Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007

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1 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007

2 Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007 Supporting Information for A Multiplexed Protein Kinase Assay Melissa D. Shults, Igor A. Kozlov, Nicholas Nelson, Bahram G. Kermani, Peter C. Melnyk, Veronika Shevchenko, Anu Srinivasan, Joseph Musmacker, John Hachmann, David L. Barker, Michal Lebl, and Chanfeng Zhao Figure S1. Assay variability is indicated by the 8 data points collected for each PKA concentration in a titration curve. Each data point represents a completely separate sequence of phosphorylation, labeling, hybridization and staining. Fits were obtained as described in Methods with all 8 data points as an input.

3 A) B) C) D) E) F) G) H) I) J) K) Figure S2. The same peptide attached to two different DNA tags gives similar responses in an enzyme titration with Src (A-C) or SGK1 (D-K). The peptide sequences are as follows: A) DEEIYEELG; B) EEEIYEEIE; C) GEGIYGVLF; D) GRPRTSSFAEG; E) RPRAASF; F) RPRAATF; G) RPR-LSSF; H) RPRLTSF; I) RPRSTSF; J) RPRTSSF; K) RPRTTSF. See methods section for data analysis.

4 Figure S3. Data from the top two CK2 substrates illustrate the effect of TBB inhibition on CK2 activity. The substrates are (a) EQSGGEGDDD and (b) EWSGGEGDDD. CK2 assays were performed with 50 µm ATP. The following IC 50 values were calculated by fitting the dose-response curves: a) 5.4 µm and b) 10 µm. The data were fit in KaleidaGraph 4.0 with the following equation: y = f(x) = a + (b/(1+10 x-c )), where c = log IC 50. Sarno and coworkers report that 10 µm TBB inhibits 85 % of CK2 activity at a similar ATP concentration of 100 µm, which correlates well with our data (Sarno, S. et al. FEBS Lett. 496, (2001)).

5 Figure S4. Kinase activity against a background of cellular protein was observed in two different commercially-available cellular extracts: a nuclear extract from PC12 cells stimulated with forskolin and another nuclear extract from Hela cells. Kinase-specific activity was verified when inhibitors for each of these enzymes were incubated in the reaction with the cell extract. By comparison with single enzyme data (Figure 3), the profile from the PC12 nuclear extract is predominantly a PKA signature and the Hela extract profile contains a CK2 signature. a) The activity profile of nuclear extract from PC12 cells (2.5 µg) stimulated with forskolin shows a strong PKA signature. It is known, however, that forskolin (N. J. de Souza, Med. Res. Rev. 1983, 3, 201) stimulates PKA activity by activating adenylate cyclase. b) This PKA signature is eliminated when the assay reaction contains the inhibitor PKI (17 µm). c) The activity profile of extract from Hela cells (2 µg) shows a strong CK2 signature. d) This CK2 signature is eliminated when the assay reaction contains the inhibitor TBB (1 mm). This concentration of TBB is required to inhibit recombinant CK2 activity when 1 m M ATP is present. The inability to detect many kinases in these mixtures may be due to the fact that the lysate preparations are unfavorable for other kinases, that other kinases are not active, that they are present below our limit of detection, or that the lysates purchased were not guaranteed for kinase activity. All reaction mixtures contained 10 nm of each conjugate and 1 mm ATP. We found that 2-3 µg lysate per 10 µl reaction was optimal for these experiments. Higher concentrations of lysate resulted in higher assay background, perhaps due to labeling of proteins that adhere to the array. The radial plots here have a maximum value of 1.

6 Supporting Methods Peptide-DNA conjugate characterization Starting peptides are of are checked for good quality by HPLC and MS and gel-purified conjugates are of high purity. 300 mau Kinase UV_VIS_3 WVL:217 nm A) B) min C) D) A) Typical HPLC profile of peptides made by Illumina s high-throughput peptide synthesis method (Waters Bondpak C 18 4 x 150 mm, 1.5 ml/min, A = water with 0.05% TFA, B = acetonitrile with 0.05% TFA, 0% B for 2.5 min followed by 0-70% B over 12.5 min.); B) typical electrospray mass spectrometry profile of peptides made by Illumina s high-throughput peptide synthesis method; C) 15% TBE-urea polyacrylamide gel analysis of conjugation reactions for 7-mer PKA substrates; lanes 1,3,5, starting oligonucleotides; lanes 2,4,6, oligo-peptide conjugates; D) 20% TBE-urea polyacrylamide gel analysis for C3, C4 and C5 after 18 months of storage: starting DNA has degraded with storage at 4C but gel shows that purified conjugates are very clean and stable for long periods of time at -20C.

7 Peptide-DNA conjugate screening To assure that each conjugate was present in the final pool, the pool was hybridized to Illumina s Sentrix Array matrix (5 nm, 20 min) followed by hybridization of the same oligonucleotides that were FAM-labeled (20 nm, 15 min). In addition, the FAM-labeled decoders were hybridized alone to determine maximal signal. When data from the pool was compared with the maximum signal, a conjugate with 75% or more of the FAM signal was considered to couple poorly, and was not included in the final pool. This corresponded to 91 conjugates out of 1250 prepared. Conjugates were also initially screened to detect background performance, and we found that 257 conjugates out of 1250 prepared gave high signal prior to phosphorylation. These conjugates were not included in the final pool. The peptide was the main contributor to this background phenomenon, and incompatibility with some peptide substrates is a potential limitation of the assay which we do not fully understand. One theme for a significant portion of these codes with high background is a sequence containing a histidine residue nearby an aspartate or glutamate residue. We suspect that, during the blocking step, histidine displaces the EDC prior to N-acyl urea formation and then this carboxylate is susceptible to dye attachment during labeling. For our purposes this was not limiting, as we could easily screen out these conjugates and still generate sufficient pool diversity. Purified Enzyme Concentrations and Additive Concentrations Enzyme Source Final Amount in 10 µl rxn Additives p60 Upstate 0.3 U PKA Calbiochem 0.25 U Abl Upstate 5 ng PKCz Upstate 25 ng 1:200 PKC lipid activator (Upstate) PKCa Upstate 5 ng 0.1 mm CaCl 2 and 1:4 PKC lipid activator (Upstate) CaMK2 Upstate 50 ng 1 mm CaCl 2 and 10 ug/ml calmodulin SGK1 Upstate 3.3 ng

