Focal Adhesion Proteins p130Cas Research Articles

Focal adhesion-derived liquid-liquid phase separations regulate mRNA translation.

Liquid-liquid phase separation (LLPS) has emerged as a major organizing principle in cells. Recent work showed that multiple components of integrin-mediated focal adhesions including p130Cas can form LLPS, which govern adhesion dynamics and related cell behaviors. In this study, we found that the focal adhesion protein p130Cas drives formation of structures with the characteristics of LLPS that bud from focal adhesions into the cytoplasm. Condensing concentrated cytoplasm around p130Cas-coated beads allowed their isolation, which were enriched in a subset of focal adhesion proteins, mRNAs and RNA binding proteins, including those implicated in inhibiting mRNA translation. Plating cells on very high concentrations of fibronectin to induce large focal adhesions inhibited message translation which required p130Cas and correlated with droplet formation. Photo-induction of p130Cas condensates using the Cry2 system also reduced translation. These results identify a novel regulatory mechanism in which high adhesion limits message translation via induction of p130Cas-dependent cytoplasmic LLPS. This mechanism may contribute to the quiescent state of very strongly adhesive myofibroblasts and senescent cells.

PTPN12/PTP-PEST Regulates Phosphorylation-Dependent Ubiquitination and Stability of Focal Adhesion Substrates in Invasive Glioblastoma Cells.

Glioblastoma (GBM) is an invasive brain cancer with tumor cells that disperse from the primary mass, escaping surgical resection and invariably giving rise to lethal recurrent lesions. Here we report that PTP-PEST, a cytoplasmic protein tyrosine phosphatase, controls GBM cell invasion by physically bridging the focal adhesion protein Crk-associated substrate (Cas) to valosin-containing protein (Vcp), an ATP-dependent protein segregase that selectively extracts ubiquitinated proteins from multiprotein complexes and targets them for degradation via the ubiquitin proteasome system. Both Cas and Vcp are substrates for PTP-PEST, with the phosphorylation status of tyrosine 805 (Y805) in Vcp impacting affinity for Cas in focal adhesions and controlling ubiquitination levels and protein stability. Perturbing PTP-PEST-mediated phosphorylation of Cas and Vcp led to alterations in GBM cell-invasive growth in vitro and in preclinical mouse models. Collectively, these data reveal a novel regulatory mechanism involving PTP-PEST, Vcp, and Cas that dynamically balances phosphorylation-dependent ubiquitination of key focal proteins involved in GBM cell invasion.Significance: PTP-PEST balances GBM cell growth and invasion by interacting with the ATP-dependent ubiquitin segregase Vcp/p97 and regulating phosphorylation and stability of the focal adhesion protein p130Cas.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3809/F1.large.jpg Cancer Res; 78(14); 3809-22. ©2018 AACR.

Bioengineering and mechanobiology: pushing (and pulling) the limits of cellular mechanics

