Role of the C-Terminal SH3 Domain and N-Terminal Tyrosine Phosphorylation in Regulation of Tim and Related Dbl-Family Proteins

Abstract

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. Although the exchange potential of many Dbl-family proteins is constitutively activated by truncation, the precise mechanisms of regulation for many Dbl-family proteins are unknown. Tim and Vav are distantly related Dbl-family proteins that are similarly regulated; their Dbl homology (DH) domains interact with N-terminal helices to exclude and prevent activation of Rho GTPases. Phosphorylation, substitution, or deletion of the blocking helices relieves this autoinhibition. Here we show that two other Dbl-family proteins, Ngef and Wgef, which like Tim contain a C-terminal SH3 domain, are also activated by tyrosine phosphorylation of a blocking helix. Consequently, basal autoinhibition of DH domains by direct steric exclusion using short Nterminal helices likely represents a conserved mechanism of regulation for the large family of Dblrelated proteins. N-Terminal truncation or phosphorylation of many other Dbl-family GEFs leads to their activation; similar autoinhibition mechanisms could explain some of these events. In addition, we show that the C-terminal SH3 domain binding to a polyproline region N-terminal to the DH domain of the Tim subgroup of Dbl-family proteins provides a unique mechanism of regulated autoinhibition of exchange activity that is functionally linked to the interactions between the autoinhibitory helix and the DH domain.The activation cycle of Rho GTPases 1 is tightly controlled by three families of proteins: GTPase-activating proteins (GAPs), guanine nucleotide dissociation inhibitors (GDIs), and guanine nucleotide exchange factors (GEFs). The Dbl family of proteins constitutes the largest group of GEFs specific for Rho GTPases. Dbl-family proteins are characterized by a Dbl homology (DH) domain, which contacts the Rho GTPase to catalyze nucleotide exchange by promoting and stabilizing an intermediate, nucleotide-free GTPase state, and an associated pleckstrin homology (PH) domain, which fine-tunes the exchange process. The 69 human Dbl-

The guanine nucleotide exchange factor Arhgef5 plays crucial roles in Src-induced podosome formation

“…This Src-dependent phosphorylation has also been reported in TIM (Yohe et al, 2008), and two tyrosine residues in the N-terminus were identified as phosphorylation sites. Phosphorylations of these residues positively regulate the GEF activity of TIM by relieving the autoinhibitory intramolecular interaction between its C-terminal SH3 domain and N-terminal polyproline region.…”

Identification of a Negative Regulatory Region for the Exchange Activity and Characterization of T332I Mutant of Rho Guanine Nucleotide Exchange Factor 10 (ARHGEF10)

“…GEF activity of several RhoGEFs of the Dbl type is regulated by the intramolecular autoinhibition. For example, Vav, Dbl, Asef, Tim, and PDZRhoGEF are negatively regulated by their own N-terminal regions (21)(22)(23)(24)(25)(26). Our results demonstrated that the region containing amino acids 212-332 of ARHGEF10 is a negatively regulatory region for its GEF activity.…”

Allosteric properties of PH domains in Arf regulatory proteins

“…[89][90][91][92][93][94] For FARP2, PH domains are central to the mechanism. 93 A Rho family GEF, FARP2 contains a catalytic Dbl homology (DH) domain and 2 PH domains.…”