8 Cdk2 Upstate 200 ng CK2 Upstate 20 ng Lck Upstate 3.3 ng EGFR Upstate 100 ng 0.6 M NH 4 SO 4 and 10 mm MnCl 2 Akt Upstate 2 ng Chk1 Upstate 10 ng Chk2 Upstate 10 ng MK2 Upstate 2 ng CK1 Upstate 20 ng p38 Upstate 250 ng Cdk5 Upstate 100 ng Aurora A BPS 4 ng Bioscience Aurora B BPS Bioscience 25 ng VEGFR2 (KDR) Flt3 EphB2 Kit Plk3 Jak2 BPS 3 ng Bioscience BPS 1.3 ng Bioscience BPS 175 ng Bioscience BPS 125 ng Bioscience BPS 10 ng Bioscience BPS 1 ng Bioscience

9 Table S1. Peptide sequences, target kinases and activities observed for pool of 900 conjugates controls. Code Peptide sequence a Kinase activity detected a 1 N/A Cy3 Control 2 N/A Cy3 Control 3 N/A Cy3 Control 4 N/A Cy3 Control 5 N/A Cy3 Control 6 N/A Cy3 Control 7 N/A Cy3 Control 8 N/A Cy3 Control 9 N/A Cy3 Control 10 N/A Cy3 Control 11 N/A FAM Control 12 N/A FAM Control 13 N/A FAM Control 14 N/A FAM Control 15 N/A FAM Control 16 N/A FAM Control 17 N/A FAM Control 18 N/A FAM Control 19 N/A FAM Control 20 N/A FAM Control 21 N/A FAM Control 22 N/A FAM Control 23 N/A FAM Control 24 N/A FAM Control 25 N/A FAM Control 26 N/A No Label 27 N/A No Label 28 N/A No Label 29 N/A No Label 30 N/A No Label 31 N/A No Label 32 N/A No Label 33 N/A No Label 34 N/A No Label 35 N/A No Label 36 N/A No Label 37 N/A No Label 38 N/A No Label 39 N/A No Label 40 N/A No Label 41 N/A No Label 42 N/A No Label 43 N/A No Label 44 N/A No Label 45 N/A No Label 46 GGGDG 47 GGGEG 48 GGGYG 49 GGGSG 50 GGGTG 51 GAGSGASGAS 52 GAGTGATGAT 53 GAGYGAYGAY EphB2 54 GGGpYG 55 GGGpYG 56 GGGpYG 57 GGGpYG 58 GGGpYG 59 GGGpYG 60 GGGpYG 61 GGGpYG 62 GGGpSG 63 GGGpSG 64 GGGpSG 65 GGGpSG 66 GGGpSG 67 GGGpSG 68 GGGpSG 69 GGGpSG 70 GGGpSG 71 GGGpSG 72 GGGpSG 73 GGGpSG 74 GGGpSG 75 GGGpSG 76 GGGpSG 77 GGGpSG 78 GGGpSG 79 GGGpTG 80 GGGpTG 81 GGGpTG 82 GGGpTG 83 GGGpTG 84 GGGpTG 85 GGGpTG 86 GGGpTG 87 GGGpTG 88 GGGpTG 89 GGGpTG 90 GGGpTG

10 91 GGGpTG 92 GGGpTG 93 GGGpTG 94 GGGpTG 95 GGGpTG 96 GGGpTG 97 GAGAGAGGAG 98 GAGHGAHGAH 99 GAGRGARGAR 100 GAGWGAWGAW 101 GAGNGANGAN 102 GAGQGAQGAQ 103 EEDGGSIFIFFAKKK CK1 104 EEDTGSIFIFFAKKK CK1, Plk3 105 EFDTGSIFIFFAKKK CK1, Plk3 106 EEDGGSIFIFFAKKK CK1 107 EFDTGSIFGFFAKKK CK1 108 GRPRAATFAEG Akt, SGK 109 RKRRNTISRS 110 RKRRNTFSSS 111 RKRRKTFSRS 112 RKRRNTFSRS SGK 113 RKRRNSISRS Akt, SGK, PKA, Aurora A/B, Cdk5 114 RKRRKSFSRS Akt, SGK, PKA, Aurora A/B, Cdk5 115 VERTNSLPPV Chk2 116 LDKAKSRPSL Aurora A/B 117 ALRRSLG Aurora B 118 ALRGSLG Aurora B 119 ALRISLG Aurora B 120 ALRPSLG Aurora B 121 ALRFSLG Aurora B 122 ALRWSLG 123 ALRKSLG Aurora B 124 ALRKSKG 125 LRRASLG Akt, PKA, Aurora A/B 126 RLQRRRGSSIPQF Akt, SGK, PKCz, PKA, Aurora A/B 127 RAGKRRPSRLVAL PKA 128 INSIRKFSIVQKT 129 IAEPMRRSVSEAALA 130 GSRSRTPSLPTPPTR 131 SRFNRRVSVCAETYN 132 NQNSRRPSRATWLSL 133 PHGSPRVSVTDDSWL Plk3, PKA, Cdk5 134 ALRRHSVA Akt, SGK, PKA, Aurora A/B 135 ALRRHTVA 136 ALRRWSVA Akt, SGK, PKA, Aurora A/B 137 ALRRWTVA 138 ALRRRSVA SGK, PKA, Aurora A/B 139 ALRRRTVA 140 ALRRFSVA Akt, SGK, PKA, Aurora A/B 141 ALRRFTVA Akt 142 ALRRETVA 143 ALRRASVA PKA, Aurora A/B 144 ALRRATVA 145 ALRRLSVA Akt, SGK, PKA, Aurora A/B, Chk2, 146 ALRRLTVA 147 LRRHSLGA PKA, Aurora A/B 148 LRRWTLGA Akt, Aurora A/B 149 LRRHTLGA 150 LRRATLGA Aurora B 151 RPRAATF Akt, SGK 152 EEQEYVQTVKSSKGG 153 RRTRVPPSRRGPDAV 154 CLPLKNASDKRNQQT 155 GRILSLPRSN 156 RRQSVRRLG 157 RRLSVRRLG Aurora B 158 RRVSVRRLG PKCz, Aurora B 159 RRFSVRRLG Aurora B 160 RRQSFRRLG 161 RRQSERRLG 162 RRQSDRRLG 163 RRESVRRLG Aurora B 164 RRDSVRRLG 165 RRRSVRRLG 166 RRGSVRRLG SGK, PKCz, PKCa 167 RRLSLRRLG 168 RRQTVRRLG 169 RRLTVRRLG 170 RRVTVRRLG 171 RRFTVRRLG 172 RRQTFRRLG 173 RRQTERRLG 174 RRQTDRRLG 175 RRQTRRRLG 176 ALRRHTVA 177 FRALSERH 178 FRALTERH 179 FRALSERT 180 FRALSERR 181 FRALSERE 182 FRALSERD 183 FRADSERH 184 FRALSDRH 185 FRALSDRT

