Dual regulation of RA-RhoGAP activity by phosphatidic acid and Rap1 during neurite outgrowth

During neurite outgrowth, Rho small G protein activity is spatiotemporally regulated to organize the neurite sprouting, extension, and branching. We have previously identified a potent Rho GTPase-activating protein (GAP), RA-RhoGAP, as a direct downstream target of Rap1 small G protein in the neurite outgrowth. In addition to the Ras-associating (RA) domain for Rap1 binding, RA-RhoGAP has the pleckstrin homology (PH) domain for lipid binding. Here, we showed that phosphatidic acid (PA) bound to the PH domain and enhanced GAP activity for Rho. RA-RhoGAP induced extension of neurite in a diacylglycerol kinase-mediated synthesis of the PA-dependent manner. Knockdown of RA-RhoGAP reduced the diacylglycerol kinase-induced neurite extension. In contrast to the effect of the RA domain, the PH domain was specifically involved in the neurite extension, not in the sprouting and branching. These results indicate that PA and Rap1 cooperatively regulate RA-RhoGAP activity for promoting neurite outgrowth.     

Resistance to inhibitors of cholinesterase-8A (Ric-8A) is critical for growth factor receptor-induced actin cytoskeletal reorganization

Heterotrimeric G proteins are critical transducers of cellular signaling. In addition to their classic roles in relaying signals from G protein-coupled receptors (GPCRs), heterotrimeric G proteins also mediate physiological functions from non-GPCRs. Previously, we have shown that Gα(13), a member of the heterotrimeric G proteins, is essential for growth factor receptor-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. These Gα(13)-mediated dorsal ruffle turnover and cell migration by growth factors acting on their receptor tyrosine kinases (RTKs) are independent of GPCRs. However, the mechanism by which RTKs signal to Gα(13) is not known. Here, we show that cholinesterase-8A (Ric-8A), a nonreceptor guanine nucleotide exchange factor for some heterotrimeric G proteins, is critical for coupling RTKs to Gα(13). Down-regulation of Ric-8A protein levels in cells by RNA interference slowed down platelet-derived growth factor (PDGF)-induced dorsal ruffle turnover and inhibited PDGF-initiated cell migration. PDGF was able to increase the activity of Ric-8A in cells. Furthermore, purified Ric-8A proteins interact directly with purified Gα(13) protein in a nucleotide-dependent manner. Deficiency of Ric-8A prevented the translocation of Gα(13) to the cell cortex. Hence, Ric-8A is critical for growth factor receptor-induced actin cytoskeletal reorganization.     

Expression and potential function of Rho family small G proteins in cells of the mammalian seminiferous epithelium

Dynamic cellular rearrangements involving the actin cytoskeleton are required of both Sertoli and germ cells during spermatogenesis. Rho family small G proteins have been implicated in the control of the actin cytoskeleton in numerous cell types. Therefore, RhoA and Rac1 were investigated in Sertoli and germ cells. RhoA and Rac1 have been detected at both the mRNA and protein levels in these cells. In addition, Sertoli cell L-selectin is shown to interact with actin binding proteins, potentially providing a link between L-selectin and Rac1 signaling. Finally, inactivation of Sertoli cell Rho family proteins yields disruption of the actin cytoskeleton.     

Involvement of 14-3-3 proteins in the second epidermal growth factor-induced wave of Rac1 activation in the process of cell migration

Immense previous efforts have elucidated the core machinery in cell migration, actin remodeling regulated by Rho family small GTPases including RhoA, Cdc42, and Rac1; however, the spatiotemporal regulation of these molecules remains largely unknown. Here, we report that EGF induces biphasic Rac1 activation in the process of cell migration, and UTKO1, a cell migration inhibitor, inhibits the second EGF-induced wave of Rac1 activation but not the first wave. To address the regulation mechanism and role of the second wave of Rac1 activation, we identified 14-3-3ζ as a target protein of UTKO1 and also showed that UTKO1 abrogated the binding of 14-3-3ζ to Tiam1 that was responsible for the second wave of Rac1 activation, suggesting that the interaction of 14-3-3ζ with Tiam1 is involved in this event. To our knowledge, this is the first report to use a chemical genetic approach to demonstrate the mechanism of temporal activation of Rac1.     

