Caveolin-1 in cell polarization and directional migration

Migration is a complex process in which cells move in a given direction either in response to changes in the extracellular environment or as a consequence of an intrinsic propensity for directional movement. Migration plays key roles in many physiological and pathological processes, including development, angiogenesis, tissue regeneration and metastasis. An important role in migration is played by caveolin-1 and caveolae. Caveolae compartmentalize intracellular signalling pathways to orchestrate cell migration. Caveolin-1 presents a polarized distribution in migrating cells and is linked to the cytoskeleton, and changes in its expression modulate migration. Although there are some discrepancies regarding the regulatory effect of caveolin-1, most studies show that it promotes cell movement and polarity. The importance of caveolin-1 has recently been reinforced by studies with Cav1(-/-) cells, which indicate that it establishes polarity during directional migration by coordinating Src kinase and Rho GTPase signalling.     

Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity.

BACKGROUND: The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. RESULTS: We show here that individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. CONCLUSIONS: Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites.     

The Rho GDI Rdi1 regulates Rho GTPases by distinct mechanisms

The small guanosine triphosphate (GTP)-binding proteins of the Rho family are implicated in various cell functions, including establishment and maintenance of cell polarity. Activity of Rho guanosine triphosphatases (GTPases) is not only regulated by guanine nucleotide exchange factors and GTPase-activating proteins but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast Saccharomyces cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1, and Rho4, and it regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase Cla4. After membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3beta homologue Ygk3, vacuolar proteases, and the proteasome. Together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases.     

Roles of Rho GTPases in leucocyte and leukaemia cell transendothelial migration

Leucocytes migrate into and out of blood vessels at multiple points during their development and maturation, and during immune surveillance. In response to tissue damage and infection, they are rapidly recruited through the endothelium lining blood vessels into the tissues. Leukaemia cells also move in and out of the bloodstream during leukaemia progression. Rho GTPases are intracellular signalling proteins that regulate cytoskeletal dynamics and are key coordinators of cell migration. Here, we describe how different members of the Rho GTPase family act in leucocytes and leukaemia cells to regulate steps of transendothelial migration. We discuss how inhibitors of Rho signalling could be used to reduce leucocyte or leukaemia cell entry into tissues.     

Inhibition of integrin-mediated cell adhesion but not directional cell migration requires catalytic activity of EphB3 receptor tyrosine kinase. Role of Rho family small GTPases

Genetic studies have shown that Eph receptor tyrosine kinases have both kinase-dependent and kinase-independent functions through incompletely understood mechanisms. We report here that ephrin-B1 stimulation of endogenous EphB kinases in LS174T colorectal epithelial cells inhibited integrin-mediated adhesion and HGF/SF-induced directional cell migration. Using 293 cells stably transfected with wild type (WT)- or kinase-deficient (KD-EphB3), we found that inhibition of integrin-mediated cell adhesion and induction of cell rounding was kinase-dependent. Unexpectedly, in two independent assays, both KD- and WT-EphB3 significantly inhibited directional cell migration. Upon ephrin-B1 stimulation, the activities of Rac1 and Cdc42 were reduced in both WT- and KD-EphB3-expressing cells that were induced to migrate. Pharmacological evidence demonstrates that a relative increase in RhoA signaling as a result of decreased Rac1/Cdc42 activities contributes to the inhibitory effects. Furthermore, EphB3-mediated inhibitory effect on cell adhesion but not migration was abolished by the integrin activating antibodies, suggesting that the inhibition of cell migration is not because of down-regulation of integrin function. These results uncover a differential requirement for EphB3 catalytic activity in the regulation of cell adhesion and migration, and suggest that while catalytic activity of EphB3 is required for inhibition of integrin-mediated cell adhesion, a distinct signaling pathway to Rho GTPases shared by WT- and KD-EphB3 receptor mediates inhibition of directional cell migration.     