11 186 FRAVSERT 187 FRAISERT 188 FRAFSERT 189 FRA LSLRH 190 FRLLSLRH 191 FRGLSLRT 192 FRNLSLRT 193 FRALTERT 194 KPQPRRRSSFGIMIK 195 GDYFRYLSEVASGDN 196 SMGTLRTSISVERQI 197 CGRPDPSSFSRAREA 198 VSAGIQTSFRTGNPT 199 GSKKKICTRKPRFMS 200 RRQSRRRLG 201 RRQSNRRLG 202 KRAKRKTAKKR 203 IK IQASFRGH 204 LGKRQTEREK 205 FFFRRSKIAV 206 PLSRTLSVAAKK 207 QKRPSQRSKYL 208 RRGRTGRGRRGIFR 209 APTKRNSSPPPSP Cdk5, Cdk2, p TPQTQSTSGRRRR 211 RRRRRKGSFRRKA Akt, SGK, PKCz, PKCa 212 FKLKRKGSFKKFA 213 TSEPVQLTPDDEDAI 214 RRTRVPPSRRGPDAV 215 VSAGIQTSFRTGNPT 216 FRAASLRH PKCz 217 FRWASLRH 218 FRHASLRH 219 FRLASLRH 220 FRVASLRH 221 FRFASLRH 222 FRGASLRH 223 FRNASLRH PKCz 224 FRPASLRH 225 FRAPSLRH 226 FRAWSLRH 227 RRPSVRRLG 228 RRWSVRRLG PKCz, Aurora B 229 RRHSVRRLG PKCz, PKCa, Aurora B 230 RRFSVRRLG SGK, PKCz, PKCa, Aurora B 231 RRQSVRRLG PKCz, PKCa, Aurora B 232 RRESVRRLG 233 RRRSVRRLG PKCz, PKCa, Aurora 234 RRVSVRRLG PKCz, PKCa, Aurora B 235 RRISVRRLG Aurora B 236 RRGSVRRLG PKCz, PKCa 237 RRLSLRRLG PKCz, PKCa, Aurora A/B 238 LRRKRSLSYS Akt, SGK, PKCz, PKA, Chk2, Cdk5, Kit 239 LRRKRSISYS Akt, SGK, PKCz, PKCa, PKA,Chk2, CaMK2, Chk1, Cdk5, Kit 240 GRPRTSSFAEG Plk3, Akt, SGK, Cdk5 241 GGRNRRLSVA Akt, SGK, PKA, Aurora A/B 242 FFRNRRLSVA Akt, SGK, PKCz, PKA, Aurora A/B 243 QQRNRRLSVA Akt, SGK, PKA, Aurora A/B 244 RRRNRRLSVA Akt, SGK, PKA, Aurora A/B, Cdk5 245 RNRLLSVA Akt, SGK 246 RERELSVA 247 RERWLSVA SGK 248 RWRELSVA 249 RPRTSSF Akt, SGK 250 RPRESSF SGK 251 RPRAATF Akt, SGK 252 RPRAASF Akt, SGK, PKA 253 RPRSSTF 254 RPRLSSF Akt, SGK 255 RPRAASF Akt, SGK 256 RPRLLSF Akt, SGK, PKA, Aurora B 257 RPRVVSF Akt, SGK 258 RPRVSSF Akt, SGK 259 RPRLSSF Akt, SGK, Aurora B 260 RPRASSF Akt, SGK 261 RPRWSSF SGK 262 RPRHSSF Akt, SGK 263 CKPVQQPSAFGSMK 264 SRTNSNASTVSGR 265 GRPRTSSGAEG Akt, SGK 266 RERKLSVA 267 RPRLLSF 268 RPRSSSF Akt, SGK, PKA 269 RPRFSSF Akt, SGK 270 GRPRTSSFAEG Plk3, SGK 271 GRPRTSTFAEG Akt, SGK 272 GRPRTTTFAEG Akt, SGK 273 GRPRTSTFAEGKK 274 GRPRTTSFAEGKK Akt, SGK 275 GRPRTTTTFAEGKK 276 RNRLLTVA Akt, SGK B

