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

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 RRKMP221 as the major BCH interaction site within BNIP-2. In comparison, deletion of either the argininepatch 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.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. When not tyrosine-phosphorylated BNIP-2 can bind to two cellular targets: Cdc42, a small GTPase and its regulator, Cdc42GAP but this binding is abrogated upon its tyrosine phosphorylation (2).Cdc42 is a member of the Rho subfamily of GTPases demonstrated to be involved in various aspects of cytoskeletal organization, regulation of the transcription of certain target genes, and the control of aspects of cell cycle progression (3-12). 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) 1 (13-15). The guanine nucleotide exchange factors catalyze the exchange of GDP on the inactive GTPase for GTP, which results in enhanced activity of the target protein. The GAPs enhance rates of GTP hydrolysis to GDP mainly by contributing catalytic arginine residues to their substrate target in trans, or by stabilizing the conformation of the inherent GTPases (15-24).Despite lacking obvious sequence homology to the canonical catalytic domain of GAP proteins, BNIP-2 was shown to possess a GAP...

Cdc42GAP regulates c-Jun N-terminal kinase (JNK)-mediated apoptosis and cell number during mammalian perinatal growth

“…† Survival rate at weaning. RhoGAP domain that was shown to be catalytically specific toward Cdc42 over other Rho GTPases (16,17) (Fig. 1 A).…”

“…Among the large number of RhoGAP family members, Cdc42GAP appears to favor Cdc42 as a substrate over other Rho GTPases in vitro (16,17), but its physiologic role has yet to be verified in vivo. We found that Cdc42GAP Ϫ/Ϫ mice have significantly reduced body and organ sizes, similar to that of the p190-B RhoGAP knockouts (18).…”

BNIP-Sα induces cell rounding and apoptosis by displacing p50RhoGAP and facilitating RhoA activation via its unique motifs in the BNIP-2 and Cdc42GAP homology domain

“…For example, an arginine-finger patch in BNIP-2 is necessary for its GAP-like activity or the other distinct region for its homophilic/heterophilic interactions (Low et al, 2000a). Likewise, a distinct region in BNIP-Sa BCH domain is also involved in conferring homophilic binding and this sequence is also required for its cell rounding and apoptotic effects (Zhou et al, 2002).…”

“…Consistent to our previous report, the DB mutant without the homophilic-interacting motif could not be precipitated by GST-BNIP-Sa whereas the DA/ext-RBD mutant still retained its ability to interact with the wildtype BNIP-Sa. To further confirm their interaction in vivo, 293T cells were co-transfected with an HA-RhoA expression plasmid together with either the FLAGtagged full-length, DA/ext-RBD mutant or the DB mutant and subjected to co-immunoprecipitation assays (Figure 4f Presence of a cryptic GAP-binding motif that overlaps with minimal Rho-binding region in the BCH domain of BNIP-Sa Our previous reports had shown that both BNIP-2 and BNIP-Sa could bind p50RhoGAP via their respective BCH domains (Low et al, 2000a;Zhou et al, 2002). In this regard, we proceeded to identify the binding motif for p50RhoGAP within the BNIP-Sa BCH domain by first subjecting the above two mutants (DA/ext-RBD and DB) and the wild-type BNIP-Sa to co-immunoprecipitation studies in vivo.…”

Active Mek2 as a regulatory scaffold that promotes Pin1 binding to BPGAP1 to suppress BPGAP1-induced acute Erk activation and cell migration

“…Transfected 293T cells were lysed with RIPA buffer [150 mM NaCl, 50 mM TrisHCl pH 7.3, 0.25 mM EDTA, 1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, 50 mM NaF, 5 mM sodium orthovanadate, protease inhibitors (Roche Applied Science)] and directly analyzed as whole-cell lysates or aliquots used for pull-down with various GST fusion proteins (20 mg) or anti-Flag M2 affinity gel (Sigma), as previously described (Low et al, 2000). Samples were run in SDS-PAGE gels followed by western blot analyses using the Pierce Pico ECL (Thermo Scientific).…”