[Inhibition of EGF and TGF-beta dependent transformation of NRK23 cells by Crk II-23 mutant].
Adaptor proteins participate in many signaling pathways from cell surface receptors. Crk protein was the first example of the adaptor protein. We have examined the function of Crk II mutant, Crk II-23. The Crk II-23 mutant contains two amino-acid substitutions in the carboxyl-terminal SH3 domain and is known to inhibit the transformation of NRK cells induced by epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta). There was no remarkable difference between Crk II and Crk II-23 in EGF-dependent binding to EGF receptor (EGFR). However, in contrast to the wild-type Crk II, the Crk II-23 mutant bound to EGFR in quiescent NIH 3T3 cells. To clarify the difference, both the Crk II and Crk II-23, proteins were expressed in E. coli and examined their binding capacity in vitro. They bound to EGFR from EGF-stimulated NIH 3T3 cells in vitro to a similar extent. Expression of Crk II-23 in NIH 3T3 cells did not affect the binding of bacterially expressed Crk II and Crk II-23 to EGFR. These results suggest that post-translational modification of Crk II-23, such as physical association to cellular proteins, induces binding of Crk II-23 to EGFR in quiescent cells. We also demonstrated that mutation of either the SH2 or the SH3 domain abolished the anti-oncogenic activity of Crk II-23, although both mutants bound to EGFR in the quiescent cells. From these results, it could be concluded that persistent signaling through Crk II-23 bound to EGFR is responsible for the suppression of transformation by EGF and TGF-beta.
Ota
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Second Department of Pathology, Hokkaido University School of Medicine, Sapporo, Japan.
D000818:Animals; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors*; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D050737:Proto-Oncogene Proteins c-crk; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors*
D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D016212:Transforming Growth Factor beta; D004815:Epidermal Growth Factor; D066246:ErbB Receptors