12 277 RPRAATF Akt, SGK 278 RPRAASF Akt, SGK 279 RPRATSF Akt, SGK 280 RPRASTF Akt, SGK 281 RPRATTF Akt, SGK 282 RPRESTF SGK 283 RPRETTF 284 RPRLLTF 285 RPRLTSF Akt, SGK 286 RPRLSTF 287 RPRLTTF Akt, SGK 288 RPRLTSF Akt, SGK 289 RPRLSTF Akt, SGK 290 RPRSTSF Akt, SGK 291 RPRTSSF Akt, SGK 292 RPRSTSF Akt, SGK 293 RPRSTTF Akt, SGK 294 RPRTTSF Akt, SGK 295 RPRTTTF Akt, SGK 296 RPRTTSF Akt, SGK 297 RPRVSTF Akt, SGK 298 RPRVTSF Akt, SGK 299 RRLSSLRA Aurora A/B 300 GAKKRNRTLTK Akt, SGK 301 RKISASEFDRPLR 302 KTFCGTPEYLAPEVRR 303 HMRSAMSGLHLVKRR 304 LKKLTRRPSFSAQ Akt, SGK, PKCz, PKA, Aurora B, Chk2, CaMK2, Chk1 305 ASARAASAAALARRR 306 LALHIRSSWSGLH 307 PLARTLSVAGLPGKK Akt, PKCz, Chk2, 308 NYLRRRLSDSN 309 LARTLSVAG Chk2, CaMK2, MK2 310 LHRTLSVAG 311 LWRTLSVAG 312 LLRTLSVAG 313 LVRTLSVAG Akt, Chk2, 314 LQRTLSVAG Akt, PKCz, Chk2, CaMK2, MK2 315 LERTLSVAG Chk2 316 LDRTLSVAG Chk2 317 LPRTLSVAG Chk2 318 LRRTLSV AG Akt, PKCz, Chk2, CaMK2, MK2 319 LARALSVAG 320 LARARSVAG 321 LARAWSVAG 322 LARAESVAG 323 LARADSVAG Chk2 324 LARTLSEAG Chk2 325 LARTLSDAG Chk2 326 LARTLSLAG 327 LARTLSIAG Chk2 328 LARTLSSAG Chk2 329 GARVFSVL 330 GARVFTVL 331 GARVLSVL 332 GARVISVL 333 GARVWSVL 334 GARVHSVL 335 GARVPSVL 336 GARVESVL 337 LARTLSVAGLPG 338 LARTLTVAG Chk2 339 LFRTLSVAG Akt, Chk2, CaMK2, MK2 340 LARAHSVAG 341 LARTLSRAG Chk2 342 LARTLSFAG Akt, PKCz, Chk2, CaMK2, MK2 343 LARTLSNAG Akt, Chk2, CaMK2 344 LARTLSQAG Chk2, CaMK2 345 GARVVSVL 346 TRQASQSGQ 347 TRQTSVSGQ 348 SRTASFSES Plk3, Akt, SGK 349 RRASTIEMP 350 GRRQSLIED Akt, PKA, Aurora A/B 351 AGRLSSMAMI 352 GRRAVSEQDA 353 VTRRISQTSQ 354 LLLQDSVDFS 355 IIRQASQAGP 356 FGRK ASGSSP 357 AGRRQSLIQD PKA, Aurora A/B 358 GRRAVSELDA 359 VIGSVSEDNS 360 FARVFSVLRE 361 ATRQTSVSGQ 362 GTRTYSLGSA 363 LTRQASQAGP 364 ITHYGSLPQK 365 GLRRQETVDALAKKK 366 KKLNRTLTVA 367 KKLNKTLSVA 368 KKLNRTLTVA 369 KRQQSFDLF CaMK2 370 KRQKSFDLF CaMK2 371 KRQLSFDLF Plk3, Chk2, CaMK2 372 KRQQSFELF Plk3, CaMK2

13 373 KRQQSFILF 374 KRQQSFDKF 375 KRQQSFDIF 376 KRQQSFDLK CaMK2 377 FRQQSFDLF 378 KRQQSIDLF CaMK2 379 AGETRFTDTRK 380 AGETRFTDTRK 381 VPGKARKKSSSQLL 382 KSDGGVKKRKSSSS Aurora B 383 VPGKARKKSSSQLL 384 PLARTLSVAGLP Akt, PKCz, Chk2, CaMK2, MK2 385 LKRYASIALEAKK Chk2, CaMK2, Chk1 386 LVRFASIALEAKK 387 LALRRYRSFSLA Plk3, Akt, SGK, PKCz, PKCa, PKA, Aurora B, Chk2, CaMK2, MK2, Chk1 388 LALRRYFSQAEY Chk2 389 LALRRYFSQAQY Chk2, MK2, Chk1 390 PKCz, Chk2, Chk1 ALKLVRYPSFVITAKKK 391 Chk2 ALKLVRYPSFVITAKKK 392 AIRRAASFRFR Akt, PKCz, PKCa, Chk2, CaMK2, Chk1 393 FIRRAASFRFR Akt, PKCz, PKCa, Chk2, CaMK2, Chk1 394 ILRRAASFRFR Akt, SGK, PKCz, PKCa, Ch k2, CaMK2, Chk1 395 ILRRAASFIFR Akt, SGK, PKCz, PKCa, Chk2, CaMK2, MK2, Chk1 396 RAKRKESIFIK 397 RLKRKESIFIK PKCz, Chk2, 398 AMRLERQDSIFYPKKK 399 AMRLERQDSIFYPKKK 400 ILRGPSWDPF 401 LSRQLSS GVS 402 FSRALSRQLS PKCz, Chk2, CaMK2, MK2 PKCz, PKCa, Chk2, CaMK2, MK2 403 KKANRTLSVA Chk2 404 KKKNRTLSVA 405 KKANRTLSVA 406 AHIQRQLSIAHH 407 AHLARQLSIAHH PKCz, Chk2, MK2 408 AHLQRQNSIAHH PKCz, Chk2, 409 AHLQRQLSVAHH PKCz, Chk2, CaMK2, MK2 410 AHFQRQLSIAHH PKCz, Chk2, MK2 411 KKLNRTLSVA PKCz, Chk2, CaMK2, MK2 412 KKFNRTLSVA PKCz, Chk2, CaMK2, MK2 413 KKLNRTLSVA Akt, SGK, PKCz, Chk2, CaMK2, MK2 414 AHLQRQLSIAHH 415 AHLQRQLSIAHH 416 ARKRRRHPSGSQTA 417 ARKRRRHPSGPPTA 418 ARKRRRHPSGPPTA 419 APRSPGRRRRK Cdk5, Cdk2 420 ARSPGRRRRK Cdk5, Cdk2 421 PKTPKKAKKL 422 GGGPRSPKKAKKL 423 GGGPRTPKKAKKL 424 GGGPESPKKAKKL 425 GGGPETPKKAKKL 426 GGGPPSPKKAKKL 427 GGGPPTPKKAKKL 428 GGGPASPKKAKKL 429 HHHRSPRKR 430 HHHRTPRKR 431 HHPRSPRKR 432 HHPRTPRKR 433 HHRRSPRKR 434 HHRRTPRKR 435 HHERSPRKR 436 HHASPRK 437 HHDSPRK 438 HHRSPRK 439 HHKSPRK 440 HHPSPRK 441 HHWSPRK 442 HHHSPRK 443 HHFSPRK 444 HHISPRK 445 HHQSPRK Cdk5, Cdk2 446 HHNSPRK 447 HHLSPRK 448 HHGSPRK Cdk5, Cdk2 449 HHSSPRK 450 HHTSPRK 451 HHATPRK 452 HHDTPRK 453 HHRTPRK 454 HHKTPRK 455 HHWTPRK 456 HHHTPRK 457 HHFTPRK 458 HHITPRK 459 HHVTPRK 460 HHQTPRK 461 HHNTPRK