X-linked inhibitor of apoptosis protein (XIAP) mediates cancer cell motility via Rho GDP dissociation inhibitor (RhoGDI)-dependent regulation of the cytoskeleton

X-linked inhibitor of apoptosis protein (XIAP) overexpression has been found to be associated with malignant cancer progression and aggression in individuals with many types of cancers. However, the molecular basis of XIAP in the regulation of cancer cell biological behavior remains largely unknown. In this study, we found that a deficiency of XIAP expression in human cancer cells by either knock-out or knockdown leads to a marked reduction in β-actin polymerization and cytoskeleton formation. Consistently, cell migration and invasion were also decreased in XIAP-deficient cells compared with parental wild-type cells. Subsequent studies demonstrated that the regulation of cell motility by XIAP depends on its interaction with the Rho GDP dissociation inhibitor (RhoGDI) via the XIAP RING domain. Furthermore, XIAP was found to negatively regulate RhoGDI SUMOylation, which might affect its activity in controlling cell motility. Collectively, our studies provide novel insights into the molecular mechanisms by which XIAP regulates cancer invasion and offer a further theoretical basis for setting XIAP as a potential prognostic marker and specific target for treatment of cancers with metastatic properties.     

Structural basis of Rnd1 binding to plexin Rho GTPase binding domains (RBDs)

Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD·Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD β3-β4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.     

Phosphorylation of doublecortin by protein kinase A orchestrates microtubule and actin dynamics to promote neuronal progenitor cell migration

Doublecortin (DCX) is a microtubule-associated protein that is specifically expressed in neuronal cells. Genetic mutation of DCX causes lissencephaly disease. Although the abnormal cortical lamination in lissencephaly is thought to be attributable to neuronal cell migration defects, the regulatory mechanisms governing interactions between DCX and cytoskeleton in the migration of neuronal progenitor cells remain obscure. In this study we found that the G(s) and protein kinase A (PKA) signal elicited by pituitary adenylate cyclase-activating polypeptide promotes neuronal progenitor cells migration. Stimulation of G(s)-PKA signaling prevented microtubule bundling and induced the dissociation of DCX from microtubules in cells. PKA phosphorylated DCX at Ser-47, and the phospho-mimicking mutant DCX-S47E promoted cell migration. Activation of PKA and DCX-S47E induced lamellipodium formation. Pituitary adenylate cyclase-activating polypeptide and DCX-S47E stimulated the activation of Rac1, and DCX-S47E interacted with Asef2, a guanine nucleotide exchange factor for Rac1. Our data reveal a dual reciprocal role for DCX phosphorylation in the regulation of microtubule and actin dynamics that is indispensable for proper brain lamination.     

Met receptors induce Sam68-dependent cell migration by activation of alternate extracellular signal-regulated kinase family members

The hepatocyte growth factor (HGF)/Met receptor signaling pathway is deregulated in diverse human malignancies and plays a central role in oncogenesis, tumor progression, and invasive cancer growth. Similarly, altered expression and splicing (i.e. inclusion of variant exon 5, "v5") of the cell adhesion marker, CD44, is associated with advanced cancer phenotypes. We sought to further understand how HGF regulates CD44v5 expression. Immortalized nontumorigenic keratinocyte (HaCaT) cells abundantly express both Met receptors and CD44v5 transmembrane glycoproteins. HGF stimulated CD44v5 protein expression and HaCaT cell migration; these events required activation of the ERK1/2 MAPK module and Sam68, a protein involved in RNA processing, splicing, and v5 inclusion. Similar to HaCaT cells, highly migratory MDA-MB-231 breast cancer cells also required Sam68 expression for HGF-induced migration. However, MDA-MB-231 cell migration occurred independently of ERK1/2 and CD44v5 expression and instead required ERK5 signaling to Sam68. Phospho-mutant, but not WT-Sam68, blocked HGF-induced cell migration in both cell types; MDA-MB-435 cells behaved similarly. These results suggest that Sam68 acts as a convergence point for ERK signaling to cell migration; blockade of phospho-Sam68 may provide a new avenue for therapeutic inhibition of metastatic cancers.     

ADP Ribosylation Factor 6 (Arf6) Acts through FilGAP Protein to Down-regulate Rac Protein and Regulates Plasma Membrane Blebbing

The small GTP-binding protein Arf6 reorganizes the actin cytoskeleton through the regulation of Rac activity. We identified FilGAP, a Rac-specific Rho GTPase-activating protein that is recruited to plasma membranes by binding to activated Arf6. FilGAP binds to Arf6 through its pleckstrin homology domain. Activated Arf6 stimulated RacGAP activity of FilGAP, and knockdown of endogenous Arf6 by siRNA suppresses FilGAP-mediated bleb formation. Mutant FilGAP lacking phosphatidylinositol 3,4,5-trisphosphate (PIP₃) binding (FilGAP R39C) binds to activated Arf6 and induces bleb formation. Moreover, bleb formation induced by wild-type FilGAP occurs in the presence of phosphatidylinositol 3-kinase inhibitors, suggesting a PIP₃-independent interaction between FilGAP and Arf6. We propose that FilGAP may function as a mediator of the regulation of Rac by Arf6.     

