Adaptor proteins CRK and CRKL associate with the serine/threonine protein kinase GCKR promoting GCKR and SAPK activation

STE20-related kinases play significant regulatory roles in a range of cellu lar responses to environmental stimuli. GCKR (also referred to as KHS1) is a serine/threonine protein kinase that has an STE20-like protein kinase dom ain and that stimulates the stress-activated protein kinase (SAPK, also ref erred to as Jun kinase or JNK) pathway. GCKR has a large C-terminal regulat ory domain that provides sites for interactions with other proteins. Adapto r proteins mediate the Interactions between signaling molecules. In this st udy we showed that the adaptor proteins Crk and CrkL associated with GCKR. When Crk-I, Crk-II, or CrkL was transiently expressed In HEK 293T cells alo ng with GCKR, each coimmunoprecipitated with GCKR. Furthermore, in the Bcr- Abl transformed cell line, K562 endogenous GCKR and CrkL coimmunoprecipitat ed, indicating a constitutive association, Detection of the CrkL-GCKR Inter action required the SH3 domains of CrkL and 2 regions in GCKR - 1 between a mino acids 387 and 395 that contains a consensus SH3 binding motif and the other between amino acids 599 and 696, Crk or CrkL overexpression increased GCKR catalytic activity, A dominant negative form of Res abolished Crk- or CrkL-induced GCKR activation, suggesting a dependence on Res activation fo r their activation of GCKR, Finally, we showed Impairment of the known abil ity of CrkL to activate the SAPK pathway by a catalytically Inactive form o f GCKR or by a GCKR antisense construct. Thus, GCKR associates with other p roteins through interactions mediated by SH2/SH3 adaptor proteins, which ca n lead to GCKR and SAPK activation. (C) 2000 by The American Society of Hematology.