14 462 HHLTPRK 463 HHGTPRK 464 HHSTPRK 465 HHTTPRK 466 GLAKSFGSPNRAY Cdk5 467 GSPNRAYTHQVVT 468 KHHKSPRRK 469 KHPKSPRRR Cdk5, Cdk2 470 KHHGSPKHR 471 KHHKSPKKK 472 KHHKSPKKH 473 KHHKSPKKR 474 KHHKSPRHR 475 KHPKSPKHR Cdk5 476 KHHKSPKHR 477 VPASPSPQRQ Cdk5, Cdk2 478 IPASPSPQRQ 479 IPTSPITTTYFFFKKK 480 IPTSPITTTYFFFKKK 481 LVEPLTPSGEAPNQK 482 SGTAGLTPPTTPPHK 483 SGTAGLTPPTTPPHK 484 LVEPLTPTGEAPNQK 485 AKPTTLSLPLSPESP 486 SGTAGLTPPTSPPHK 487 SGTAGLTPPTSPPHK 488 TLSLPLTPESPNDP 489 LPLTPESPNDPKGS 490 LSLPLTPESPNDPK 491 LPLTPESPNDPKGS 492 LTPESPNDPKGSPF 493 TTLSLPLTPESPND 494 TPPTTPPHKANQDN 495 LTPPTTPPHKANQD 496 AGLTPPTTPPHKAN 497 SGTAGLTPPTTPPG 498 TLSLPLSPESPNDP 499 LPLSPESPNDPKGS 500 LSLPLSPESPNDPK 501 LTPETPNDPKGSPF 502 TTLSLPLTPETPND 503 TPPTSPPHKANQDN 504 LTPPTSPPHKANQD 505 AGLTPPTSPPHKAN 506 SGTAGLTPPTSPPG 507 EVTSTSQSPHSPD 508 QSPHSPDSSQSSL 509 SLPLTPESPNDPK 510 GLTPPTTPPHKAN 511 PTTPPHKANQDNP 512 IPTSPITTTYFFF 513 EVTSTSQTPHSPD 514 QSPHTPDSSQSSL 515 PETPND PKGSPFE 516 PTSPPHKANQDNP 517 KKLRRTLSVA Akt, SGK, PKCz, Chk2, CaMK2, MK2 518 KKLRRSLSVA SGK, Chk2, CaMK2 519 APRTPGGRR 520 GAGPQSPIG Cdk5 521 PGPQSPGSPL Cdk5, Cdk2 522 GAGGPMSPIG 523 GAGIPQSPIG Cdk5, Cdk2, p GAGIPQSPWG 525 GAGIPQSPYG Cdk5 526 GAGVPQSPIG 527 GAGVPQSPWG 528 GAGVPQSPYG 529 GAGGPGSPIG 530 GAGGPGSPWG 531 GAGGPGSPYG 532 GAGVPQSPLG 533 GAGLAISPGG 534 GAGQPPSPAG 535 GAGTSQSPRG 536 GAGSPRSPDG 537 GAGPLASPVG 538 GAGSVTSPSG 539 GAGADQSPTG 540 GAGQTPSPTG 541 GAGSPGSPLG 542 KRRFSFRLF 543 FRRFSFFLL 544 FRRFSFFIF 545 FRRFSIFLF 546 FRRFSFFLF PKA 547 FRMMSFFLF 548 FRRFSIFRLF 549 RRRADDSDDDDD CK2 550 RRRADDTDDDDD CK2 551 LETEEEGVPST 552 LETEEEGVPTT 553 EDNSEDEISNL 554 EDNTEDEISNL 555 EDNSEDEITNL Plk3 556 EDNTEDEITNL 557 EEGSPGEGDDD 558 EEGSWGEGDDD Plk3 559 EEGSKGEGDDD 560 EEGSRGEGDDD

15 561 EEGSFGEGDDD Plk3 562 EEGSLGEGDDD 563 EEGSVGEGDDD Plk3 564 EEGSIGEGDDD Plk3 565 EEGSEGEGDDD 566 EEGSDGEGDDD CK2 567 EEGSGGEGDDD 568 EEGSGPEGDDD CK2 569 EEGSGWEGDDD Plk3 570 EEGSGHEGDDD 571 EEGSGEEGDDD 572 RRRDDDSDDD CK2 573 RRRDDDTDDD 574 ADTESEDEED Plk3 575 ADTETEDEED 576 EGSGGEGDDD 577 EGSGGEGDDD 578 EESGGEGDDD 579 EDSGGEGDDD CK2 580 ERSGGEGDDD 581 EKSGGEGDDD 582 EWSGGEGDDD CK2 583 EHSGGEGDDD 584 EFSGGEGDDD 585 EPSGGEGDDD 586 ENSGGEGDDD 587 EQSGGEGDDD CK2 588 GADDSDGA 589 GADDTDGA 590 AQLSTSEENS 591 VPLSRTLS 592 ISLSSSEESI 593 GEQQQTEDEL 594 ISEDNSEDEI Plk3, CK2 595 SVEPPLSQETFSDL Plk3 596 TGPLSPGPFGS Cdk5, p LSKIGSTENL 598 ANAPVSALGE 599 IHATPSPPVD 600 DSGIHSGATTTAPSL 601 AAEELDSRAGSPQ 602 SVPPSPSLSG Cdk5 603 SVPPSPSLSG 604 SVPPTPSLSG Cdk5 605 SVPPSLSLSG 606 SVPTPSLSG 607 SVPSSSLSG 608 SVPSTSLSG 609 SVPSPSLSG 610 SVPSPSLSG 611 SVPSWSLSG 612 SVPSHSLSG 613 SVPSESLSG 614 SVPSDSLSG 615 SVPSRSLSG 616 SVPSFSLSG 617 SVWSPSLSG 618 SVESPSLSG 619 SVDSPSLSG Plk3 620 SVRSPSLSG 621 SVHSPSLSG 622 SVFSPSLSG 623 SVTSPSLSG 624 SVSSPSLSG 625 SVVSPSLSG 626 SVLSPSLSG 627 SVISPSLSG 628 GASRLLEGE 629 GASTLLEGE 630 GASSLLEGE 631 GASWLLEGE 632 GASFLLEGE Plk3 633 GASDLLEGE 634 GASDLLEGE 635 GASLLDEGE 636 GASLLEEGE 637 GASLLREGE 638 GASLLSEGE 639 GASLLTEGE 640 GASLLGEGE 641 GASLLQEGE 642 GASLLYEGE Plk3, EphB2 643 GASLDDEGE Plk3 644 GASLRREGE 645 GASLLLEGE 646 GASLLNEGE 647 APMSCDKSTQTPSP 648 CDKSTQTPSPPCQA 649 STQTPSPPCQAFNH 650 TEGRPPSPPPTSTP 651 RPPSPPPTSTPEKC 652 GRPPSPPPTSTPEK 653 HFWSSLSPVAPLSP 654 LSSIHFWSSLSPVA 655 SSLSPVAPLSPARL 656 WSSLSPVAPLSPAR 657 GAASPPAASPFLGL 658 GGAASPPAASPFLG 659 HIASPPNFRLTHDI 660 TGDTPGAEDDEEDD

