Transactivation of Abl by the Crk II adapter protein requires a PNAY sequence in the Crk C-terminal SH3 domain.
To gain a better understanding of how Crk II regulates the function of the Abl tyrosine kinase, we explored the function of the C-terminal linker and SH3 domain, a region of Crk II that is still poorly understood. Molecular modeling, tryptophan fluorescence, and covariation sequence alignment indicate that the Crk-SH3-C has a unique binding groove and RT loop not observed in typical SH3 domains. Based on these models, we made a series of mutations in the linker and in residues predicted to destabilize the putative binding pocket and RT loop. In Abl transactivation assays, Y222F and P225A mutations in the linker resulted in strong transactivation of Abl by Crk II. However, mutations predicted to be at the surface of the Crk SH3-C were not activators of Abl. Interestingly, combinations of activating mutations of Crk II with mutations in the highly conserved PNAY sequence in the SH3-C inactivated the activating mutations, suggesting that the SH3-C is necessary for activation. Our data provide insight into the role of highly conserved residues in the Crk-SH3-C, suggesting a mechanism for how the linker and the Crk-SH3-C function in the transactivation of the Abl tyrosine kinase.
Reichman
Charles
C
Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA.
Singh
Kamalendra
K
Liu
Yan
Y
Singh
Sukhwinder
S
Li
Hong
H
Fajardo
J Eduardo
JE
Fiser
Andras
A
Birge
Raymond B
RB
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation; D014982:Xenopus laevis; D018909:src Homology Domains
D050737:Proto-Oncogene Proteins c-crk; D016315:Proto-Oncogene Proteins c-abl