RACK1 regulates Src activity and modulates paxillin dynamics during cell migration

Receptor for Activated C Kinase, RACK1, is an adaptor protein that regulates signaling via Src and PKC-dependent pathways, and has been implicated in cell migration. In this study we demonstrate novel functions for RACK1 in regulating adhesion dynamics during cell migration. We report that cells lacking RACK1 are less motile and show reduced dynamics of paxillin and talin at focal complexes. To investigate the role of the RACK1/Src interactions in adhesion dynamics, we used RACK1 in which the putative Src binding site has been mutated (RACK Y246F). RACK1-deficient cells showed enhanced c-Src activity that was rescued by expression of wild type RACK1, but not by RACK Y246F. Expression of wild type RACK1, but not RACK Y246F, was also able to rescue the adhesion and migration defects observed in the RACK1-deficient cells. Furthermore, our findings indicate that RACK1 functions to regulate paxillin phosphorylation and that its effects on paxillin dynamics require the Src-mediated phosphorylation of tyrosine 31/118 on paxillin. Taken together, these findings support a novel role for RACK1 as a key regulator of cell migration and adhesion dynamics through the regulation of Src activity, and the modulation of paxillin phosphorylation at early adhesions.     

Cadherin engagement regulates Rho family GTPases.

The formation of cell-cell adherens junctions is a cadherin-mediated process associated with reorganization of the actin cytoskeleton. Because Rho family GTPases regulate actin dynamics, we investigated whether cadherin-mediated adhesion regulates the activity of RhoA, Rac1, and Cdc42. Confluent epithelial cells were found to have elevated Rac1 and Cdc42 activity but decreased RhoA activity when compared with low density cultures. Using a calcium switch method to manipulate junction assembly, we found that induction of cell-cell junctions increased Rac1 activity, and this was inhibited by E-cadherin function-blocking antibodies. Using the same calcium switch procedure, we found little effect on RhoA activity during the first hour of junction assembly. However, over several hours, RhoA activity significantly decreased. To determine whether these effects are mediated directly through cadherins or indirectly through engagement of other surface proteins downstream from junction assembly, we used a model system in which cadherin engagement is induced without cell-cell contact. For these experiments, Chinese hamster ovary cells expressing C-cadherin were plated on the extracellular domain of C-cadherin immobilized on tissue culture plates. Whereas direct cadherin engagement did not stimulate Cdc42 activity, it strongly inhibited RhoA activity but increased Rac1 activity. Deletion of the C-cadherin cytoplasmic domain abolished these effects.     

Cell migration: Rho GTPases lead the way

Rho GTPases control signal transduction pathways that link cell surface receptors to a variety of intracellular responses. They are best known as regulators of the actin cytoskeleton, but in addition they control cell polarity, gene expression, microtubule dynamics and vesicular trafficking. Through these diverse functions, Rho GTPases influence many aspects of cell behavior. This review will focus specifically on their role in cell migration.     

The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading.

Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor-induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.     

RhoD regulates endosome dynamics through Diaphanous-related Formin and Src tyrosine kinase

Early endosomes move bidirectionally between the cell periphery and the interior through a mechanism regulated by the low molecular weight GTPase RhoD. Here, we identify a novel splice variant of human Diaphanous, hDia2C, which specifically binds to RhoD and is recruited onto early endosomes. Expression of RhoD and hDia2C induces a striking alignment of early endosomes along actin filaments and reduces their motility. This activity depends on the membrane recruitment and activation of c-Src kinase, thus uncovering a new role in endosome function. Our results define a novel signal transduction pathway, in which hDia2C and c-Src are sequentially activated by RhoD to regulate the motility of early endosomes through interactions with the actin cytoskeleton.     

Drosophila C-terminal Src kinase negatively regulates organ growth and cell proliferation through inhibition of the Src, Jun N-terminal kinase, and STAT pathways

Src family kinases regulate multiple cellular processes including proliferation and oncogenesis. C-terminal Src kinase (Csk) encodes a critical negative regulator of Src family kinases. We demonstrate that the Drosophila melanogaster Csk ortholog, dCsk, functions as a tumor suppressor: dCsk mutants display organ overgrowth and excess cellular proliferation. Genetic analysis indicates that the dCsk(-/-) overgrowth phenotype results from activation of Src, Jun kinase, and STAT signal transduction pathways. In particular, blockade of STAT function in dCsk mutants severely reduced Src-dependent overgrowth and activated apoptosis of mutant tissue. Our data provide in vivo evidence that Src activity requires JNK and STAT function.     

