Regulation of the transforming immortalized mammary protein and its homologs by auto-inhibition and tyrosine phosphorylation

Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho-family GTPases and typically possess tandem Dbl (DH) and pleckstrin homology (PH) domains that act in concert to catalyze exchange. The exchange activities of many Dbl-proteins are regulated by phosphorylation or constitutively activated by truncations preceding their DH domains. However, exact mechanisms of regulation remain poorly understood. Here we show that a sub-group of Dbl-family proteins, including Tim, Ngef, and Wgef, are auto-inhibited by a highly conserved helix immediately N-terminal to the DH domain that directly occludes the catalytic interface of the DH domain to prevent GTPase activation. Similar to the distantly related Vav isozymes, auto-inhibition is relieved by truncation, mutation, or phosphorylation of the auto-inhibitory helix. Furthermore, substitutions within a highly conserved surface of the DH domain designed to disrupt interactions with the auto-inhibitory helix also fully activates the exchange process. Therefore, the regulated auto-inhibition of DH domains by direct steric exclusion using short N-terminal segments likely represents a general mode of regulation within the large family of Dbl-family proteins. The C-terminal SH3 domain binding to a polyproline region N-terminal to the DH domain of this subgroup of Dbl-family proteins provides a unique mechanism of regulated auto-inhibition of exchange activity that is functionally linked to the interactions between the auto-inhibitory helix and the DH domain.