Evidence for a Novel Cdc42GAP Domain at the Carboxyl Terminus of BNIP-2

Boon Chuan Low,Kah Tong Seow,Graeme R Guy

doi:10.1074/jbc.275.19.14415

Boon Chuan Low, Kah Tong Seow + Show 1 more

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https://doi.org/10.1074/jbc.275.19.14415

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Abstract

We recently identified BNIP-2, a previously cloned Bcl-2- and E1B-associated protein, as a putative substrate of the FGF receptor tyrosine kinase and showed that it possesses GTPase-activating activity toward Cdc42 despite the lack of homology to previously described catalytic domains of GTPase-activating proteins (GAPs). BNIP-2 contains many arginine residues at the carboxyl terminus, which includes the region of homology to the noncatalytic domain of Cdc42GAP, termed BNIP-2 and Cdc42GAP homology (BCH) domain. Using BNIP-2 glutathione S-transferase recombinants, it was found that its BCH bound Cdc42, and contributed the GAP activity. This domain was predicted to fold into alpha-helical bundles similar to the topology of the catalytic GAP domain of Cdc42GAP. Alignment of exposed arginine residues in this domain helped to identify Arg-235 and Arg-238 as good candidates for catalysis. Arg-238 matched well to the arginine "finger" required for enhanced GTP hydrolysis in homodimerized Cdc42. Site-directed mutagenesis confirmed that an R235K or R238K mutation severely impaired the BNIP-2 GAP activity without affecting its binding to Cdc42. From deletion studies, a region adjacent to the arginine patch ((288)EYV(290) on BNIP-2) and the Switch I and Rho family-specific "Insert" region on Cdc42 are involved in the binding. The results indicate that the BCH domain of BNIP-2 represents a novel GAP domain that employs an arginine patch motif similar to that of the Cdc42-homodimer.

Highlights

Ras superfamily GTPase proteins act as molecular switches for signal transduction pathways to control cell growth, differentiation, and motility
We have identified several arginines as key residues within the BNIP-2 BNIP-2 and Cdc42GAP homology (BCH) domain that are responsible for the GTPase-activating proteins (GAPs) activity of the protein
This region containing the arginines bears no similarity to the arginine motifs employed by the “cradle-fold” structural topology in the RasGAP or Cdc42GAP/ RhoGAP catalytic structures (3, 14 –22) or to the leucine repeat folds of RanGAP binding to Ran [23]

Summary

Introduction

Ras superfamily GTPase proteins act as molecular switches for signal transduction pathways to control cell growth, differentiation, and motility. Structural and biochemical studies show that all GAPs, bearing no close overall sequence homology to each other, exert their effect either by contributing catalytic residues in-trans, by lowering the activation energy for GTP hydrolysis, or by stabilizing the conformation of the inherent GTPases These mechanisms are employed by 120-kDa RasGAP and the 50-kDa RhoGAP/Cdc42GAP through a highly conserved arginine “finger” catalytic motif and by similar binding topology (3, 14 –22). BNIP-2 was shown to possess a “GAP-like” activity toward Cdc42 [26] This protein contains no sequence homology to the canonical catalytic domain of a GAP, but it shares a highly conserved sequence with a region in the amino-terminal, noncatalytic half of Cdc42GAP [26, 27].

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