Rho GTPases in animal cell mitosis

The Rho GTPases have been thought to influence cell morphogenesis through remodeling of the actin cytoskeleton. Consistently, downstream targets such as the mDia family of formins and the WASP family proteins induce actin nucleation and polymerization, and another set of downstream effectors, the ROCK family protein kinases, are involved in regulation of actomyosin contractility. However, evidence has now accumulated that Rho GTPases also regulate local dynamics of microtubules. The mDia family proteins, for example, function downstream of Rho to stabilize and align microtubules in interphase cells. Concomitantly, the role of Rho GTPases in animal cell division, once thought to be limited to cytokinesis, has now been shown to extend to mitosis. Recent work indicates that they may function during both spindle orientation and chromosome congression. However, their involvement is cell-type-specific, raising arguments for and against a mitotic role for Rho GTPases.     

Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase

Fluid shear stress activates a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinase (ERK), by mechanisms dependent on cholesterol in the plasma membrane in bovine aortic endothelial cells (BAEC). Caveolae are microdomains of the plasma membrane that are enriched with cholesterol, caveolin, and signaling molecules. We hypothesized that caveolin-1 regulates shear activation of ERK. Because caveolin-1 is not exposed to the outside, cells were minimally permeabilized by Triton X-100 (0.01%) to deliver a neutralizing, polyclonal caveolin-1 antibody (pCav-1) inside the cells. pCav-1 then bound to caveolin-1 and inhibited shear activation of ERK but not c-Jun NH(2)-terminal kinase. Epitope mapping studies showed that pCav-1 binds to caveolin-1 at two regions (residues 1-21 and 61-101). When the recombinant proteins containing the epitopes fused to glutathione-S-transferase (GST-Cav(1-21) or GST-Cav(61-101)) were preincubated with pCav-1, only GST-Cav(61-101) reversed the inhibitory effect of the antibody on shear activation of ERK. Other antibodies, including m2234, which binds to caveolin-1 residues 1-21, had no effect on shear activation of ERK. Caveolin-1 residues 61-101 contain the scaffolding and oligomerization domains, suggesting that binding of pCav-1 to these regions likely disrupts the clustering of caveolin-1 or its interaction with signaling molecules involved in the shear-sensitive ERK pathway. We suggest that caveolae-like domains play a critical role in the mechanosensing and/or mechanosignal transduction of the ERK pathway.     

Protein-tyrosine phosphatase PTPD1 regulates focal adhesion kinase autophosphorylation and cell migration

PTPD1 is a cytosolic nonreceptor tyrosine phosphatase and a positive regulator of the Src-epidermal growth factor transduction pathway. We show that PTPD1 localizes along actin filaments and at adhesion plaques. PTPD1 forms a stable complex via distinct molecular modules with actin, Src tyrosine kinase, and focal adhesion kinase (FAK), a scaffold protein kinase enriched at adhesion plaques. Overexpression of PTPD1 promoted cell scattering and migration, short hairpin RNA-mediated silencing of endogenous PTPD1, or expression of PTPD1 mutants lacking either catalytic activity (PTPD1(C1108S)) or the FERM domain (PTPD1(Delta1-325)) significantly reduced cell motility. PTPD1 and Src catalytic activities were both required for epidermal growth factor-induced FAK autophosphorylation at its active site and for downstream propagation of ERK1/2 signaling. Our findings demonstrate that PTPD1 is a component of a multivalent scaffold complex nucleated by FAK at specific intracellular sites. By modulating Src-FAK signaling at adhesion sites, PTPD1 promotes the cytoskeleton events that induce cell adhesion and migration.     

