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Our results identify p190 RhoGAPs as effectors of Rnd proteins and demonstrate a novel mechanism by which Rnd proteins function as antagonists of RhoA.

Previous work indicates that RhoA phosphorylation on Ser188 by cAMP or cGMP-dependent kinases inhibits its activity. However, these studies lacked the possibility to directly study phosphorylated RhoA activity in vivo. Therefore, we created RhoA proteins containing phosphomimetic residues in place of the cAMP/cGMPdependent kinase phosphorylation site. RhoA phosphorylation or phosphomimetic substitution did not affect Rho guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity in vitro but promoted binding to the Rho guanine-dissociation inhibitor as measured by exchange factor competition assays. The in vitro similarities between RhoA phosphomimetic proteins and phosphorylated RhoA allowed us to study function of phosphorylated RhoA in vivo. RhoA phosphomimetic proteins display depressed GTP loading when transiently expressed in NIH 3T3 cells. Stable-expressing RhoA and RhoA(S188A) clones spread significantly slower than mock-transfected or RhoA(S188E) clones. RhoA(S188A) clones were protected from the morphological effects of a cAMP agonist, whereas phosphomimetic clones exhibit stress fiber disassembly similar to control cells. Together, these data provide in vivo evidence that addition of a charged group to Ser 188 upon phosphorylation negatively regulates RhoA activity and indicates that this occurs through enhanced Rho guanine-dissociation inhibitor interaction rather than direct perturbation of guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity.

Type I collagen stimulation of pro-matrix metalloproteinase (pro-MMP)-2 activation by ovarian cancer cells involves ␤ 1 integrin receptor clustering; however, the specific cellular and biochemical events that accompany MMP processing are not well characterized. Collagenolysis is not required for stimulation of pro-MMP-2 activation, and denatured collagen does not elicit an MMP-2 activation response. Similarly, DOV13 cells bind to intact collagen utilizing both ␣ 2 ␤ 1 and ␣ 3 ␤ 1 integrins but interact poorly with collagenase-treated or thermally denatured collagen. Antibody-induced clustering of ␣ 3 ␤ 1 strongly promotes activation of pro-MMP-2, whereas ␣ 2 ␤ 1 integrin clustering has only marginal effects. Membrane-type 1 (MT1)-MMP is present on the DOV13 cell surface as both an active 55-kDa TIMP-2-binding species and a stable catalytically inactive 43-kDa form. Integrin clustering stimulates cell surface expression of MT1-MMP and co-localization of the proteinase to aggregated integrin complexes. Furthermore, cell surface proteolysis of the 55-kDa MT1-MMP species occurs in the absence of active MMP-2, suggesting MT1-MMP autolysis. Cellular invasion of type I collagen matrices requires collagenase activity, is blocked by tissue inhibitor of metalloproteinases-2 (TIMP-2) and collagenase-resistant collagen, is unaffected by TIMP-1, and is accompanied by pro-MMP-2 activation. Together, these data indicate that integrin stimulation of MT1-MMP activity is a rate-limiting step for type I collagen invasion and provide a mechanism by which this activity can be down-regulated following collagen clearance.The MMP family is composed of at least 25 zinc-dependent extracellular endopeptidases whose activities are regulated predominantly by expression as inactive precursors, or zymogens (1-3). Although precise physiological activators of MMPs 1 are unknown, a variety of serine proteinases and other MMPs in the extracellular milieu execute the initial propeptide cleavage events in vitro (2). An exception to serine protease activation is pro-MMP-2 (72-kDa gelatinase A), which lacks the necessary basic amino acid cleavage sites in its pro-domain (4). A primary mechanism of pro-MMP-2 activation involves zymogen association with the cell surface via formation of a ternary complex containing tissue inhibitor of metalloproteinase (TIMP)-2 and membrane type 1-MMP (MT1-MMP, MMP-14) (3-6). Following trimeric complex formation, it is hypothesized that a neighboring MT1-MMP molecule that is not associated with TIMP-2 cleaves pro-MMP-2 at the Asn 37 -Leu 38 peptide bond within the pro-domain (7). Intermediately processed MMP-2 (Leu 38 -MMP-2) undergoes further concentration-dependent autolytic cleavage(s) to generate mature enzymes that can be released into the soluble phase or remain surface-associated (8). Although biological mechanisms of active MMP-2 release from the cell surface are not well characterized and dissociation kinetics provide little insight, cellular binding affinities may shift following pro-MMP-2 cleavage.Culturing ...

Rho proteins cycle between an inactive, GDP-bound state and an active, GTP-bound state. Activation of these GTPases is mediated by guanine nucleotide exchange factors (GEFs), which promote GDP to GTP exchange. In this study we have characterized XPLN, a Rho family GEF. Like other Rho GEFs, XPLN contains a tandem Dbl homology and pleckstrin homology domain topography, but lacks homology with other known functional domains or motifs. XPLN protein is expressed in the brain, skeletal muscle, heart, kidney, platelets, and macrophage and neuronal cell lines. In vitro, XPLN stimulates guanine nucleotide exchange on RhoA and RhoB, but not RhoC, RhoG, Rac1, or Cdc42. Consistent with these data, XPLN preferentially associates with RhoA and RhoB. The specificity of XPLN for RhoA and RhoB, but not RhoC, is surprising given that they share over 85% sequence identity. We determined that the inability of XPLN to exchange RhoC is mediated by isoleucine 43 in RhoC, a position occupied by valine in RhoA and RhoB. When expressed in cells, XPLN activates RhoA and RhoB, but not RhoC, and stimulates the assembly of stress fibers and focal adhesions in a Rho kinase-dependent manner. We also found that XPLN possesses transforming activity, as determined by focus formation assays. In conclusion, here we describe a Rho family GEF that can discriminate between the closely related RhoA, RhoB, and RhoC, possibly giving insight to the divergent functions of these three proteins.