Function of activation loop tyrosine phosphorylation in the mechanism of c-Kit auto-activation and its implication in sunitinib resistance.
The activation of receptor tyrosine kinases (RTKs) is tightly regulated through a variety of mechanisms. Kinetic studies show that activation of c-Kit RTK occurs through an inter-molecular autophosphorylation. Phosphopeptide mapping of c-Kit reveals that 14-22 phosphates are added to each mol of wild-type (WT) c-Kit during the activation. Phosphorylation sites are found on the JM, kinase insert (KID), c-terminal domains and the activation loop (A-loop), but only the sites on the JM domain contribute to the kinase activation. The A-loop tyrosine (Y(823)) is not phosphorylated until very late in the activation (>90% completion), indicating that the A-loop phosphorylation is not required for c-Kit activation. A sunitinib-resistant mutant D816H that accelerates auto-activation by 184-fold shows no phosphorylation on the A-loop tyrosine after full activation. A loss-of-phosphorylation mutation Y823F remains fully competent in auto-activation. Similar to WT and D816H, the unactivated Y823F mutant binds sunitinib and imatinib with high affinity (K(D) = 5.9 nM). But unlike the WT and D816H where the activated enzymes lose the ability to bind the two drugs, activated Y823F binds the two inhibitors effectively. These observations suggest that the A-loop of activated Y823F remains flexible and can readily adopt unactivated conformations to accommodate DFG-out binders.
Pfizer Research Technology Center, 620 Memorial Drive, Cambridge, MA 02139, USA. jdinitto@msn.com
Jonathan P
JP
DiNitto
Gayatri D
GD
Deshmukh
Yan
Y
Zhang
Suzanne L
SL
Jacques
Rocco
R
Coli
Joseph W
JW
Worrall
Wade
W
Diehl
Jessie M
JM
English
Joe C
JC
Wu
D019943:Amino Acid Substitution; D000970:Antineoplastic Agents / Q000378:metabolism*; D001549:Benzamides; D020134:Catalytic Domain; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D000068877:Imatinib Mesylate; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D008832:Microchemistry / Q000379:methods; D008954:Models, Biological; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000502:physiology*; D010879:Piperazines / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D019009:Proto-Oncogene Proteins c-kit / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism; D011758:Pyrroles / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000077210:Sunitinib
D000970:Antineoplastic Agents; D001549:Benzamides; D004791:Enzyme Inhibitors; D007211:Indoles; D050505:Mutant Proteins; D010879:Piperazines; D011743:Pyrimidines; D011758:Pyrroles; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D000068877:Imatinib Mesylate; D019009:Proto-Oncogene Proteins c-kit; D000077210:Sunitinib