Molecular cloning of IBP, a SWAP-70 homologous GEF, which is highly expressed in the immune system

Abstract

Rho GTPases play a fundamental role in a variety of biological processes ranging from the reorganization of the actin cytoskeleton to the regulation of cell proliferation. The activation of Rho GTPases is regulated by guanine nucleotide exchange factors (GEFs) belonging to the Dbl family of proteins. The hallmark of this large family of GEFs is the presence of a tandem DH-PH module in which a pleckstrin-homology (PH) domain is located at the C-terminus of a Dbl-homology (DH) domain. Recent studies have demonstrated that SWAP-70 constitutes a novel class of Rac-GEF, in which the PH domain is located at the N-terminus, rather than the C terminus, of the DH domain. Here we report the molecular cloning of human IBP (IRF-4 binding protein), a new member of this novel family of GEFs. The IBP gene maps to human chromosome 6p21.31 centromeric to the MHC locus. Isolation of the murine IBP cDNA reveals a very high degree of homology with the human IBP cDNA suggesting that IBP is evolutionarily conserved. The 5′ portion of the murine IBP cDNA is furthermore identical to the Def-6 cDNA fragment, which was identified in the course of a search for genes differentially expressed in the murine hematopoietic system. IBP is broadly expressed in the immune system and can be detected in both T and B cell compartments in contrast to SWAP-70 whose expression is primarily restricted to B cells. Taken together these findings indicate that IBP is a novel type of GEF, which participates in the activation of Rho GTPases in lymphoid tissues.

Introduction

The Rho subfamily of small GTPases, which include RhoA, Rac1 and Cdc42, control a wide range of biological processes, from cell survival and proliferation to motility and invasion 1, 2, 3. Like all small GTPases, Rho GTPases function as molecular switches that cycle between an inactive GDP-bound form and an active GTP-bound form. Guanine nucleotide exchange factors (GEFs) promote the formation of the GTP-bound state, and therefore these proteins represent one of the major classes of regulators that control the activation state of Rho GTPases 4, 5, 6. About 50 different GEFs for Rho GTPases have been identified to date in eukaryotes. These GEFs usually contain a sequence of ∼200 amino acids termed the Dbl-homology (DH) domain, responsible for catalyzing the GDP/GTP exchange reactions, followed by a C-terminal pleckstrin-homology (PH) domain necessary for proper intracellular localization and function. The expression of many Rho- GEF proteins is confined to specific cell-types, suggesting that the activity of Rho GTPases can be controlled in a tissue-restricted manner, possibly contributing to the unique biologic properties of different cells.

Recent studies have revealed that SWAP-70 is a unique type of Rac-GEF in which the DH domain is flanked at its N-terminus, rather than at its C-terminus, by a PH domain [7]. Interestingly, the DH domain of SWAP-70 exhibits only a very low degree of homology to that of other known Rho-GEFs like Vav1 and Tiam1 further suggesting that this class of GEFs may activate a unique subset of Rho GTPases-mediated functions. SWAP-70 expression is predominantly confined to activated mature B lymphocytes although low levels can also be detected in mast cells and fibroblasts 8, 9. Consistent with its expression pattern, mice deficient in SWAP-70 primarily display alterations in antibody production and defects in mast cell development 10, 11. Furthermore, consistent with its Rac-GEF activity, SWAP-70-deficient kidney fibroblasts exhibit impaired membrane ruffling upon growth factor stimulation [7]. The existence of additional members of this unique family of GEFs has been suggested by the fact that, during studies geared to identifying genes differentially expressed in the murine haematopoietic system, a cDNA fragment exhibiting homology to SWAP-70 cDNA was isolated [12]. This murine cDNA fragment was termed Def-6 (differentially expressed in FDCP-Mix), since its expression was shown to be downregulated in the course of the differentiation of a myeloid progenitor cell line (FDCP-Mix A4) toward either myeloid or erythroid lineages.

Here we report the molecular cloning of a human cDNA encoding a novel SWAP-70- homologous protein, which we have termed IBP. In this report, we also describe the expression pattern of human IBP. The 5′ portion of the human IBP cDNA displays a very high degree of sequence homology to murine Def-6. This finding thus suggests that IBP represents the human orthologue of Def-6. Consistent with this notion the gene encoding human IBP is located on chromosome 6p21.31 in a syntenic region to murine chromosome 17 where the murine Def-6 gene is located. The IBP protein exhibits a significant homology with SWAP-70 and, like SWAP-70, it contains a putative EF-hand motif at the N-terminus, and a central PH domain flanked at its C-terminus by a DH domain [13]. A systematic investigation of the tissue distribution of IBP reveals that IBP is expressed in both central and peripheral lymphoid tissues.