16 661 DATGDTPGAEDDEE 662 RSSSPLKTGEQTPP 663 SWQDRVSRSSSPLK 664 SRSSSPLKTGEQTP 665 GEQTPPHEHICLSD 666 CLSSPERAEPPGGG 667 SGYFSFDTDRSPA 668 CDKSTQTPSPPCQ 669 LGGGAASPPAASP 670 ESGGLGGGAASPP 671 ASPFLGLHIASPP 672 IDATGDTPGAEDD 673 SSSPLKTGEQTPP 674 RLEATEEIYLTPV 675 IYLTPVQRPPDAA 676 EEIYLTPVQRPPD 677 GFDCLSSPERAEP 678 EEEEGFDCLSSPE 679 NCASVSSPYESAI 680 VELILSPRSK Cdk5, Cdk2 681 LGGLTSPGLS 682 FAPVASPAAP 683 GSYPLSPLSD 684 APAAPSPGSS 685 AKNIVTPRTP 686 GDLPLSPSAF 687 EILSRRPSYRK PKCz, PKCa, Chk2, 688 IRRLSTRRR PKCz, PKCa, Aurora B 689 IRRLSTRRR PKCz, PKCa, Aurora B 690 AKRRRLSSLRA Akt, SGK, Aurora A/B 691 VRKRTLRRL 692 PLSRTLSVAA 693 PLSRTLSVAA Akt, PKCz, Chk2, CaMK2 694 IAKRRRLSSLRAS 695 PLGPLAGSPVIAA 696 HHKLVLPSNTPNV 697 SGISSVPTPSPLG 698 KAYGNGYSSNGNT 699 GLAQFQLSQEVFA 700 GLAQFQLSQEVFA 701 GFAQFQLSQEVFA 702 GLAFFQLSQEVFA 703 GLAQFFLSQEVFA 704 GLAQFQLSQQVFA 705 ASELPASQPQPFSA 706 DLLMYLSQLVQALK 707 KVSPYLSQFQQDKQ 708 HSTPPSAYGSVKA 709 NMTPYRSPPPYVPP Cdk5 710 GAGAGYIYGSFK 711 YLYGSFK PKCz, Src, Kit 712 YKYGSFK 713 YHYGSFK PKCz, Kit 714 YFYGSFK PKCz 715 YPYGSFK 716 GGIYGSFKKK PKCz, Lck 717 QEENRRDSWSYINSK Aurora A/B, Jak2 718 ARKRERTYTFG SGK, Chk2, Cdk5, Kit 719 LARRRYRSFSLA Plk3, Akt, SGK, PKCz, PKA, Aurora B, CaMK2, Chk1, Cdk5, Kit 720 LVRYASIALEAKK Chk2, Kit 721 LVRYASIALTAKK 722 LALRRYFSQAEF Chk2, CaMK2, MK2, Chk1, Kit 723 LAAARYFSQAEYAKK EphB2 724 LVKYASIALEAKK 725 RMLARAESFFYA Plk3, SGK, PKCz, Chk2, CaMK2, MK2, Chk1, EphB2 726 RFLARYYSQAQQ Chk2, MK2 727 RFFARYYSQAQQ 728 RLKRYESIFIK PKCz, Chk2, 729 RIKRYESIFIK PKCz, Chk2, Kit, Flt3 730 RAKRYESIFIK Kit, Flt3 731 RAKRYESIFIR PKCz, Chk2, EphB2, Kit, Flt3 732 YEYYAASIFIFFAKKK 733 YYDAASIFGYFAKKK CK1, VEGFR2, Kit, Flt3, Jak2 734 YYDAASIFIFFAKKK CK1, Plk3, VEGFR2, EphB2, Kit, Flt3 735 YEDAASIFIFFAKKK CK1, Kit 736 YYDDASIFIFFAKKK 737 YYDAASIYIFFAKKK 738 YYDAASIIFFFAKKK CK1, VEGFR2, Kit, Flt3, Jak2 739 YIYGSFK Lck, Src 740 GIYGSFK Lck, Src 741 FIYGSFK Lck, Src 742 RIYGSFK Lck, Src 743 YIGGSFK 744 YIFGSFK 745 YIKGSFK 746 YIRGSFK 747 YIHGSFK 748 YGYGSFK 749 YVYGSFK 750 SRKVGPGYLGSGG 751 FPTSTSLSPFYLR 752 PPTNGNGYEEDMGYR