The Minisymposium on Bioengineering and Mechanobiology focused on engineering approaches to investigate the mechanical signaling of cells and tissues and was cochaired by Adam J. Engler (University of California, San Diego) and Celeste M. Nelson (Princeton University). The program moved thematically from the mechanics of the nucleus to those of the cell cortex and finally to the mechanical behaviors of tissues. Jan Lammerding (Cornell University) described work probing the role of the nuclear envelope proteins, lamin A and C, in the structure and mechanical properties of the nuclei of individual cells and whole tissues. Fibroblasts isolated from patients harboring mutations in the LMNA gene that cause muscular dystrophies had softer nuclei than controls. Expressing these mutated forms of the LMNA gene in otherwise lamin A/C–deficient mouse embryonic fibroblasts also yielded softer nuclei and defects in coupling of the nucleus to the cytoskeleton. Importantly, applying strain to the body wall musculature of genetically modified Drosophila melanogaster showed that lamin mutations altered nuclear mechanics within intact muscle tissues. Ben Fabry (University of Erlangen–Nuremberg) demonstrated that the binding between the focal adhesion proteins p130Cas and vinculin is important for mechanotransduction. Mechanical stress is coupled to p130Cas through vinculin and initiates the activation of downstream signaling through pathways such as the extracellular signal–regulated kinase (ERK1/2) pathway. Phosphorylation of p130Cas on residue Y12 or mutation of this residue to a phosphomimicking glutamate prevents vinculin binding, reduces the localization of p130Cas to focal adhesion sites, and suppresses ERK1/2 signaling in response to mechanical stretch. Yee Seir Kee (Robinson and Iglesias Laboratories, Johns Hopkins University School of Medicine) used micropipette aspiration and compression approaches to investigate how assembly of the cleavage furrow responds to mechanical stress in Dictyostelium. During cytokinesis, cell shape is controlled by a mechanosensory system that includes the myosin II motor protein and the actin cross-linker, cortexillin I. Accumulation of myosin II at the cleavage furrow is governed by a regulatory network composed of IQGAP1, IQGAP2, kinesin-6, and INCENP. Feedback loops present within this network are responsible for regulating the levels of myosin II at the cleavage furrow. Celeste Nelson (Princeton University) presented results suggesting that cells respond to the mechanical properties of their surrounding microenvironment, in part by regulating the subcellular localization of small GTPases and the assembly and activation of NADPH oxidase. Mammary epithelial cells underwent epithelial–mesenchymal transition (EMT) on stiff, but not soft, substrata. Cells on stiff substrata apportioned Rac1b, a splice variant of Rac1, to the plasma membrane, at which it formed an activated complex with NADPH oxidase and permitted the generation of reactive oxygen species. The complex did not form on soft substrata, and blocking membrane localization of Rac1b prevented EMT on stiffer microenvironments. Dylan Tyler Burnette (Lippincott–Schwartz Laboratory, National Institutes of Health) was the recipient of the Merton Bernfield Memorial Award, which is given every year to one graduate student or postdoctoral fellow in recognition of his or her meritorious achievements in cell biology research. Burnette discussed his efforts in expanding the imaging toolbox to the nanometer scale using conventional fluorophores, in an approach dubbed bleaching/blinking-assisted localization microscopy (BaLM; Burnette et al., 2011 ). Point localization–based superresolution images with a resolution on the scale of tens of nanometers can be produced from commonly used fluorescent molecules by taking advantage of their intrinsic bleaching and blinking. Four-color images can be generated via BaLM with standard electron-multiplying charge-coupled device cameras by using multiple fluorophores in a single cell and can resolve individual motor proteins along intact cytoskeletal networks. Adam Engler (University of California, San Diego) reported on the differences in differentiation response between adipose-derived and bone marrow–derived stem cells. Both types of stem cells are sensitive to the stiffness of their surrounding microenvironment and differentiate down a muscle lineage when cultured on extracellular matrix of a specific compliance mimicking that of muscle in vivo. The adipose-derived stem cells (ASCs), however, express markers of muscle differentiation at 10-fold higher levels than do the bone marrow–derived stem cells. Furthermore, a fraction of ASCs cultured under these conditions form multinucleated myotubes, which could be maintained when replated on non­permissive substrata.

Insights into the Importance of Mechanosensing through the Focal Adhesion Protein p130Cas

The Src substrate p130Cas has previously been identified as one of several focal adhesion proteins implicated in mechanosensing, the process by which cells feel changes in their physical environment. As a protein that has also been implicated in enhanced cell migration, actin dynamics, and focal adhesion turnover, the manner by which p130Cas orchestrates such sensing is becoming of increasing interest. In vitro studies have suggested that the central substrate domain may relay mechanosensing information via spring-like stretching of this domain. However, in vivo detection of such a mechanism has not been possible to date. To gain greater insight into this problem, we used polyacrylamide substrates of varying rigidities and observed the abilities of mouse embryonic fibroblasts (MEFs) to adhere, spread, and form focal adhesions by spinning disk confocal microscopy. By comparing cells lacking and stably re-expressing p130Cas, we addressed the following questions: 1) How sensitive is p130Cas to these mechanical changes (i.e. at what rigidity does p130Cas first become tyrosine phosphorylated)? 2) Is p130Cas critical to the ability of the cell to sense these changes (analyzed by tracking changes in focal adhesion characteristics as well as cell spread)? and 3) What is the mechanism underlying the ability of p130Cas to transduce mechanical signals (i.e. is there a spring-like domain within p130Cas)? Our multi-parameter experimental strategy allowed us to analyze increases in cell spread and focal adhesion organization as substrate rigidity was altered from ∼100 Pa to 69 GPa. Interestingly, we observed that MEFs deficient for p130Cas displayed changes in cell spread and focal adhesion rearrangement at lower rigidity scales than was observed in wild-type counterparts, illustrating a need for p130Cas to sensitize cells to substrate rigidity.

