Multifunctional Roles for the PH Domain of Dbs in Regulating Rho GTPase Activation

Abstract

Dbl family members are guanine nucleotide exchange factors specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. Dbs, a Dbl family member specific for Cdc42 and RhoA, exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. In this study, the PH domain of Dbs was mutated to impair selectively either guanine nucleotide exchange or phosphoinositide binding in vitro and resulting physiological alterations were assessed. As anticipated, substitution of residues within the PH domain of Dbs integral to the interface with GTPases reduced nucleotide exchange and eliminated the ability of Dbs to transform NIH 3T3 cells. More interestingly, substitutions within the PH domain that prevent interaction with phosphoinositides yet do not alter in vitro activation of GTPases also do not transform NIH 3T3 cell and fail to activate RhoA in vivo despite proper subcellular localization. Therefore, the PH domain of Dbs serves multiple roles in the activation of GTPases and cannot be viewed as a simple membrane-anchoring device. In particular, the data suggest that binding of phosphoinositides to the PH domain within the context of membrane surfaces may direct orientations or conformations of the linked DH and PH domains to regulate GTPases activation.The Rho family GTPases 1 are an essential subset of the Ras superfamily of small molecular weight GTPases. Like Ras, Rho family GTPases cycle between GDP-and GTP-bound forms. When GDP-bound, Rho proteins are inactive and do not functionally couple to their downstream effectors. However, when GTP-bound, Rho GTPases elicit profound effects on the organization of the actin cytoskeleton, in addition to tightly regulating the activation state of transcription factors such as the serum response factor, c-Jun, and NF-B (1-3). GDP/GTP cycling within Rho proteins is primarily accomplished through the actions of two classes of regulatory proteins. GTPase activating proteins promote the inactive, GDP-bound form of Rho proteins by enhancing their intrinsic GTPase ability to convert bound GTP to GDP. In contrast, the actions of guanine nucleotide exchange factors (GEFs) upon Rho GTPases results in Rho activation by exchanging their bound GDP for GTP.Members of the Dbl family of oncoproteins act as GEFs exclusively for Rho GTPases (RhoGEFs) and, like constitutively activated members of Rho GTPases, can exhibit potent transformation potential within various cell types upon overexpression or constitutive activation (4 -6). Dbl family members invariably contain a Dbl homology (DH) domain in tandem with an adjacent, carboxyl-terminal pleckstrin homology (PH) domain. However, outside of this region, Dbl-related proteins share little sequence conservation and typically possess a variety of protein-signaling domains, presumably reflecting diversity in regulation and cellular function.The invariant positioning of PH domains immediately carboxyl-terminal to DH domains strongly implies a unique functional couplin...

A PH Domain in the Arf GTPase-activating Protein (GAP) ARAP1 Binds Phosphatidylinositol 3,4,5-Trisphosphate and Regulates Arf GAP Activity Independently of Recruitment to the Plasma Membranes

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Leukocyte transendothelial migration: A local affair

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