The proto-Dbl protein is the prototype member of a large family of guanine nucleotide exchange factors (GEFs) for Rho GTPases (8
). Oncogenic activation of proto-Dbl occurs by truncation of the amino-terminal 497 residues (41
), resulting in constitutively active carboxyl-terminal sequences that include a Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain, the conserved motifs of the Dbl family. Many members of this family, including Vav, Ect2, Tim, Ost, Dbs, Lbc, Lfc, Lsc, and Net, possess transformation or invasion ability, similar to onco-Dbl upon activation. In many cases, the DH-PH module represents the minimum structural unit that is required for cell transformation (8
A large body of evidence has helped establish that the biological functions of Dbl family members are intimately dependent upon their ability to interact with and activate Rho GTPases and that the cellular effects of Dbl-like proteins, including actin cytoskeletal reorganization, cell growth stimulation, and transformation, are likely the consequences of coordinated action of their immediate downstream substrates, the Rho family GTPases (8
). The evidence includes the findings that Dbl family oncoprotein-induced foci are morphologically similar to those transformed by constitutively activated Rho GTPases but distinct from that seen when cells are transformed by Ras, Raf, or Src (23
); coexpression of Dbl family members with dominant negative mutants of Rho family GTPases blocks their transforming activity (20
); mutants of the GEFs that are no longer able to interact or activate Rho protein substrates behave dominant-negatively in cells (46
); and many cellular activities induced by Dbl family proteins, such as actin cytoskeleton reorganization, c-Jun kinase (JNK) activation, SRF transcriptional activation, and NF-κB activation, are associated with the activation of signaling pathways known to be mediated by the Rho GTPase effector targets (24
). Therefore, the ability to interact and activate Rho proteins is essential for Dbl family functions.
Current biochemical and structural data have pointed to the conserved structural motif of the Dbl family, the DH domain, as the primary interactive site with Rho GTPases (2
). The DH domain does not have significant sequence homology with other subtypes of small GTPase activators such as the Cdc25 domain and Sec7 domain, which are specific to Ras and ARF, respectively (6
), indicating that the DH-Rho protein interaction employs a distinct mechanism (9
). Deletions or mutations within the DH domain have been reported to result in loss of GEF activity and cellular functions by the GEFs (20
), suggesting that an intact DH domain, likely its Rho GTPase-interactive ability, is critical for the cellular effects of Dbl family members.
The invariable location of a PH domain immediately C-terminal to the DH domain of the Dbl family GEFs suggests a functional interdependence between the two domains. Indeed, a regulatory role of the PH domain in the function of Dbl family members has been recognized. Derivatives of the Dbl family members onco-Dbl, Lbc, Lfc, and Dbs that are truncated within the PH domain are impaired in their transforming activity (38
). In these cases, the PH domain was found to promote the translocation of the Dbl family proteins to the plasma membrane or cytoskeleton, where the Rho GTPase substrates reside. It is therefore likely that the PH domain of the Dbl proteins, acting similarly to the SH2/SH3 domains in the Ras pathway (10
), serves to bring the catalytic DH domain to specific intracellular locations to effectively activate the Rho GTPases.
Many members of the Dbl family appear to exist in an inactive, basal state prior to full activation. The incoming upstream signals, such as the heterotrimeric G-protein Gα and Gβγ subunits, protein tyrosine or serine/threonine kinases, adaptor or scaffolding proteins, and phosphoinositol lipids, may contribute in varying degrees to GEF activation processes (12
). The best-understood example of self-regulation among the family members is the proto-Vav protein. The N-terminal autoinhibitory extension of proto-Vav forms an α-helix that binds in the DH domain active site through direct contact with the Rho GTPase binding pocket, blocking access to GTPases (3
). Phosphorylation of Tyr174, which is an integral part of the autoinhibition interface, by Syk or Src-like kinases causes the N-terminal peptide to become unstructured and released from the DH domain, resulting in proto-Vav activation (3
). The yeast Dbl family member Cdc24, which is a Cdc42-specific GEF, forms a protein complex with the scaffolding molecule Far1 and the Gβγ subunits to mediate the mating response of Saccharomyces cerevisiae
). Mammalian p115RhoGEF becomes activated as a Rho GEF upon Gα13
binding to its N-terminal RGS domain, suggesting that the coupling between a Gα and p115Rho GEF may relieve the intrinsic constraint of the DH domain (19
). Moreover, phosphorylation of the Rac1-specific GEF Tiam1 by Ca2+
/calmodulin-dependent protein kinase II has been shown to lead to its translocation to the plasma membrane and activation (13
), possibly by interference of the PH domain function of Tiam1, which has previously been demonstrated to determine its subcellular location (45
). These cases suggest that the Dbl family GEFs employ a diverse range of self-regulatory mechanisms to maintain themselves in the basal state.
Proto-Dbl activation occurs through truncation of N-terminal 497 amino acids (42
), suggesting that the N-terminal half of the molecule contains a negative regulatory element(s) for the C-terminal DH-PH functional module. A previous database search found limited similarities between the N terminus of proto-Dbl and the intermediate filament protein vimentin, spanning a 300-amino-acid region which was predicted to consist of an extended α-helical coiled-coil structure (41
). However, where the inhibitory function resides upstream of the DH domain (residues 498 to 690) and how the N terminus exerts the inhibitory function remain unclear. In the present article, we report the finding that proto-Dbl protein involves an intramolecular interaction between the N terminus and the PH domain to maintain an autoinhibited, inactive state. The N- and C-terminal domain interaction effectively limits the access of the Rho GTPase substrates RhoA and Cdc42 to the catalytic site of the DH domain and masks the intracellular targeting function of the PH domain, resulting in suppression of its GEF function and a unique perinuclear localization pattern in cells. Such an autoinhibition state prevents proto-Dbl from transforming cells, and presents a basal mode that could be subject to modulation by a variety of upstream signals.