are shown in blue, bold type. The phosphorylation residue number is given after the sequence.

6

Uniprot FGFR1_HUMAN FGFR2_HUMAN FGFR3_HUMAN FGFR4_HUMAN RON_HUMAN MET_HUMAN KIT_HUMAN FLT3_HUMAN

C-terminal extension sequence CWHAVPSQRPTFKQLVEDLDRIVALTSNQEYLDLSMP CWHAVPSQRPTFKQLVEDLDRILTLTTNEEYLDLSQP CWHAAPSQRPTFKQLVEDLDRVLTVTSTDEYLDLSAP CWHAAPSQRPTFKQLVEALDKVL-LAVSEEYLDLRLT CWEADPAVRPTFRVLVGEVEQIVSALLGDHYVQLPAT CWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNAT CWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANC CWAFDSRKRPSFPNLTSFLGCQLADAEEAMYQNVDGR

Site Y766 Y769 Y760 Y754 Y1353 Y1349 Y936 Y955

Fig. S6 Sequence alignment of C-terminal extensions of FGFR2 and related tyrosine receptor kinases. Sequence alignment of FGFR2 region of autophosphorylation site Y769 (PDB: 3CLY) and the same region in the indicated receptor tyrosine kinases. All of these proteins are annotated in UniProt as autophosphorylated at the highlighted tyrosine sites (bold, red type). The phosphorylation residue number is given after the sequence.

Uniprot CLK1_HUMAN CLK2_HUMAN CLK3_HUMAN CLK4_HUMAN

N-terminal region HRRKR-TRSVEDDEEGHLI HSSRR-AKSVEDDAEGHLI QSSKRSSRSVEDDKEGHLV HRRKR-SRSIEDDEEGHLI

Site S140 S142 S283 S138

Fig. S7. Sequence alignment of the N-terminal regions of CLK family members. The N-terminus of CLK2, CLK1, CLK3 and CLK4 are highly conserved. The serine phosphorylation sites (bold red) in CLK1, CLK3, and CLK4 are not specified as phosphorylated in UniProt. Other phosphorylation sites (one in CLK1 and one in CLK4) are shown in bold blue, annotated as phosphorylated sites in UniProt.

Uniprot KCC2A_HUMAN KCC2B_HUMAN KCC2G_HUMAN KCC2D_HUMAN

C-terminal tail sequence MHRQETVDCLKK MHRQETVECLKK MHRQETVECLRK MHRQETVDCLKK

Site T286 T287 T287 T287

Fig. S8. Sequence alignment of the C-terminal tails of CaMKII family members. Sequence alignment of 4 human CaMKII isoforms shows that the autophosphorylated threonine and C-terminal tail regions are highly conserved.

Uniprot EGFR_HUMAN ERBB2_HUMAN ERBB4_HUMAN ERBB3_HUMAN

C-terminal tail sequence DSNFYRALMDEEDMDDVVDADEYLIPQQG DSTFYRSLLEDDDMGDLVDAEEYLVPQQG DSKFFQNLLDEEDLEDMMDAEEYLVPQAF PHGLTNKKLEEVELEPELDLDLDLEAEED

Site Y1016 Y1023 Y1022 -

Fig. S9. Sequence alignment of the C-terminal tails of EGFR, ErbB2, ErbB3, and ErbB4. Sequence alignment of EGFR C-terminal tail shows that the phosphorylation site is conserved in ERBB2 and ERBB4. The same region in ERBB3 region is quite different from that in the other kinases and does not contain a tyrosine at this site.

7

Table S1. LCK autophosphorylation experimental data. Data from four independent experiments are shown. µ(Myc_Mut/Myc_WT)

a

µ(pLCK_Mut/Myc_Mut)

b

c

WT

1.000

0.446

µ(pLck_Mut/Myc_Mut)/ c pLCK_WT/Myc_WT) 1.000

T445V

0.739

0.092

0.207

(0.073-0.589)

N446A

0.971

0.208

0.277

(0.168-0.456)

P447A

0.749

1.764

2.344

(1.680-3.271)

P447G

1.320

0.780

1.752

(0.910-3.372)

P447L

1.108

0.830

1.864

(0.838-4.146)

Q451E

1.166

0.416

0.935

(0.336-2.560)

Mutant

CI -

a

µ(Mut/WT) is the proper average of the ratios of the Myc level of the mutant and the wildtype in the same blot on the same day, calculated as a geometric mean by transforming to a log scale and transforming back with the anti-log:

⎛1 4 ⎛ Myc ( Mut )i ⎞ ⎞ µ = exp ⎜ ∑ log ⎜ ⎟⎟ ⎝ Myc (WT )i ⎠ ⎠ ⎝ 4 i=1 b

µ(pLCK/Myc) is the geometric mean of the pLCK/Myc ratios for each transfection. i.e.,

⎛1 4 ⎛ pLCK ( Mut )i ⎞ ⎞ µ = exp ⎜ ∑ log ⎜ ⎟⎟ ⎝ Myc ( Mut )i ⎠ ⎠ ⎝ 4 i=1 c

The 95% confidence interval (CI) for (pLCK/Myc)(Mut) / (pLCK/Myc)(WT) is calculated as