Evidence for two CRIB domains in phospholipase D2 (PLD2) that the enzyme uses to specifically bind to the small GTPase Rac2

Phospholipase D (PLD) and small GTPases are vital to cell signaling. We report that the Rac2 and the PLD2 isoforms exist in the cell as a lipase-GTPase complex that enables the two proteins to elicit their respective functionalities. A strong association between the two molecules was demonstrated by co-immunoprecipitation and was confirmed in living cells by FRET with CFP-Rac2 and YFP-PLD2 fluorescent chimeras. We have identified the amino acids in PLD2 that define a specific binding site to Rac2. This site is composed of two CRIB (Cdc42-and Rac-interactive binding) motifs that we have named "CRIB-1" and "CRIB-2" in and around the PH domain in PLD2. Deletion mutants PLD2-ΔCRIB-1/2 negate co-immunoprecipitation with Rac2 and diminish the FRET signal in living cells. The PLD2-Rac2 association was further confirmed in vitro using affinity-purified recombinant proteins. Binding was saturable with an apparent K(d) of 3 nm and was diminished with PLD2-ΔCRIB mutants. Furthermore, PLD2 bound more efficiently to Rac2-GTP than to Rac2-GDP or to a GDP-constitutive Rac2-N17 mutant. Increasing concentrations of recombinant Rac2 in vitro and in vivo during cell adhesion inhibit PLD2. Conversely, Rac2 activity is increased in the presence of PLD2-WT but not in PLD2-ΔCRIB. We propose that in activated cells PLD2 affects Rac2 in an initial positive feedback, but as Rac2-GTP accumulates in the cell, this constitutes a "termination signal" leading to PLD2 inactivation.     

Type 5 G protein beta subunit (Gbeta5) controls the interaction of regulator of G protein signaling 9 (RGS9) with membrane anchors

The R7 family of regulators of G protein signaling (RGS) proteins, comprising RGS6, RGS7, RGS9, and RGS11, regulate neuronal G protein signaling pathways. All members of the R7 RGS form trimeric complexes with the atypical G protein β subunit, Gβ5, and membrane anchor R7BP or R9AP. Association with Gβ5 and membrane anchors has been shown to be critical for maintaining proteolytic stability of the R7 RGS proteins. However, despite its functional importance, the mechanism of how R7 RGS forms complexes with Gβ5 and membrane anchors remains poorly understood. Here, we used protein-protein interaction, co-localization, and protein stability assays to show that association of RGS9 with membrane anchors requires Gβ5. We further establish that the recruitment of R7BP to the complex requires an intact interface between the N-terminal lobe of RGS9 and protein interaction surface of Gβ5. Site-directed mutational analysis reveals that distinct molecular determinants in the interface between Gβ5 and N-terminal Dishevelled, EGL-10, Pleckstrin/DEP Helical Extension (DEP/DHEY) domains are differentially involved in R7BP binding and proteolytic stabilization. On the basis of these findings, we conclude that Gβ5 contributes to the formation of the binding site to the membrane anchors and thus is playing a central role in the assembly of the proteolytically stable trimeric complex and its correct localization in the cell.     

HSP90 and HSP70 proteins are essential for stabilization and activation of WASF3 metastasis-promoting protein

Inactivation of HSP90 and HSP70 leads to loss of invasion in a variety of cancer cell types, presumably as a result of destabilization of, as yet, undefined clients of these molecular chaperones that influence this phenotype. The WASF3 gene has been shown to be up-regulated in high-grade tumors and its down-regulation leads to loss of invasion and metastasis. WASF3 phosphorylation by ABL kinase is essential for its ability to regulate invasion. Mass spectroscopy analysis now shows that HSP90 is present in the WASF3 immunocomplex from prostate cancer cells. Inactivation of HSP90 in these and other cell types does not affect WASF3 stability but prevents its phosphoactivation as a result of destabilization of ABL. HSP70 was also found in the WASF3 immunocomplex and inactivation of HSP70 results in destabilization of WASF3 through proteasome degradation. Knockdown of WASF3, HSP90, and HSP70 individually, all lead to loss of invasion but as knockdown of WASF3 in the presence of robust expression of HSP90/70 has the same effect, it seems that the influence these chaperone proteins have on invasion is mediated, at least in part, by their control over the critical invasion promoting capacity of the WASF3 protein. Overexpression of HSP70 in WASF3 null cells does not enhance invasion. These observations suggest that targeting HSP90/70 may have efficacy in reducing cancer cell invasion.     

