Dok-1 is a common substrate of many protein tyrosine kinases (PTKs). It recruits rasGAP and other SH2-containing proteins and negatively regulates Ras-Erk signalling downstream of PTKs. However, the mechanisms of its inhibitory effect are yet unclear. Here, a series of C-terminal deletion mutants of Dok-1 delineated the core domain for the inhibition of Erk from 334 to 346 amino acid, which contains two SH2-binding motifs having Tyr-336 or Tyr-340. The Dok-1 mutants having tyrosine-to-phenylalanine (YF) substitution(s) at Tyr-336 and/or Tyr-340 lost their inhibitory effect on Ras and Erk downstream of Src-like PTK, Lyn or Fyn, whereas the rasGAP-binding of each mutant remained intact. However, the Dok-1 mutant having YF substitutions at the rasGAP-binding sites (Tyr-295 and Tyr-361) also showed incapability of Ras and Erk inhibition. Moreover, the Dok-1 mutant having YF substitutions at Tyr-336 and Tyr-340 showed an impaired inhibitory effect on v-Abl-induced transformation of NIH-3T3 cells. These results demonstrate that Tyr-336 and Tyr-340 of Dok-1 are dispensable for rasGAP-binding but essential for inhibition of Ras-Erk signalling and cellular transformation downstream of PTKs. Thus, Dok-1 probably recruits as yet unidentified molecule(s), which, in concert with rasGAP, negatively regulate Ras-Erk signalling.
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