title: Adaptor proteins CRK and CRKL associate with the serine/threonine protein kinase GCKR promoting GCKR and SAPK activation.
abstract: 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.
authors.affiliation: B Cell Molecular Immunology Section, Laboratory of Immunoregulation, NIAID, National Institutes of Health, Bethesda, MD 20892-1876, USA.
authors.forename: C S
authors.initials: CS
authors.lastname: Shi
authors.forename: J
authors.initials: J
authors.lastname: Tuscano
authors.forename: J H
authors.initials: JH
authors.lastname: Kehrl
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D002460:Cell Line; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D011905:Genes, ras; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D020014:K562 Cells; D046911:Macromolecular Substances; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D050737:Proto-Oncogene Proteins c-crk; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011993:Recombinant Fusion Proteins; C404144:MAP4K5 protein, human; D017346:Protein Serine-Threonine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)