CONSTITUTIVE ASSOCIATION OF EGF RECEPTOR WITH THE CRKII-23 MUTANT THAT INHIBITS TRANSFORMATION OF NRK CELLS BY EGF AND TGF-BETA

Crk belongs to the adapter proteins that participate in many signallin g pathways from cell surface receptors. We have characterised the CrkI I-23 mutant that inhibits the transformation of NRK cells induced by e pidermal growth factor (EGF) and transforming growth factor (TGF)-beta . To study the biochemical difference, cDNAs of the wild-type CrkII an d the CrkII-23 mutant were introduced stably into NIH 3T3 cells expres sing EGF receptor (EGFR). Both CrkII and CrkII-23 were phosphorylated on tyrosine upon EGF simulation with similar time course and dose depe ndency. Whereas the wild-type CrkII bound to EGFR only after EGF stimu lation, CrkII-23 bound to EGFR from before stimulation. Mutation in th e Src homology (SH) 2 or amino terminal SH3 domain did not abolish the binding of CrkII-23 to EGFR in the quiescent cells, suggesting that t he binding is mediated by a novel mechanism. These CrkII-23-derived mu tants, however, did not suppress transformation of NRK cells by EGF an d TGF-beta. Hence, both the SH2 and amino-terminal SH3 domains are req uired to inhibit transformation of NRK cells. These results suggest th at persistent signalling from CrkII-23 bound to EGFR suppresses transf ormation by EGF and TGF-beta in NRK23 cells. (C) 1998 Elsevier Science Inc.