The docking protein p130Cas regulates cell sensitivity to proteasome inhibition

BackgroundThe focal adhesion protein p130Cas (Cas) activates multiple intracellular signaling pathways upon integrin or growth factor receptor ligation. Full-length Cas frequently promotes cell survival and migration, while its C-terminal fragment (Cas-CT) produced upon intracellular proteolysis is known to induce apoptosis in some circumstances. Here, we have studied the putative role of Cas in regulating cell survival and death pathways upon proteasome inhibition.ResultsWe found that Cas-/- mouse embryonic fibroblasts (MEFs), as well as empty vector-transfected Cas-/- MEFs (Cas-/- (EV)) are significantly resistant to cell death induced by proteasome inhibitors, such as MG132 and Bortezomib. As expected, wild-type MEFs (WT) and Cas-/- MEFs reconstituted with full-length Cas (Cas-FL) were sensitive to MG132- and Bortezomib-induced apoptosis that involved activation of a caspase-cascade, including Caspase-8. Cas-CT generation was not required for MG132-induced cell death, since expression of cleavage-resistant Cas mutants effectively increased sensitivity of Cas-/- MEFs to MG132. At the present time, the domains in Cas and the downstream pathways that are required for mediating cell death induced by proteasome inhibitors remain unknown. Interestingly, however, MG132 or Bortezomib treatment resulted in activation of autophagy in cells that lacked Cas, but not in cells that expressed Cas. Furthermore, autophagy was found to play a protective role in Cas-deficient cells, as inhibition of autophagy either by chemical or genetic means enhanced MG132-induced apoptosis in Cas-/- (EV) cells, but not in Cas-FL cells. Lack of Cas also contributed to resistance to the DNA-damaging agent Doxorubicin, which coincided with Doxorubicin-induced autophagy in Cas-/- (EV) cells. Thus, Cas may have a regulatory role in cell death signaling in response to multiple different stimuli. The mechanisms by which Cas inhibits induction of autophagy and affects cell death pathways are currently being investigated.ConclusionOur study demonstrates that Cas is required for apoptosis that is induced by proteasome inhibition, and potentially by other death stimuli. We additionally show that Cas may promote such apoptosis, at least partially, by inhibiting autophagy. This is the first demonstration of Cas being involved in the regulation of autophagy, adding to the previous findings by others linking focal adhesion components to the process of autophagy.

Pressure activates colon cancer cell adhesion via paxillin phosphorylation, Crk, Cas, and Rac1.

Physical forces can activate colon cancer cell adhesion, critical for metastasis. Paxillin is phosphorylated by FAK and required for pressure-stimulated adhesion. However, whether paxillin acts as an inert scaffolding protein or whether paxillin phosphorylation is required is unknown. Transfection with paxillin point-phosphorylation mutants demonstrated that phosphorylation at tyrosines 31 and 118 together is necessary for pressure-stimulated adhesion. We further evaluated potential paxillin partners. Reducing the adaptor protein Crk or the focal adhesion protein p130Cas blocked pressure-stimulated adhesion. Furthermore, Crk and p130Cas both displayed increased co-immunoprecipitation with paxillin in response to increased pressure, except in cells transfected with a Y31Y118 paxillin mutant. Inhibiting the small GTPase Rac1 also abolished pressure-stimulated adhesion, and reducing paxillin by siRNA blocked Rac1 phosphorylation by pressure. Thus, paxillin phosphorylation at tyrosines 31 and 118 together is necessary for pressure-induced adhesion. Paxillin, Crk and Cas form a trimeric complex that activates Rac1 and mediates this effect.

