Dok-3 sequesters Grb2 and inhibits the Ras-Erk pathway downstream of protein-tyrosine kinases

Adaptor proteins are essential in coordinating recruitment and, in a few cases, restraint of various effectors during cellular signaling. Dok-1, Dok-2 and Dok-3 comprise a closely related family of adaptor, which negatively regulates mitogen-activated protein kinase Erk downstream of protein-tyrosine kinases (PTKs). Recruitment of p120 rasGAP, a potent inhibitor of Ras, by Dok-1 and Dok-2 appears critical in the negative regulation of the Ras-Erk pathway. However, as Dok-3 does not bind rasGAP, it has been unclear how Dok-3 inhibits Erk downstream of PTKs. Here, we identified Grb2 as a Dok-3-binding protein upon its tyrosine phosphorylation. This interaction required the intact binding motifs of the Grb2 SH2 domain, and a mutant (Dok-3-FF) having a Tyr/Phe substitution at these motifs failed to inhibit Ras and Erk activation downstream of a cytoplasmic PTK Src. Because Grb2 forms a stable complex with Sos, a crucial activator of Ras, these data suggest that Dok-3 restrains Grb2 and inhibits the ability of the Grb2-Sos complex to activate Ras. Indeed, forced expression of Dok-3, but not Dok-3-FF, inhibited the recruitment of the Grb2-Sos complex to Shc downstream of Src, which is an essential event for activation of the Ras-Erk pathway. These findings indicate that Dok-3 sequesters Grb2 from Shc and inhibits the Ras-Erk pathway downstream of PTKs.