17 753 GPPEPGPYAQPSVNT 754 GPPEPGPYAQPSVNT 755 VDSEGHLYTVPIREQ Kit 756 PVYNQPVYNQPVGAA 757 SSGGSGNYGTIGRSS EphB2 758 TSSLPEGYYEEAVPV Plk3, EphB2 759 PERNEGVYTAIAVQE 760 PLQNGGMYMVGHSHL 761 ASANSVVYASQQQML 762 QKGQESEYGNITYPP Plk3, EphB2 763 TTANVVYYVGENVVN 764 QQQAENPYGQVPMPP 765 GEAIYAAPFAKKK Abl, EphB2, Kit, Flt3 766 GEAIY AAPFA Abl 767 RRLIEDAIYAARG 768 ADPSNIYASPWFW Abl, EphB2, Kit, Flt3 769 GGGIYAAPKKS 770 PPTNGNGYEEDMGYR EphB2 771 PLQNGGMYMVGHSHL 772 GPPEPGPYAQPSVNT Kit 773 GPPEPGPYAQPSVNT 774 PVYNQPVYNQPVGAA 775 SSGGSGNYGTIGRSS EphB2 776 QQQAENPYGQVPMPP Src,VEGFR, Kit, Flt3, EphB2 777 AEVIYAAPF Abl, Lck, Src, VEGFR2, Kit, Flt3, Jak2, EphB2 778 RRLIEDAIYAARG Lck, Kit, EphB2 779 GGVIYAAPF Abl, Kit, Flt3, EphB2 780 GPVIYAAPF Abl, Kit, Flt3, EphB2 781 GHVIYAAPF Abl, Kit, EphB2 782 GRVIYAAPF Abl, Kit, Flt3, EphB2 783 GDVIYAAPF Abl, Lck, Src, EphB2, VEGFR, Kit, Flt3, Jak2, EphB2 784 GEVIYAAPF Abl, Lck, VEGFR, Kit, Flt3, EphB2 785 GYVIYAAPF 786 GIYGGPG 787 GVYGGPG 788 GLYGGPG 789 GIYGGFG Src 790 GVYGGFG 791 AEVIYAAPF Abl, Lck, Src, VEGFR, EphB2, Kit, Flt3, Jak2, 792 AEVVYAAPF Abl, Kit, Flt3, EphB2 793 AEVLYAAPF Abl, Kit, Flt3, Jak2, EphB2 794 LEPGPYAQPS 795 FTGDTYTAHA 796 GADGVYAASG 797 AADGIYAASG Src, EphB2 798 FKQPIYIVME VEGFR, EphB2, Kit, Flt3, Jak2 799 GEGIYGVLF Lck, Src, EphB2, Kit 800 GEGVYGVLF 801 GEGLYGVLF Lck, Src, EphB2 802 GEGFYGVLF Lck, Src, VEGFR, EphB2, Kit, Flt3, Jak2 803 GEGIYAVLF 804 GEGIYGVVF Lck, Src, EphB2 805 GEGIYGVFF 806 GEGIYGVIF 807 GEGIYGVLL Lck, Src, EphB2 808 GEGIYGVLV 809 GEGIYGVLI 810 GEGIYGVLF Lck, Src, EphB2 811 GEGIYGVLF 812 GEGIYGVLF 813 GEGVYGVVF Lck, Src, EphB2 814 GEGVYGVFF Lck, Src, EphB2 815 GEGVYGVIF 816 GEGVYGVLL Lck, Src, EphB2 817 GEGVYGVLV 818 GEGVYGVLI Lck, Src, EphB2 819 GEGLYGVVF EphB2 820 GEGLYGVFF 821 GEGLYGVIF Lck, Src, EphB2 822 GEGLYGVLL Lck, Src, EphB2 823 GEGLYGVLV 824 GEGLYGVLI EphB2 825 GERIYGVLF Lck, Src, EphB2 826 GEKIYGVLF 827 GEEIYGVLF Lck, Src 828 GEDIYGVLF 829 GEHIYGVLF 830 GEWIYGVLF 831 GENIYGVLF 832 GEQIYGVLF 833 GEL IYGVLF Lck, Src 834 GEVIYGVLF 835 EEGIYGVLF Lck, Src, EphB2 836 DEGIYGVLF 837 REGIYGVLF Lck, Src, EphB2 838 KEGIYGVLF Lck, Src, EphB2 839 HEGIYGVLF Lck, Src, EphB2 840 GEPIYGVLF 841 PEGIYGVLF Lck, Src, EphB2 842 WEGIYGVLF Lck, Src, EphB2 843 NEGIYGVLF 844 QEGIYGVLF 845 LEGIYGVLF Lck, Src, EphB2

18 846 AEVIYGVFF Lck, Src, Flt3 847 EGIYNELQ 848 VMAEAYSEIG 849 IQEGLYNELQ 850 DEEIYEELG Lck, Src, EphB2, Kit, Flt3, EGFR 851 DEEIYGELE Lck, Src, EphB2,Kit 852 DEEIYGELG Abl, Lck, Src, EphB2, Kit, Flt3 853 DEEIYEELG Src, EphB2, Kit 854 EEEIYGEFE Lck, Src, EphB2, Kit, Flt3 855 VYIYGSFK Kit 856 VTIEDDDYSPPSKRP 857 LFMADDAYSHHSGVD 858 GQRDSSYYWEIEASE EphB2, Kit 859 NSFNNPAYYVLEGVP Kit, Flt3, Jak2 860 YIYGSFK 861 EEEIYGEFD 862 EEEVYGEFD Lck, Src, EphB2 863 EEELYGEFD Lck, Src, EphB2, Kit 864 EEEIYEEFD Lck, Src, EphB2, Kit, Flt3 865 EEEIYDEFD Lck, Src, EphB2 866 EEEIYGEVD Lck, Src, EphB2, Kit, Flt3 867 DDGVYEID 868 DDGVYELD Lck, Src, EphB2 869 DDGVYELE Lck, Src, EphB2 870 EEGVYELE 871 APASAYGSVK 872 GGIYGQFKKK Lck, Src 873 GGIYGNFKKK 874 GGIYGGFKKK 875 GGIYGFFKKK 876 GGIYGLFKKK 877 GGIYGYFKKK Lck, Src, Kit 878 GGIYGIFKKK 879 GGIYGHFKKK 880 GGIYGRFKKK 881 GAGGIYWHHY 882 KKKKYKKKKYKKKK 883 EEYPYGEVD Src, Kit 884 EEEYPYELE 885 GEDNEYTARQ EphB2, Kit 886 APETVYEVAG EphB2 887 VREAEYEPET 888 ISSPVYQDAV 889 LSTGIYEALE EphB2, Kit 890 FEETPYSYPT EphB2, Kit 891 APPSAYGSVK 892 VPPSAYGSVK 893 LEEEAYGWMD Kit 894 FSNPTYSVMR Kit 895 AADDEYAPKQ 896 VTEDQYSLVE Plk3, Kit 897 IRENEYMPMA EphB2, Kit, Jak2 898 FLEKKYVRRD Kit 899 GLEKKYVRRD Kit 900 AGDSSYKNIH Plk3, Kit 901 LEDNEYTARE 902 GEPIYMFFFAKKK Abl, Lck, Src, EphB2, VEGFR, Kit, Flt3, Jak2 903 GEPIYMFFFAKKK Lck, Src, EphB2, VEGFR, Kit, Flt3, Jak2 904 GEVIYIFFFAKKK 905 GEPIYIFLFAKKK EphB2, VEGFR, Kit, Flt3, Jak2 906 GEPIYIFFF 907 MEEIYGIFF 908 LEEVYGIFF Lck, Src, EphB2, Kit, Flt3 909 LEEIYEIFF 910 LEEIYGIFE Lck, Src, EphB2 911 LEEIYGIFF 912 MENVYGIFF 913 MEEIYEIFF Lck, Src, EphB2, Kit, Flt3 914 MEEVYGIFF Lck, Src, EphB2, Kit, Flt3 915 MENIYGIFF 916 MEEIYGIFF 917 FEEHVYSFPN Kit 918 YDYVHL EphB2, VEGFR, Kit, Flt3, Jak2 919 AEEIYEEIE EphB2 920 AEEIYEEIE EphB2, Kit 921 EEEIYEEIE Lck, Src, EphB2, Kit, Flt3 922 EEEIYEEIE Lck, Src, EphB2, Kit, Flt3 923 EEEEYEEIV EphB2 924 DEEEYEEIV EphB2, Kit 925 EEEEYEVEV 926 EEEEYEVEE 927 EEEDYEEIV EphB2, Kit 928 EEEEYEEIV EphB2, Kit 929 EEEEYFELV Jak2 930 DEEEYFELV 931 REEEYFELV Kit, Jak2 932 AEEEYFELV VEGFR2, Kit, Flt3, Jak2, EphB2 933 EDEEYFELV 934 EEDEYFELV 935 EDDEYFELV VEGFR2, Kit, Flt3, EGFR, Jak2, EphB2