AND-34 Activates Phosphatidylinositol 3-Kinase and Induces Anti-Estrogen Resistance in a SH2 and GDP Exchange Factor–Like Domain-Dependent Manner

AND-34, a 95-kDa protein with modest homology to Ras GDP exchange factors, associates with the focal adhesion protein p130Cas. Overexpression of AND-34 confers anti-estrogen resistance in breast cancer cell lines, a property linked to its ability to activate Rac. Here, we show that both the GDP exchange factor-like domain and the SH2 domain of AND-34 are required for Rac activation and for resistance to the estrogen receptor (ER) antagonist ICI 182,780. As phosphatidylinositol 3-kinase (PI3K) signaling can regulate Rac activation, we examined the effects of AND-34 on PI3K. Overexpression of AND-34 in MCF-7 cells increased PI3K activity and augmented Akt Ser(473) phosphorylation and kinase activity. Inhibition of PI3K with LY294002 or a dominant-negative p85 construct blocked AND-34-mediated Rac and Akt activation. Although R-Ras can activate PI3K, transfection with constitutively active R-Ras failed to induce Rac activation and AND-34 overexpression failed to induce R-Ras activation. Treatment of either vector-only or AND-34-transfected ZR-75-1 cells with ICI 182,780 markedly diminished ERalpha levels, suggesting that AND-34-induced anti-estrogen resistance is likely to occur by an ERalpha-independent mechanism. Treatment of a ZR-75-1 breast cancer cell line stably transfected with AND-34 plus 2 micromol/L LY294002 or 10 micromol/L NSC23766, a Rac-specific inhibitor, abrogated AND-34-induced resistance to ICI 182,780. Our studies suggest that AND-34-mediated PI3K activation induces Rac activation and anti-estrogen resistance in human breast cancer cell lines.

PTP-PEST controls motility through regulation of Rac1.

The cytoplasmic protein tyrosine phosphatase, PTP-PEST, associates with the focal adhesion proteins p130cas and paxillin and has recently been implicated in cell migration. In this study, we investigated the mechanism by which PTP-PEST regulates this phenomenon. We find that PTP-PEST is activated in an adhesion-dependent manner and localizes to the tips of membrane protrusions in spreading fibroblasts. We show that the catalytic activity of PTP-PEST is a key determinant for its effects on motility. Overexpression of PTP-PEST, but not a catalytically inactive form, impairs haptotaxis, cell spreading and formation of membrane protrusions in CHOK1 cells. In addition, overexpression of PTP-PEST in Rat1 fibroblasts perturbs membrane ruffling and motility in response to PDGF stimulation. The expression level of PTP-PEST modulates the activity of the small GTPase, Rac1. PTP-PEST overexpression suppresses activation of Rac1 in response to both integrin-mediated adhesion or growth factor stimulation. In contrast, fibroblasts that lack PTP-PEST expression show enhanced Rac1 activity. Co-expression of constitutively active Rac1 with PTP-PEST overcomes the inhibition of cell spreading and migration indicating that PTP-PEST acts by antagonizing Rac1 activation. Our data suggest a model in which PTP-PEST is activated by integrins and localized to regions where it can control motile events at the leading edge through inhibition of the small GTPase Rac1.

Binding of 14-3-3beta regulates the kinase activity and subcellular localization of testicular protein kinase 1.