Interaction of protein kinase C isozymes with Rho GTPases

Evidence is provided for direct protein-protein interactions between protein kinase C (PKC) alpha, betaI, betaII, gamma, delta, epsilon, and zeta and members of the Rho family of small GTPases. Previous investigations, based on the immunoprecipitation approach, have provided evidence consistent with a direct interaction, but this remained to be proven. In the study presented here, an in vitro assay, consisting only of purified proteins and the requisite PKC activators and cofactors, was used to determine the effects of Rho GTPases on the activities of the different PKC isoforms. It was found that the activity of PKCalpha was potently enhanced by RhoA and Cdc42 and to a lesser extent by Rac1, whereas the effects on the activities of PKCbetaI, -betaII, -gamma, -delta, -epsilon, and -zeta were much reduced. These results indicate a direct interaction between PKCalpha and each of the Rho GTPases. However, the Rho GTPase concentration dependencies for the potentiating effects on PKCalpha activity differed for each Rho GTPase and were in the following order: RhoA > Cdc42 > Rac1. PKCalpha was activated in a phorbol ester- and Ca(2+)-dependent manner. This was reflected by a substantial decrease in the phorbol ester concentration requirements for activity in the presence of Ca(2+), which for each Rho GTPase was induced within a low nanomolar phorbol ester concentration range. The activity of PKCalpha also was found to be dependent on the nature of the GTP- or GDP-bound state of the Rho GTPases, suggesting that the interaction may be regulated by conformational changes in both PKCalpha and Rho GTPases. Such an interaction could result in significant cross-talk between the distinct pathways regulated by these two signaling elements.     

Rho GTPases和细胞凋亡

细胞凋亡涉及细胞骨架的形态学改变,Rho GTPases在细胞骨架改变中起着至关重要的作用。近年来的研究揭示了Rho蛋白家族在肌动蛋白(actin)聚合、解聚及actin-myosin的分子调节机制。同时越来越多的研究表明,Rho GTPases在巨噬细胞吞噬凋亡小体中也发挥了关键作用。本综述就Rho GTPases信号途径在细胞凋亡中细胞骨架的结构改变及凋亡小体被吞噬过程中的作用进行具体讨论。     

Role of Rho kinase in sphingosine 1-phosphate-mediated endothelial and smooth muscle cell migration and differentiation

The role of sphingosine 1-phosphate (S1P)-induced Rho kinase (ROCK) activation in the angiogenic responses of pulmonary artery-derived endothelial cells (PAEC) and smooth muscle cells (PASMC) was examined. S1P, a biologically active phospholipid that regulates angiogenesis, promoted PAEC chemotaxis and capillary morphogenesis; furthermore, this activity was unaltered by pretreatment with the pharmacological inhibitor of ROCK, H1152. In contrast, S1P (500 nM) significantly inhibited spontaneous PASMC chemotaxis and differentiation; however, this inhibition was eradicated upon H1152 pretreatment. Similarly, PASMCs transfected with ROCK II siRNA diminished S1P-induced inhibition of the development of multi-cellular structures. Analysis by RT-PCR identified the presence of S1P₁ and S1P₃ receptors on both PAECs and PASMCs, while S1P₂ receptor expression was confined to only PASMCs. Consistent with this observation, the S1P₁ and S1P₃ receptor antagonist, VPC23019, virtually abolished the S1P-initiated PAEC differentiation but did not impede the S1P-induced inhibition of PASMC differentiation. However, the S1P₂ receptor antagonist, JTE013, had no effect on S1P-mediated differentiation of PAECs but abolished the S1P-induced inhibition of PASMC function. Co-cultured endothelial and smooth muscle cells differentiated into “neovascular-like” networks, which were significantly inhibited by S1P. The inhibition of co-culture differentiation in both PAECs and PASMCs was negated by H1152 pretreatment. However, when smooth muscle cells were added to S1P-initiated endothelial cell networks, additional S1P treatment did not inhibit the cellular networks generated by these cells. In conclusion, S1P-induced PAEC angiogenic responses are regulated by S1P₁ and/or S1P₃ receptors independent of Rho kinase activation, whereas S1P₂ receptor-mediated curtailment of PASMC function by S1P.