CRKI (SH2-SH3) and CRKII (SH2-SH3-SH3) are splicing isoforms of the oncoprotein CRK that regulate transcription and cytoskeletal reorganization for cell growth and motility by linking tyrosine kinases to small G proteins. CRKI shows substantial transforming activity, whereas the activity of CRKII is low, and phosphorylated CRKII has no biological activity whatsoever. The molecular mechanisms underlying the distinct biological activities of the CRK proteins remain elusive. We determined the solution structures of CRKI, CRKII and phosphorylated CRKII by NMR and identified the molecular mechanism that gives rise to their activities. Results from mutational analysis using rodent 3Y1 fibroblasts were consistent with those from the structural studies. Together, these data suggest that the linker region modulates the binding of CRKII to its targets, thus regulating cell growth and motility.
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We thank M. Tsuda (Hokkaido University) for preparation of time-lapse images, and H. Hanafusa and T. Iwahara (Osaka Bioscience Institute) for valuable suggestions on this work. D. Baltimore (California Institute of Technology) developed BOSC23 packing cells used in the protein-expression system. This work was supported by Grants-in-Aid for Scientific Research and the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science and Culture of Japan. Y.K. is a research fellow of the Japan Society for the Promotion of Science.
The authors declare no competing financial interests.
Chemical shift difference between full-length pCRKII, pCRKII1–228 and isolated cSH3210–304. (PDF 190 kb)
Overlay of the 1H-15N HSQC spectra of wild-type CRKII and ISC mutants. (PDF 1635 kb)
Steady state NOE of CRKII in free and bound state of SOS-derived VPP peptide. (PDF 112 kb)
Overlay of the 1H-15N HSQC spectra of wild-type CRKII and CRKII(W275K). (PDF 867 kb)
Time–lapse microscopical analysis of the wound-healing assay of CRKI-expressing cells. (MOV 2889 kb)
Time–lapse microscopical analysis of the wound-healing assay of CRKII-expressing cells. (MOV 3201 kb)
Time–lapse microscopical analysis of the wound-healing assay of CRKII(Δ226–237)-expressing cells. (MOV 2997 kb)
Time-lapse microscopic analysis of the wound-healing assay of CRKII(m226–237)-expressing cells. (MOV 2417 kb)
Time-lapse microscopic analysis of the wound-healing assay of mock cells. (MOV 2457 kb)
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Kobashigawa, Y., Sakai, M., Naito, M. et al. Structural basis for the transforming activity of human cancer-related signaling adaptor protein CRK. Nat Struct Mol Biol 14, 503–510 (2007) doi:10.1038/nsmb1241
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