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase that phosphorylates cofilin and induces actin cytoskeletal reorganization. The kinase activity of TESK1 is stimulated by integrin-mediated signaling pathways, but the mechanism of regulation has remained unknown. By using the yeast two-hybrid system, we identified 14-3-3beta to be the binding protein of TESK1. Specific interaction between TESK1 and 14-3-3beta became evident in in vitro and in vivo co-precipitation assays. 14-3-3beta interacts with TESK1 through the C-terminal region of TESK1 and in a manner dependent on the phosphorylation of Ser-439 within an RXXSXP motif. Binding of 14-3-3beta inhibited the kinase activity of TESK1. During cell spreading on fibronectin, the TESK1/14-3-3beta interaction significantly decreased, in a time course that inversely correlated with increase in TESK1 kinase activity. Thus, the dissociation of 14-3-3beta from a TESK1/14-3-3beta complex is likely to be involved in the integrin-mediated TESK1 activation. In HeLa cells, TESK1, together with 14-3-3beta, accumulated at the cell periphery when cells were plated on fibronectin, whereas they were diffusely distributed in the cytoplasm in the case of non-stimulated cells. We propose that 14-3-3beta plays important roles in regulating the kinase activity of TESK1 and localizing TESK1 to cell adhesion sites following integrin stimulation.

P130cas is a cellular target protein for tyrosine nitration induced by peroxynitrite.

We found that the exposure of human neuroblastoma SH-SY5Y cells to the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) induced tyrosine nitration of a 130-kDa protein, and prevented tyrosine phosphorylation of the 130-kDa protein. The focal adhesion protein p130cas was identified as a component of the 130-kDa protein using specific antibody. These results suggest that p130cas is a new target protein for nitration induced by SIN-1.

Identification of p130Cas as a substrate of Yersinia YopH (Yop51), a bacterial protein tyrosine phosphatase that translocates into mammalian cells and targets focal adhesions.

A number of pathogenic bacteria utilize type III secretion pathways to translocate virulence proteins into host eukaryotic cells. We identified a host target of YopH, a protein tyrosine phosphatase that is translocated into mammalian cells by Yersiniae. A catalytically inactive 'substrate-trapping' mutant, YopHC403S, was used as a probe to determine where YopH substrates localize in eukaryotic cells. Immunofluorescence microscopy demonstrated that YopHC403S localized to focal adhesions in human epithelial cells infected with Y. pseudotuberculosis. YopHC403S stabilized focal adhesions, as shown by its dominant-negative effect on focal adhesion disassembly mediated by YopE, a translocated protein which disrupts actin stress fibers. Conversely, YopH destabilized focal adhesions, even in the absence of YopE, as shown by loss of phosphotyrosine staining. Immunoprecipitation revealed that YopHC403S was trapped in a complex with a hyperphosphorylated 125-135 kDa protein, identified by immunoblotting as the focal adhesion protein p130Cas. YopHC403S bound directly to p130Cas in a phosphotyrosine-dependent manner in vitro. Translocation of YopH into cells plated on fibronectin resulted in rapid and selective dephosphorylation of p130Cas. These results demonstrate that YopH targets focal adhesions in host cells and that p130Cas, a docking protein for multiple SH2 domains, is a direct substrate of this enzyme in vivo.

Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases.

Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. Among these are the focal adhesion proteins p130cas (Cas) and focal adhesion kinase (FAK). Here we identify the kinase(s) mediating integrin-induced Cas phosphorylation and characterize protein-protein interactions mediated by phosphorylated Cas. We found that expression of a constitutively active FAK in fibroblasts results in a consecutive tyrosine phosphorylation of Cas. This effect required the autophosphorylation site of FAK, which is a binding site for Src family kinases. Integrin-mediated phosphorylation of Cas was not, however, compromised in fibroblasts lacking FAK. In contrast, adhesion-induced tyrosine phosphorylation of Cas was reduced in cells lacking Src, whereas enhanced phosphorylation of Cas was observed Csk- cells, in which Src kinases are activated. These results suggest that Src kinases are responsible for the integrin-mediated tyrosine phosphorylation of Cas. FAK seems not to be necessary for phosphorylation of Cas, but when autophosphorylated, FAK may recruit Src family kinases to phosphorylate Cas. Cas was found to form complexes with Src homology 2 (SH2) domain-containing signaling molecules, such as the SH2/SH3 adapter protein Crk, following integrin-induced tyrosine phosphorylation. Guanine nucleotide exchange factors C3G and Sos were found in the Cas-Crk complex upon integrin ligand binding. These observations suggest that Cas serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion.