RAP1-RAC1 Signaling Has an Important Role in Adhesion and Migration in HNSCC.
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ABSTRACT:
SUBMITTER: Liu M
PROVIDER: S-EPMC7346744 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ObjectiveThe goal of this study was to investigate the potential crosstalk between Rap1 and Rac1, 2 small GTPases central to platelet activation, particularly downstream of the collagen receptor GPVI.Methods and resultsWe compared the activation response of platelets with impaired Rap signaling (double knock-out; deficient in both the guanine nucleotide exchange factor, CalDAG-GEFI, and the Gi-coupled receptor for ADP, P2Y12), to that of wild-type platelets treated with a small-molecule Rac inhibitor, EHT 1864 (wild-type /EHT). We found that Rac1 is sequentially activated downstream of Rap1 on stimulation via GPVI. In return, Rac1 provides important feedback for both CalDAG-GEFI- and P2Y12-dependent activation of Rap1. When analyzing platelet responses controlled by Rac1, we observed (1) impaired lamellipodia formation, clot retraction, and granule release in both double knock-out and EHT 1864-treated wild-type platelets; and (2) reduced calcium store release in EHT 1864-treated wild-type but not double knock-out platelets. Consistent with the latter finding, we identified 2 pools of Rac1, one activated immediately downstream of GPVI and 1 activated downstream of Rap1.ConclusionsWe demonstrate important crosstalk between Rap1 and Rac1 downstream of GPVI. Whereas Rap1 signaling directly controls sustained Rac1 activation, Rac1 affects CalDAG-GEFI- and P2Y12-dependent Rap1 activation via its role in calcium mobilization and granule/ADP release, respectively.
| S-EPMC3262085 | biostudies-literature
Project description:After injury, residual epithelial cells coordinate contextual clues from cell-cell and cell-matrix interactions to polarize and migrate over the wound bed. Protrusion formation, cell body translocation and rear retraction is a repetitive process that allows the cell to move across the substratum. Fundamental to this process is the assembly and disassembly of focal adhesions that facilitate cell adhesion and protrusion formation. Here, we identified syndecan-1 as a regulator of focal adhesion disassembly in migrating lung epithelial cells. Syndecan-1 altered the dynamic exchange of adhesion complex proteins, which in turn regulates migration speed. Moreover, we provide evidence that syndecan-1 controls this entire process through Rap1. Thus, syndecan-1 restrains migration in lung epithelium by activating Rap1 to slow focal adhesion disassembly.
| S-EPMC3533394 | biostudies-literature
Project description:Rap1 is a potent inside-out signal that increases LFA-1 adhesive activity. In this study, we have defined the cytoplasmic region of the alphaL and beta2 integrin that are required for Rap1-stimulated adhesion and subsequent migration on ICAM-1. Human LFA-1 bearing truncated and point-mutated alphaL and beta2 cytoplasmic regions were reconstituted in mouse IL-3-dependent proB cells, BAF/3. Truncation of the alphaL, but not beta2 subunit cytoplasmic region, abolished Rap1V12-dependent adhesion to ICAM-1. The alanine substitution of two lysine residues (K1097/K1099) in the alphaL subunit was found to be critical in adhesion induced by Rap1V12, but not PMA. This mutation suppressed Rap1V12-induced LFA-1 conformation changes and ligand-binding affinity. The K1097/K1099 mutation also impaired binding to ICAM-1 induced by TCR cross-linking or SDF-1. In contrast, the alanine substitution for tyrosine in the beta2 subunit endocytosis motif inhibited internalization of LFA-1, and severely impaired detachment at the cell rear, which resulted in long-elongated cell shapes. This result demonstrates that internalization of LFA-1 is a critical step in the deadhesion process. Our study revealed novel requirements of amino acid residues of the LFA-1 cytoplasmic region in the response to the inside-out signaling and the subsequent deadhesion process.
| S-EPMC194904 | biostudies-literature
Project description:INTRODUCTION:The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. METHODS:MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and ?1-integrin, we examined activation of the ?1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and ?1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. RESULTS:JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the ?1-integrin substrate fibronectin. This was accompanied by reduced protein expression of ?1-integrin and its binding partners ?V- and ?5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and ?1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF-6 and the Rap1 activator PDZ-GEF2 in MCF7 cells and in primary cultures from breast cancer patients. CONCLUSIONS:Our findings provide compelling evidence of a novel role for JAM-A in driving breast cancer cell migration via activation of Rap1 GTPase and ?1-integrin. We speculate that JAM-A over-expression in some breast cancer patients may represent a novel therapeutic target to reduce the likelihood of metastasis.
| S-EPMC3219194 | biostudies-literature
Project description:BackgroundCell migration requires coordinated formation of focal adhesions (FAs) and assembly and contraction of the actin cytoskeleton. Nonmuscle myosin II (MII) is a critical mediator of contractility and FA dynamics in cell migration. Signaling downstream of the small GTPase Rac1 also regulates FA and actin dynamics, but its role in regulation of MII during migration is less clear.ResultsWe found that Rac1 promotes association of MIIA with FA. Live-cell imaging showed that, whereas most MIIA at the leading edge assembled into dorsal contractile arcs, a substantial subset assembled in or was captured within maturing FA, and this behavior was promoted by active Rac1. Protein kinase C (PKC) activation was necessary and sufficient for integrin- and Rac1-dependent phosphorylation of MIIA heavy chain (HC) on serine1916 (S1916) and recruitment to FA. S1916 phosphorylation of MIIA HC and localization in FA was enhanced during cell spreading and ECM stiffness mechanosensing, suggesting upregulation of this pathway during physiological Rac1 activation. Phosphomimic and nonphosphorylatable MIIA HC mutants demonstrated that S1916 phosphorylation was necessary and sufficient for the capture and assembly of MIIA minifilaments in FA. S1916 phosphorylation was also sufficient to promote the rapid assembly of FAs to enhance cell migration and for the modulation of traction force, spreading, and migration by ECM stiffness.ConclusionsOur study reveals for the first time that Rac1 and integrin activation regulates MIIA HC phosphorylation through a PKC-dependent mechanism that promotes MIIA association with FAs and acts as a critical modulator of cell migration and mechanosensing.
| S-EPMC4302036 | biostudies-literature
Project description:CD38 is a transmembrane exoenzyme that is associated with poor prognosis in chronic lymphocytic leukemia (CLL). High CD38 levels in CLL cells are linked to increased cell migration, but the molecular basis is unknown. CD38 produces nicotinic acid adenine dinucleotide phosphate and adenosine 5'-diphosphate-ribose, both of which can act to increase intracellular Ca2+ levels. Here we show that CD38 expression increases basal intracellular Ca2+ levels and stimulates CLL cell migration both with and without chemokine stimulation. We find that CD38 acts via intracellular Ca2+ to increase the activity of the Ras family GTPase Rap1, which is in turn regulated by the Ca2+-sensitive Rap1 guanine-nucleotide exchange factor RasGRP2. Both Rap1 and RasGRP2 are required for CLL cell migration, and RasGRP2 is polarized in primary CLL cells with high CD38 levels. These results indicate that CD38 promotes RasGRP2/Rap1-mediated CLL cell adhesion and migration by increasing intracellular Ca2+ levels.
| S-EPMC6039665 | biostudies-literature
Project description:Cell migration is critical for several physiological and pathophysiological processes. It depends on the coordinated action of kinases, phosphatases, Rho-GTPases proteins, and Ca2+ signaling. Interestingly, ubiquitination events have emerged as regulatory elements of migration. Thus, the role of proteins involved in ubiquitination processes could be relevant to a complete understanding of pro-migratory mechanisms. KCTD5 is a member of Potassium Channel Tetramerization Domain (KCTD) proteins that have been proposed as a putative adaptor for Cullin3-E3 ubiquitin ligase and a novel regulatory protein of TRPM4 channels. Here, we study whether KCTD5 participates in cell migration-associated mechanisms, such as focal adhesion dynamics and cellular spreading. Our results show that KCTD5 CRISPR/Cas9- and shRNA-based depletion in B16-F10 cells promoted an increase in cell migration and cell spreading, and a decrease in the focal adhesion area, consistent with an increased focal adhesion disassembly rate. The expression of a dominant-negative mutant of Rho-GTPases Rac1 precluded the KCTD5 depletion-induced increase in cell spreading. Additionally, KCTD5 silencing decreased the serum-induced Ca2+ response, and the reversion of this with ionomycin abolished the KCTD5 knockdown-induced decrease in focal adhesion size. Together, these data suggest that KCTD5 acts as a regulator of cell migration by modulating cell spreading and focal adhesion dynamics through Rac1 activity and Ca2+ signaling, respectively.
| S-EPMC7600296 | biostudies-literature
Project description:The small GTPase Rap1 affects cell adhesion and cell motility in numerous developmental contexts. Loss of Rap1 in the Drosophila wing epithelium disrupts adherens junction localization, causing mutant cells to disperse, and dramatically alters epithelial cell shape. While the adhesive consequences of Rap1 inactivation have been well described in this system, the effects on cell signaling, cell fate specification, and tissue differentiation are not known. Here we demonstrate that Egfr-dependent cell types are lost from Rap1 mutant tissue as an indirect consequence of DE-cadherin mislocalization. Cells lacking Rap1 in the developing wing and eye are capable of responding to an Egfr signal, indicating that Rap1 is not required for Egfr/Ras/MAPK signal transduction. Instead, Rap1 regulates adhesive contacts necessary for maintenance of Egfr signaling between cells, and differentiation of wing veins and photoreceptors. Rap1 is also necessary for planar cell polarity in these tissues. Wing hair alignment and ommatidial rotation, functional readouts of planar cell polarity in the wing and eye respectively, are both affected in Rap1 mutant tissue. Finally, we show that Rap1 acts through the effector Canoe to regulate these developmental processes.
| S-EPMC2730837 | biostudies-literature
Project description:The hemidesmosomal transmembrane component collagen XVII (ColXVII) plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine Col17a1⁻/⁻ keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. Col17a1⁻/⁻ keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits β4 and β1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K) activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the β4 integrin subunit and the focal adhesion kinase (FAK) as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.
| S-EPMC3914815 | biostudies-literature
Project description:T-lymphocyte adhesion plays a critical role in both inflammatory and autoimmune responses. The small GTPase Rap1 is the key coordinator mediating T-cell adhesion to endothelial cells, antigen-presenting cells, and virus-infected cells. We describe a signaling pathway, downstream of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor, leading to Rap1-mediated adhesion. We identified a role for the Rap1 guanine nucleotide exchange factor C3G in the regulation of T-cell adhesion and showed that this factor is required for both T-cell receptor (TCR)-mediated and CTLA-4-mediated T-cell adhesion. Our data indicated that C3G translocates to the plasma membrane downstream of TCR signaling, where it regulates activation of Rap1. We also showed that CTLA-4 receptor signaling mediates tyrosine phosphorylation in the C3G protein, and that this is required for augmented activation of Rap1 and increased adhesion mediated by leukocyte function-associated antigen type 1 (LFA-1). Zap70 is required for C3G translocation to the plasma membrane, whereas the Src family member Hck facilitates C3G phosphorylation. These findings point to C3G and Hck as promising potential therapeutic targets for the treatment of T-cell-dependent autoimmune disorders.
| S-EPMC3958029 | biostudies-literature