19 936 EEEDYFELV 937 EEEIYFELV Src, VEGFR2, Flt3, Jak2, EphB2 938 EEEEYVELV 939 EEEEYIELV EphB2 940 EEEEYFFLV 941 EEEEYFDLV 942 EEEEYFELV Flt3, Jak2 943 EEEEYFEIV Flt3, Jak2, EphB2 944 EEEEYFEFV 945 EEEEYFEVV 946 DDDDYFELV Jak2, EphB2 947 DDDDYFDLV 948 EEEEYFFFV 949 EEEEYFELV VEGFR2, Kit, Flt3, EGFR, Jak2, EphB2 950 EEEEYFELV VEGFR2, Flt3, Jak2 951 EEEEYFELV VEGFR2, Kit, Flt3, EGFR,Jak2, EphB2 952 EEEEYVFIV VEGFR2, Kit, Flt3, Jak2, EphB2 953 EEEEYIDFV 954 EEEEYLELV Jak2 955 EEEEYRELV Kit 956 EEEEYKELV Kit 957 EEEEYNELV 958 EEEEYVELV EphB2 959 EEEEYSELV EphB2 960 EEEEYTELV Kit, Flt3, Jak2, EphB2 961 EEEEYYELV VEGFR2, Kit, Flt3, Jak2, EphB2 962 EEEQYFELV 963 EEENYFELV 964 EEEPYFELV Kit, Flt3, Jak2 965 ANPGFYVEAN EphB2, Kit 966 VVETTYADFI Plk3, Src, EphB2, Kit, Flt3 967 IHKSGYLSSE 968 LFEARYQQPF Kit 969 GEGSAYEEVP Plk3, EphB2, Kit 970 AAEPDYGALY EphB2 971 VPVPEYINQS Kit, Jak2 972 IENAEYLRVA EphB2, VEGFR, Kit, Flt3, Jak2 973 LEEQEYVQTV 974 FDNPDYQQDF 975 GRLQDYEEKT 976 AEEQEYIKTV 977 VQNPVYHNQP 978 IVDEMYREAP 979 GSVQNPVYHNQPL 980 LLLSNPAYRLLLA 981 AEEEYFFIF 982 AEEEYFFIF Lck, Src, Kit, Flt3 983 AEEEYVFLF EphB2, VEGFR, Kit, Flt3, EGFR, Jak2 984 AAEEYFFLF EphB2, VEGFR, Kit, Flt3, Jak2 985 AEEEYFFIF 986 AEEEYFFLF 987 YVQLPATYSNLGP Jak2 988 KKKSPGEYVNIEFG EphB2, VEGFR, Kit, Flt3, Jak2 989 KKSRGDYMTMQIG Kit, Jak2 990 KKSRGDYMTMQIG 991 AEEEYFFFF 992 AEENYFFEF EphB2, VEGFR, Kit, Flt3, Jak2 993 AEEEYFFFF 994 AEEEYMMMM EphB2, VEGFR, Kit, Flt3, Jak2 995 EEEEYVFIE EphB2, VEGFR, Kit, Flt3, EGFR,Jak2 996 EEEEYVFFA EphB2, VEGFR, Kit, Flt3, EGFR,Jak2 997 EEEEYVFIA EphB2, VEGFR, Kit, Flt3, EGFR,Jak2 998 EEEEYVFVA EphB2, VEGFR, Kit, Flt3, EGFR,Jak2 999 EEEEYEFIE EphB2, Kit 1000 EEEEYVFIE 1001 EEEEYVFIG EphB2, VEGFR, Kit, Flt3, EGFR,Jak2 [a] Peptide sequences were derived from several sources: known kinase phosphorylation consensus sequences, peptide library results, and a phosphorylation site database (see references below). We attempted to target 50 kinases from these sources, their location is indicated on the kinome tree below. Because the pool evolved along with the assay, there are a larger portion of substrates that respond to the AGC and Src family kinases. As profiling emerged as the most powerful application, and relative phosphorylation efficiencies of substrates could not be determined, we quickly expanded the pool to include fewer substrates for as many kinases as possible. [b] Phosphorylation was positively identified if the signal was greater than three times the noise in codes above the background signal.

20 Table S1 references: [1] Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., Cantley, L. C. Determination of the specific substrate sequence motifs of protein kinase C isozymes. J. Biol. Chem. 1997, 272, [2] Songyang, Z. et al. Use of an oriented peptide library to determine optimal substrates of protein kinases. Curr. Biol. 1994, 4, [3] Songyang, Z. et al. Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature 1995, 373, [4] Songyang, Z. et al. A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1. Mol. Cell. Biol. 1996, 16, [5] O'Neill, T. et al. Determination of substrate motifs for human Chk1 and hcds1/chk2 by the oriented peptide library approach. J. Biol. Chem. 2002, 277, [6] Pinna, L. A., Ruzzene, M. How do protein kinases recognize their substrates? Biochim. Biophys. Acta 1996, 1314, [7] Diella, F. et al. Phospho.ELM: a database of experimentally verified phosphorylation sites in eukaryotic proteins. BMC Bioinformatics 2004, 5, 79.