The BNIP-2 and Cdc42GAP Homology Domain of BNIP-2 Mediates Its Homophilic Association and Heterophilic Interaction with Cdc42GAP

Boon Chuan Low,Kah Tong Seow,Graeme R Guy

doi:10.1074/jbc.m004897200

Abstract

We recently showed that BNIP-2 is a putative substrate of the fibroblast growth factor receptor tyrosine kinase and it possesses GTPase-activating activity toward the small GTPase, Cdc42. The carboxyl terminus of BNIP-2 shares high homology to the non-catalytic domain of Cdc42GAP, termed BCH (for BNIP-2 and Cdc42GAP homology) domain. Despite the lack of obvious homology to any known catalytic domains of GTPase-activating proteins (GAPs), the BCH domain of BNIP-2 bound Cdc42 and stimulated the GTPase activity via a novel arginine-patch motif similar to that employed by one contributing partner in a Cdc42 homodimer. In contrast, the BCH domain of Cdc42GAP, although it can bind Cdc42, is catalytically inactive. This raises the possibility that these domains might have other roles in the cell. Using glutathione S-transferase recombinant proteins, immunoprecipitation studies, and yeast two-hybrid assays, it was found that BNIP-2 and Cdc42GAP could form homo and hetero complexes via their conserved BCH domains. Molecular modeling of the BNIP-2 BCH homodimer complex and subsequent deletion mutagenesis helped to identify the region (217)RRKMP(221) as the major BCH interaction site within BNIP-2. In comparison, deletion of either the arginine-patch (235)RRLRK(239) (necessary for GAP activity) or region (288)EYV(290) (a Cdc42 binding sequence) had no effect on BCH-BCH interaction. Extensive data base searches showed that the BCH domain is highly conserved across species. The results suggest that BCH domains of BNIP-2 and Cdc42GAP represent a novel protein-protein interaction domain that could potentially determine and/or modify the physiological roles of these molecules.

Highlights

We recently identified BNIP-2, a previously cloned Bcl-2 and adenovirus E1B-interacting protein (1), as a putative substrate of the fibroblast growth factor receptor tyrosine kinase
Since currently we have not established the site(s) in the Cdc42GAP-BCH domain that is involved in its homophilic and heterophilic interaction, it remains to be seen whether deletion of such binding regions in Cdc42GAP could lead to an increase in its GTPase-activating proteins (GAPs) activity as was seen for BNIP-2
The present study examined the interaction between BNIP-2 and Cdc42GAP by using in vitro and in vivo binding experiments and demonstrated that their homologous BCH domains primarily mediate both homophilic and heterophilic interaction between the proteins

Summary

Introduction

We recently identified BNIP-2, a previously cloned Bcl-2 and adenovirus E1B-interacting protein (1), as a putative substrate of the fibroblast growth factor receptor tyrosine kinase. GTPases cycle between the inactive, GDP-bound form and the active GTP-bound form The equilibrium between these two states is controlled at least by two major classes of regulators, the guanine nucleotide exchange factors and the GTPase-activating proteins (GAPs) (13–15). We recently identified that this unexpected GAP activity is mediated by several key arginine residues within the COOH terminus of BNIP-2 (25) that constitute an apparently catalytic motif similar to the “arginine finger” demonstrated in Cdc homodimers (26, 27). The COOH-terminal region of BNIP-2 shares a high degree of sequence homology with a region at the NH2terminal, non-catalytic half of Cdc42GAP, which we termed the BCH (BNIP-2 and Cdc42GAP homology) domain (25) Both BNIP-2 and Cdc42GAP BCH domains can bind Cdc, but only the BCH domain of BNIP-2 functions as a GAP toward Cdc as Cdc42GAP lacks the arginine-finger motif (25). In this context phosphatidylinositol 4-phosphate seems to be a target phospholipid similar to phosphatidylinositol 3-phosphate and both may play a role in intracellular trafficking

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Results

Conclusion