The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity

GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little is known about the normal function of the receptor. We found that the large N terminus (NT) of GPR56 is cleaved from the rest of the receptor during processing but remains non-covalently associated with the seven-transmembrane region of the receptor, as indicated by coimmunoprecipitation of the two GPR56 fragments from both transfected cells and native tissue. We also found that truncation of the GPR56 NT results in constitutive activation of receptor signaling, as revealed by increased GPR56-stimulated signaling upon transfection of HEK-293 cells with truncated GPR56, greatly enhanced binding of β-arrestins by truncated GPR56 relative to the full-length receptor, extensive ubiquitination of truncated GPR56, and cytotoxicity induced by truncated GPR56 that could be rescued by cotransfection of cells with β-arrestin 2. Furthermore, we found that the GPR56 NT is capable of homophilic trans-trans interactions that enhance receptor signaling activity. On the basis of these findings, we suggest a model of receptor activation in which the large N terminus of GPR56 constrains receptor activity but N-terminal interactions (GPR56 NT with an extracellular ligand and/or GPR56 NT homophilic trans-trans associations) can remove this inhibitory influence of the N terminus to activate receptor signaling.     

Hedgehog signaling and Tre1 regulate actin dynamics through PI(4,5)P2 to direct migration of Drosophila embryonic germ cells

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Identification of critical residues in G(alpha)13 for stimulation of p115RhoGEF activity and the structure of the G(alpha)13-p115RhoGEF regulator of G protein signaling homology (RH) domain complex

RH-RhoGEFs are a family of guanine nucleotide exchange factors that contain a regulator of G protein signaling homology (RH) domain. The heterotrimeric G protein Gα(13) stimulates the guanine nucleotide exchange factor (GEF) activity of RH-RhoGEFs, leading to activation of RhoA. The mechanism by which Gα(13) stimulates the GEF activity of RH-RhoGEFs, such as p115RhoGEF, has not yet been fully elucidated. Here, specific residues in Gα(13) that mediate activation of p115RhoGEF are identified. Mutation of these residues significantly impairs binding of Gα(13) to p115RhoGEF as well as stimulation of GEF activity. These data suggest that the exchange activity of p115RhoGEF is stimulated allosterically by Gα(13) and not through its interaction with a secondary binding site. A crystal structure of Gα(13) bound to the RH domain of p115RhoGEF is also presented, which differs from a previously crystallized complex with a Gα(13)-Gα(i1) chimera. Taken together, these data provide new insight into the mechanism by which p115RhoGEF is activated by Gα(13).     

A conserved protein interaction interface on the type 5 G protein beta subunit controls proteolytic stability and activity of R7 family regulator of G protein signaling proteins

Regulators of G protein signaling (RGS) proteins of the R7 subfamily limit signaling by neurotransmitters in the brain and by light in the retina. They form obligate complexes with the Gβ5 protein that are subject to proteolysis to control their abundance and alter signaling. The mechanisms that regulate this proteolysis, however, remain unclear. We used genetic screens to find mutations in Gβ5 that selectively destabilize one of the R7 RGS proteins in Caenorhabditis elegans. These mutations cluster at the binding interface between Gβ5 and the N terminus of R7 RGS proteins. Equivalent mutations within mammalian Gβ5 allowed the interface to still bind the N-terminal DEP domain of R7 RGS proteins, and mutant Gβ5-R7 RGS complexes initially formed in cells but were then rapidly degraded by proteolysis. Molecular dynamics simulations suggest the mutations weaken the Gβ5-DEP interface, thus promoting dynamic opening of the complex to expose determinants of proteolysis known to exist on the DEP domain. We propose that conformational rearrangements at the Gβ5-DEP interface are key to controlling the stability of R7 RGS protein complexes.     

mda-9/Syntenin protein positively regulates the activation of Akt protein by facilitating integrin-linked kinase adaptor function during adhesion to type I collagen