( )

µ = exp µlog

σ ⎞ ⎛ CI ± = exp ⎜ µlog ± 1.96 log ⎟ ⎝ N⎠ where µ log and σlog are the mean and standard deviation of

⎛ pLCK ( Mut )i log ⎜ ⎝ Myc ( Mut )i

pLCK (WT )i ⎞ . Myc (WT )i ⎟⎠

8

Table S2. 58 domain-swapping PDB dimers in 17 distinct crystal forms from ProtCID. CF No.1 10 1 9 13 13 2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 14 11 12 15 16 8 3 3 3 7 6 6 17 17 17 17 5 5 5 177

Space Group P 1 21 1 P 21 21 21 P 1 P 32 2 1 P 32 2 1 P 31 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 32 2 1 P 1 21 1 P 1 21 1 P 43 21 2 P 43 21 2 P 1 P 32 2 1 P 32 2 1 P 32 2 1 C 2 2 21 P 21 21 21 P 21 21 21 P 61 2 2 P 61 2 2 P 61 2 2 P 61 2 2 I 2 2 2 I 2 2 2 P 1 21 1

PDB 3OHT 4C3P 2BMC 3DJ5 3DJ6 4AF3 2CN5 2W0J 2W7X 2WTC 2WTD 2WTI 2WTJ 2XBJ 2XK9 2XM8 2XM9 2YCF 2YCQ 2YCR 2YCS 2YIQ 2YIR 2YIT 4A9R 4A9S 4A9T 4A9U 4BDA 4BDB 4BDC 4BDD 4BDE 4BDF 4BDG 4BDH 4BDI 4BDJ 4BDK 2CN8 2Y4P 3ZXT 3BQR 2J90 2JAM 2AC3 2AC5 2HW7 3ALO 2VWI 3DAK 2J51 2JFL 2JFM 2UV2 2J7T 4BC6 4EQU 58

Surface Area2 2712 2019 1791 2235 2269 1613 1338 1282 1241 1182 1281 1204 1239 1227 1319 879 1191 1257 1317 1343 1278 1308 1394 1276 1211 1154 1207 1194 1222 1263 1261 1246 1240 1228 1256 1245 1242 1265 1219 1161 1383 1407 1918 1941 2356 1499 1474 1393 2193 1942 2070 1737 2040 1804 1769 1734 1487 1737 1520

Auth. BA3 A2 A2B2 A2 A A AB A2 A A2 A A A A A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A A A A2 A A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 AB A A A AB A4 A4 A2 A2 A2 A2 A2 A2 A2

PISA BA4 A2 A2B2 A2 A2 A2 A2B2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A4 A2B A2 A2 A2 A2B2 A4 A2 A2 A2 A2 A2 A2 A2 A2

In Auth.5 1 1 1 0 0 0 1 0 1 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 1 1 1 1 1 1 1 1 40

In PISA6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 58

UniProt Code A9UJZ9_SALSA AURKA_HUMAN AURKA_HUMAN AURKA_MOUSE AURKA_MOUSE AURKB_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN CHK2_HUMAN DAPK1_HUMAN DAPK1_HUMAN DAPK3_HUMAN DAPK3_HUMAN KCC1G_HUMAN MKNK2_HUMAN MKNK2_HUMAN MKNK2_HUMAN MP2K4_HUMAN OXSR1_HUMAN OXSR1_HUMAN SLK_HUMAN SLK_HUMAN SLK_HUMAN SLK_HUMAN STK10_HUMAN STK10_HUMAN STK10_HUMAN 13

GT Hydrogen Bond Yes Yes Yes

Yes Yes Yes Yes

Yes Yes 9

1

CF No: crystal form number in ProtCID database. Surface Area: the accesible surface area. 3 Auth. BA: the biological assembly defined by authors. 4 PISA BA: the biological assembly defined by PISA program. 5 In Auth.: whether the dimer exists in author-defined biological assembly (1=Yes; 0=No). 6 In PISA: whether the dimer exists in PISA-defined biological assembly (1=Yes; 0=No). 7 The last row gives the counts in each column, except for the surface area, which is instead the average value. 2

9

Data file S1. Coordinates of kinase autophosphorylation complexes. The zip file includes PDB-format files of 25 1166 autophosphorylation complexes in table 1 as well as the D:A interface of 3LVP (file: IGF1R_3lvp_2.pdb, with Tyr disordered). Data file S2. Models of asymmetric autophosphorylation complexes of IGF1R and LCK. The zip file includes the PDB files of the models of IGF1R and LCK in Fig. 2 of the main text. The IGF1R model was built by superposition of an active monomer from PDB: 1K3A (chain A, DFG-in) onto one monomer of the the IGF1R (PDB: 3D94) autophosphorylation complex. The LCK model was built by superposition of an active monomer from PDB: 1QPC (chain A, DFG-in) to one monomer of the LCK (PDB: 2PL0) autophosphorylation complex. Both complexes were optimized by energy minimization with the program Gromacs (see main text).

10