Concerted Regulation of Cell Dynamics by BNIP-2 and Cdc42GAP Homology/Sec14p-like, Proline-rich, and GTPase-activating Protein Domains of a Novel Rho GTPase-activating Protein, BPGAP1

Xun Shang,Yi Ting Zhou,Boon Chuan Low

doi:10.1074/jbc.m304514200

Xun Shang, Yi Ting Zhou + Show 1 more

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https://doi.org/10.1074/jbc.m304514200

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Abstract

RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility. Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domain-containing, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP. BP-GAP1 selectively enhanced RhoA GTPase activity in vivo although it also interacted strongly with Cdc42 and Rac1. "Pull-down" and co-immunoprecipitation studies indicated that it formed homophilic or heterophilic complexes with other BCH domain-containing proteins. Fluorescence studies of epitope-tagged BPGAP1 revealed that it induced pseudopodia and increased migration of MCF7 cells. Formation of pseudopodia required its BCH and GAP domains but not the proline-rich region, and was differentially inhibited by coexpression of the constitutively active mutant of RhoA, or dominant negative mutants of Cdc42 and Rac1. However, the mutant without the proline-rich region failed to confer any increase in cell migration despite the induction of pseudopodia. Our findings provide evidence that cell morphology changes and migration are coordinated via multiple domains in BPGAP1 and present a novel mode of regulation for cell dynamics by a RhoGAP protein.

Highlights

Cells undergo dynamic changes as part of their adaptation and response to stimuli
The proline-rich region was required for ensuring cell migration following the morphological changes induced by both GTPase-activating proteins (GAPs) and BNIP-2 and Cdc42GAP Homology (BCH) domains. These results indicate the unique interplay by different domains of BPGAP1 in exerting cell dynamics and confirm that changes in cell morphology is a prerequisite but not necessarily the only determinant for cell migration, it requires the input of other factor(s) as well
Some of them include the Pleckstrin homology domain, Src homology-3 domain, Fes/ CIP4 homology domain, Rho guanine nucleotide exchange factor domain, and the p21 Rho binding domain. One of these classes is represented by several putative members that resemble the organization of the Cdc42GAP protein. They are typified by the presence, at the proximal N terminus, of the newly identified BNIP-2 and Cdc42GAP homology (BCH)/Sec14p-like domain that we first described in the BNIP-2 family (20 –23) and a well conserved GAP domain at its distal C terminus

Summary

Introduction

Cells undergo dynamic changes as part of their adaptation and response to stimuli. These include their abilities to proliferate, differentiate, or execute death. The GTPbound form assumes an active conformation that allows interaction with downstream effectors, the “on-switch,” whereas its conversion to the GDP-bound form keeps the proteins in an “off-switch” mode and renders the GTPase inactive The balance of these two forms determines the final execution of the pathway. P200RhoGAP targets RhoA and Rac but not Cdc42 [18] while p115RhoGAP confines its action mainly to RhoA [19] It appears that there is no specific GAP for a single GTPase. Most of these GAPs possess multiple signaling modules that could couple their activities to other signaling pathways This could have far reaching consequences for the regulation of Rho and other small GTPase signals, and remains to be seen how, where, and when any subsets or combinations of these cellular counterparts will co-exist and exert their effects

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