IRAK4 Kinase Activation and Cytokine Induction SUPPLEMENTAL DATA

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Ernst Bjerke
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1 SUPPLEMENTAL DATA Interleukin 1/Toll-Like Receptor Induced Autophosphorylation Activates Interleukin 1 Receptor- Associated 4 and Controls Cytokine Induction in a Cell-Type Specific Manner Leah Cushing, Wayne Stochaj, Marshall Siegel, Robert Czerwinski, Ken Dower, Quentin Wright, Margaret Hirschfield, Jean-Laurent Casanova, Capucine Picard, Anne Puel, Lih-Ling Lin, Vikram R. Rao 1

2 SUPPLEMENTAL FIGURE 1 (S1): Ion chromatograms of Peak B trypsin digests. Method: Purified and autophosphorylated IRAK 4 kinase domain proteins were each thermally denatured and then digested overnight at 37 C with sequencing grade trypsin (Promega). The peptides where then analyzed by HPLC ESI-MS/MS. The reversed phase HPLC utilized a C18 Waters Symmetry 300 column (1 x 150 mm, 3μ particle size) with an Agilent 1100 HPLC system with a flow rate of 65 μl/min and solvent gradient initially 2% A and 98% B to 10% A and 90% B over a period of 90 minutes where A is water with 0.1% formic acid and B is acetonitrile with 0.1% formic acid. The ESI-MS/MS data were acquired using a Waters-Micromass q-tof Micro mass spectrometer. The doubly charged parent ions of the trypsin fragments were analyzed with the cone voltage set to 16 V, the argon pressure in the collision cell set to 3.5 x 10-5 mm and the collision energy set to 26 V. SUPPLEMENTAL FIGURE 2 (S2): Chromatograms of phosphopeptides. Stoichiometry and assignment of di- and mono- phosphorylated T3, T19, and T20 peptides. SUPPLEMENTARY TABLE 1 (ST1): Kinome occupancy of primary human monocytes incubated with 10 μm of Compound1. Percentage occupancy of indicated kinase by Compound 1 is determined by KiNativ assay by ActivX. 2

3 Figure S1 3

4 Figure S2 4

5 Table ST1 % Occupancy IRAK PIP4K2C 75.9 AurB 71 AurA 70.6 IRAK ARAF 53.5 MPSK SGK 48 JAK1 domain PAK STLK PLK TNK PI4KB 31.5 CDC2 31 DGKA 30.6 NEK CASK 27.8 AMPKa1,AMPKa CaMKK OSR BRAF 26 IRAK MLKL 25.3 PAN3 25 ILK 24.4 PIK3C SGK Erk p70s6k 23.7 MLK RSKL KHS PEK 22.4 CDK7 22 5

6 % Occupancy PCTAIRE2,PCTAIRE PHKg SNRK 19.3 Wnk1,Wnk2,Wnk LATS ULK p38d,p38g 17.5 ATR 17.1 PYK HPK FER 15.7 BTK 15.5 p38a 15.2 MAPKAPK MAP3K GCK 13.9 MSK1 domain1 13 PIP5K CaMK1a 12.8 NDR NEK CDK LATS ABL,ARG 11 LOK 10.6 LKB ZAP Wnk1,Wnk2 10 MARK3,MARK4 9.8 PKR 9.4 p70s6kb 9.3 TAK1 9.3 CDK11,CDK8 9.2 RSK1 domain1 9.2 MAP3K2,MAP3K

7 % Occupancy PIP4K2A 8.4 NDR1 7.9 CHK2 7.8 SLK 7.8 JNK1,JNK2,JNK3 7.7 MASTL 6.7 ULK1 5.4 IKKe 5.3 FRAP 5.2 RSK1 domain2 4.7 TBK1 3.5 MLK4 3.4 RSK1,RSK2,RSK3 domain1 2.8 MAP2K1 2 MAP3K6 1.7 MARK3 1.7 KSR1,KSR2 0.8 MAP3K5 0.8 YANK3 0.6 FES 0.5 NEK1 0.2 KSR1 0 NEK8-1.1 NuaK2-1.1 NEK3-1.2 RIPK3-1.2 CSK -1.6 MAP2K6-1.6 PRP4-2.9 PIP4K2B -3.1 PFTAIRE1-4 MRCKb -4.3 ACK -6.5 ZAK -6.9 MAP3K

8 % Occupancy FYN,SRC,YES -7.9 LRRK2-8.9 MAP2K3-8.9 CK1g2-9.6 MAST SMG MAP3K MAP2K CDK CaMK2d STLK RSK2 domain PKD CDK5-15 IRE CaMK1d PI4KA,PI4KAP TAO Erk2-17 TLK MST PIK3CD SYK MST CaMK2g MARK2,MARK NEK DNAPK BARK MST4,YSK1-22 COT GPRK6-23 MAP2K MSK2 domain ZC1/HGK,ZC2/ TNIK,ZC3/MINK

9 % Occupancy NEK6,NEK p38a PKN1-27 TLK Erk HCK LYN GSK3A -30 eef2k RSK2 domain PKD GSK3B MST PIK3CB MAP3K TYK2 domain IKKb MAP2K1,MAP2K PRPK IKKa FGR PITSLRE MAP2K TAO1,TAO DGKQ JAK2 domain PIK3CG -59 JAK1 domain DNAPK ITPK GCN

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