The integrin-linked kinase (ILK)-PINCH1-α-parvin (IPP) complex functions as a signaling platform for integrins that modulates various cellular processes. ILK functions as a central adaptor for the assembly of IPP complex. We report here that mda-9/syntenin, a positive regulator of cancer metastasis, regulates the activation of Akt (also known as protein kinase B) by facilitating ILK adaptor function during adhesion to type I collagen (COL-I) in human breast cancer cells. COL-I stimulation induced the phosphorylation and plasma membrane translocation of Akt. Inhibition of mda-9/syntenin or expression of mutant ILK (E359K) significantly blocked the translocation of both ILK and Akt to the plasma membrane. mda-9/syntenin associated with ILK, and this association was increased at the plasma membrane by COL-I stimulation. Knockdown of mda-9/syntenin impaired COL-I-induced association of ILK with Akt and plasma membrane targeting of ILK-Akt complex. These results demonstrated that mda-9/syntenin regulates the activation of Akt by controlling the plasma membrane targeting of Akt via a mechanism that facilitates the association of Akt with ILK at the plasma membrane during adhesion to COL-I. On a striking note, inhibition of mda-9/syntenin impaired COL-I-induced plasma membrane translocation of the IPP complex and assembly of integrin β1-IPP signaling complexes. Thus, our study defines the role of mda-9/syntenin in ILK adaptor function and describes a new mechanism of mda-9/syntenin for regulation of cell migration.     

The epidermal growth factor receptor mediates tumor necrosis factor-alpha-induced activation of the ERK/GEF-H1/RhoA pathway in tubular epithelium

Tumor necrosis factor (TNF)-α induces cytoskeleton and intercellular junction remodeling in tubular epithelial cells; the underlying mechanisms, however, are incompletely explored. We have previously shown that ERK-mediated stimulation of the RhoA GDP/GTP exchange factor GEF-H1/Lfc is critical for TNF-α-induced RhoA stimulation. Here we investigated the upstream mechanisms of ERK/GEF-H1 activation. Surprisingly, TNF-α-induced ERK and RhoA stimulation in tubular cells were prevented by epidermal growth factor receptor (EGFR) inhibition or silencing. TNF-α also enhanced phosphorylation of the EGFR. EGF treatment mimicked the effects of TNF-α, as it elicited potent, ERK-dependent GEF-H1 and RhoA activation. Moreover, EGF-induced RhoA activation was prevented by GEF-H1 silencing, indicating that GEF-H1 is a key downstream effector of the EGFR. The TNF-α-elicited EGFR, ERK, and RhoA stimulation were mediated by the TNF-α convertase enzyme (TACE) that can release EGFR ligands. Further, EGFR transactivation also required the tyrosine kinase Src, as Src inhibition prevented TNF-α-induced activation of the EGFR/ERK/GEF-H1/RhoA pathway. Importantly, a bromodeoxyuridine (BrdU) incorporation assay and electric cell substrate impedance-sensing (ECIS) measurements revealed that TNF-α stimulated cell growth in an EGFR-dependent manner. In contrast, TNF-α-induced NFκB activation was not prevented by EGFR or Src inhibition, suggesting that TNF-α exerts both EGFR-dependent and -independent effects. In summary, in the present study we show that the TNF-α-induced activation of the ERK/GEF-H1/RhoA pathway in tubular cells is mediated through Src- and TACE-dependent EGFR activation. Such a mechanism could couple inflammatory and proliferative stimuli and, thus, may play a key role in the regulation of wound healing and fibrogenesis.     

Dopamine D1-histamine H3 receptor heteromers provide a selective link to MAPK signaling in GABAergic neurons of the direct striatal pathway

Previously, using artificial cell systems, we identified receptor heteromers between the dopamine D(1) or D(2) receptors and the histamine H(3) receptor. In addition, we demonstrated two biochemical characteristics of the dopamine D(1) receptor-histamine H(3) receptor heteromer. We have now extended this work to show the dopamine D(1) receptor-histamine H(3) receptor heteromer exists in the brain and serves to provide a novel link between the MAPK pathway and the GABAergic neurons in the direct striatal efferent pathway. Using the biochemical characteristics identified previously, we found that the ability of H(3) receptor activation to stimulate p44 and p42 extracellular signal-regulated MAPK (ERK 1/2) phosphorylation was only observed in striatal slices of mice expressing D(1) receptors but not in D(1) receptor-deficient mice. On the other hand, the ability of both D(1) and H(3) receptor antagonists to block MAPK activation induced by either D(1) or H(3) receptor agonists was also found in striatal slices. Taken together, these data indicate the occurrence of D(1)-H(3) receptor complexes in the striatum and, more importantly, that H(3) receptor agonist-induced ERK 1/2 phosphorylation in striatal slices is mediated by D(1)-H(3) receptor heteromers. Moreover, H(3) receptor-mediated phospho-ERK 1/2 labeling co-distributed with D(1) receptor-containing but not with D(2) receptor-containing striatal neurons. These results indicate that D(1)-H(3) receptor heteromers work as processors integrating dopamine- and histamine-related signals involved in controlling the function of striatal neurons of the direct striatal pathway.