Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 776-782
Adaptor proteins CRK and CRKL associate with the serine/threonine
protein kinase GCKR promoting GCKR and SAPK activation
Chong-Shan Shi,
Joseph Tuscano, and
John H. Kehrl
From the B Cell Molecular Immunology Section, Laboratory of
Immunoregulation, NIAID, National Institutes of Health, Bethesda, MD,
and the Department of Medicine, UC Davis Cancer Center, University of
California-Davis, Sacramento, CA.
STE20-related kinases play significant regulatory roles in a range
of cellular responses to environmental stimuli. GCKR (also referred to
as KHS1) is a serine/threonine protein kinase that has an STE20-like
protein kinase domain and that stimulates the stress-activated protein
kinase (SAPK, also referred to as Jun kinase or JNK) pathway. GCKR has
a large C-terminal regulatory domain that provides sites for
interactions with other proteins. Adaptor proteins mediate the
interactions between signaling molecules. In this study 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 along with
GCKR, each coimmunoprecipitated with GCKR. Furthermore, in the Bcr-Abl
transformed cell line, K562 endogenous GCKR and CrkL
coimmunoprecipitated, indicating a constitutive association. Detection
of the CrkL-GCKR interaction required the SH3 domains of CrkL and 2 regions in GCKR
1 between amino 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 Ras abolished Crk- or CrkL-induced GCKR
activation, suggesting a dependence on Ras activation for their
activation of GCKR. Finally, we showed impairment of the known ability
of CrkL to activate the SAPK pathway by a catalytically inactive form
of GCKR or by a GCKR antisense construct. Thus, GCKR associates with
other proteins through interactions mediated by SH2/SH3 adaptor
proteins, which can lead to GCKR and SAPK activation.
