Small guanine nucleotide triphosphatases (GTPases) constitute a large superfamily of molecular switches that regulate important signaling networks in cell growth, cell dynamics, and tissue/organ development. For examples, the Ras family members are involved in cell proliferation, Rho family as regulators of cell dynamics, Arf family on intracellular trafficking, whereas Ran family controls nucleus export/import (Bishop and Hall, 2000
; Etienne-Manneville and Hall, 2002
). These pathways are activated by certain classes of guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs).
GAP domain catalyzes the conversion of the active GTP-bound form of small GTPases to their inactive GDP-bound state through a canonical “arginine finger” motif (Sprang, 1997
). Thus far, at least 53 distinct proteins harboring the GAP domain have been identified from the human genome database (Moon and Zheng, 2003
). To date, there is no specific GAP for a single GTPase; instead, there exists a GAP that recognizes more than one GTPases, and a single GTPase can be a target of multiple GAPs. Furthermore, in vitro substrate profile can vary compared with the in vivo results (Ridley et al., 1993
). Although it has been well established that GAP-containing proteins usually function to negatively regulate their cognate GTPase substrates, some are believed to function as an effector; for example, the RasGAP Neurofibromatosis 1 (Yunoue et al., 2003
) and TcGAP that is involved in insulin-stimulated glucose transport (Chiang et al., 2003
), whereas others potentiate the action of the protein they reside in, such as the RhoGAP domain in the regulatory subunits of phosphatidylinositol 3-kinase, p85, which interacts specifically with Rac1 and Cdc42 to stimulate its kinase activity in vitro, without detectable GAP activity (Zheng et al., 1994
). Furthermore, GAPs can be subjects of signaling cross talk by providing multiple signaling modules linked to other signaling pathways such as tyrosine kinase, phosphoi-nositites, and serine/threonine kinases. The recent findings that p190-B RhoGAP unexpectedly regulates body size of mice by affecting insulin-mediated CREB transcriptional signaling and that Rho GTPases regulate a switch between adipogenesis and myogenesis immediately open up a wealth of new and exciting prospects for testing functions of other GAPs in vivo (Sordella et al
). All these point to the complexity in the nature of GAP and small GTPases regulation and highlighting the need to address the function of GAPs (and other regulators) in totality, including the roles of other domains they carry. Being the key signal transducers with multiple motifs and differential substrate specificities, GAPs are poised to coordinately interact with numerous molecules to converge or diverge signals inside the cells.
We have recently identified a novel RhoGAP, termed BPGAP1, that harbors three distinctive protein domains, the BNIP-2 and Cdc42GAP homology (BCH) domain that we first described (Low et al
), a proline-rich region (PRR) and a functional GAP domain (Shang et al., 2003
). BPGAP1 induces the formation of pseudopodia at the cell periphery in a process that required its BCH domain and the GAP domain. Furthermore, we previously showed that these two domains collaborate with the PRR to bring about enhanced cell motility. These results suggest that changes in cell morphology are coupled to determinant(s) in cell migration.
To further elucidate the molecular mechanism underlying cell dynamics control by BPGAP1, we used protein precipitations and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and identified cortactin, a cortical actin binding protein as a bona fide partner of BPGAP1. Cortactin has been shown to be involved in various intracellular signaling leading to membrane ruffling, endocytosis, and motility via reorganization of actin cytoskeleton (Weed and Parsons, 2001
). In vitro and in vivo protein interaction studies confirmed that cortactin interacted directly and specifically with BPGAP1 in a constitutive manner that required its Src homology (SH)3 domain binding to the proline-rich motif of BPGAP1 and such interaction facilitated translocation of cortactin to the membrane periphery for enhanced cell migration. These results provide the first evidence that a RhoGAP functionally interacts with cortactin and represents a novel determinant in the regulation of cell dynamics.