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1.
Cell Commun Signal ; 16(1): 73, 2018 11 03.
Article in English | MEDLINE | ID: mdl-30390666

ABSTRACT

BACKGROUND: p130 Crk-associated substrate (p130CAS; also known as BCAR1) is a scaffold protein that modulates many essential cellular processes such as cell adhesion, proliferation, survival, cell migration, and intracellular signaling. p130Cas has been shown to be highly expressed in a variety of human cancers of epithelial origin. However, few data are available regarding the role of p130Cas during normal epithelial development and homeostasis. METHODS: To this end, we have generated a genetically modified mouse in which p130Cas protein was specifically ablated in the epidermal tissue. RESULTS: By using this murine model, we show that p130Cas loss results in increased cell proliferation and reduction of cell adhesion to extracellular matrix. In addition, epidermal deletion of p130Cas protein leads to premature expression of "late" epidermal differentiation markers, altered membrane E-cadherin/catenin proteins localization and aberrant tyrosine phosphorylation of E-cadherin/catenin complexes. Interestingly, these alterations in adhesive properties in absence of p130Cas correlate with abnormalities in progenitor cells balance resulting in the amplification of a more committed cell population. CONCLUSION: Altogether, these results provide evidence that p130Cas is an important regulator of epidermal cell fate and homeostasis.


Subject(s)
Cell Adhesion , Cell Differentiation , Crk-Associated Substrate Protein/deficiency , Crk-Associated Substrate Protein/genetics , Epidermis/metabolism , Gene Deletion , Homeostasis/genetics , Animals , Cell Proliferation , Extracellular Matrix/metabolism , Keratinocytes/cytology , Mice , Mice, Inbred C57BL , Phenotype
2.
Immunol Cell Biol ; 94(10): 981-993, 2016 11.
Article in English | MEDLINE | ID: mdl-27359298

ABSTRACT

The immunological synapse formed between a T-cell and an antigen-presenting cell is important for cell-cell communication during T-cell-mediated immune responses. Immunological synapse formation begins with stimulation of the T-cell receptor (TCR). TCR microclusters are assembled and transported to the center of the immunological synapse in an actin polymerization-dependent process. However, the physical link between TCR and actin remains elusive. Here we show that lymphocyte-specific Crk-associated substrate (Cas-L), a member of a force sensing protein family, is required for transport of TCR microclusters and for establishing synapse stability. We found that Cas-L is phosphorylated at TCR microclusters in an actin polymerization-dependent fashion. Furthermore, Cas-L participates in a positive feedback loop leading to amplification of Ca2+ signaling, inside-out integrin activation, and actomyosin contraction. We propose a new role for Cas-L in T-cell activation as a mechanical transducer linking TCR microclusters to the underlying actin network and coordinating multiple actin-dependent structures in the immunological synapse. Our studies highlight the importance of mechanotransduction processes in T-cell-mediated immune responses.


Subject(s)
Actins/metabolism , Crk-Associated Substrate Protein/metabolism , Immunological Synapses/metabolism , Polymerization , Animals , Calcium/metabolism , Cell Adhesion , Crk-Associated Substrate Protein/deficiency , Integrins/metabolism , Lymphocyte Activation/immunology , Mice, Inbred C57BL , Models, Immunological , Phosphorylation , Protein Transport , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolism
3.
BMC Biol ; 9: 73, 2011 Oct 28.
Article in English | MEDLINE | ID: mdl-22034875

ABSTRACT

BACKGROUND: The 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. RESULTS: We 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. CONCLUSION: Our 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.


Subject(s)
Crk-Associated Substrate Protein/metabolism , Fibroblasts/enzymology , Proteasome Inhibitors , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis/drug effects , Autophagy/drug effects , Caspases/metabolism , Crk-Associated Substrate Protein/deficiency , Cytoprotection/drug effects , Doxorubicin/pharmacology , Embryo, Mammalian/cytology , Enzyme Activation/drug effects , Fibroblasts/cytology , Fibroblasts/drug effects , Heat-Shock Proteins/metabolism , Leupeptins/pharmacology , Mice , Proteasome Endopeptidase Complex/metabolism , Sequestosome-1 Protein , Signal Transduction/drug effects
4.
J Cell Biol ; 178(7): 1295-307, 2007 Sep 24.
Article in English | MEDLINE | ID: mdl-17875741

ABSTRACT

Remodeling of dendritic spines is believed to modulate the function of excitatory synapses. We previously reported that the EphA4 receptor tyrosine kinase regulates spine morphology in hippocampal pyramidal neurons, but the signaling pathways involved were not characterized (Murai, K.K., L.N. Nguyen, F. Irie, Y. Yamaguchi, and E.B. Pasquale. 2003. Nat. Neurosci. 6:153-160). In this study, we show that EphA4 activation by ephrin-A3 in hippocampal slices inhibits integrin downstream signaling pathways. EphA4 activation decreases tyrosine phosphorylation of the scaffolding protein Crk-associated substrate (Cas) and the tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) and also reduces the association of Cas with the Src family kinase Fyn and the adaptor Crk. Consistent with this, EphA4 inhibits beta1-integrin activity in neuronal cells. Supporting a functional role for beta1 integrin and Cas inactivation downstream of EphA4, the inhibition of integrin or Cas function induces spine morphological changes similar to those associated with EphA4 activation. Furthermore, preventing beta1-integrin inactivation blocks the effects of EphA4 on spines. Our results support a model in which EphA4 interferes with integrin signaling pathways that stabilize dendritic spines, thus modulating synaptic interactions with the extracellular environment.


Subject(s)
Dendritic Spines/enzymology , Integrin beta1/metabolism , Receptor, EphA4/metabolism , Signal Transduction , Animals , Cell Adhesion/drug effects , Crk-Associated Substrate Protein/chemistry , Crk-Associated Substrate Protein/deficiency , Dendritic Spines/drug effects , Enzyme Activation/drug effects , Ephrin-A3/pharmacology , Hippocampus/drug effects , Hippocampus/metabolism , Humans , Mice , Nerve Tissue Proteins/metabolism , Phosphoproteins/metabolism , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Protein Binding/drug effects , Proto-Oncogene Proteins c-crk/metabolism , Proto-Oncogene Proteins c-fyn/metabolism , Rats , Signal Transduction/drug effects , Substrate Specificity/drug effects , src Homology Domains
5.
Mol Biol Cell ; 17(11): 4698-708, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16914515

ABSTRACT

Salmonella typhimurium colonizes the intestinal epithelium by injecting an array of effector proteins into host cells that induces phagocytic uptake of attached bacteria. However, the host molecules targeted by these effectors remain poorly defined. Here, we demonstrate that S. typhimurium induces formation of focal adhesion-like complexes at sites of bacterial attachment and that both focal adhesion kinase (FAK) and the scaffolding protein p130Cas are required for Salmonella uptake. Entry of Salmonella into FAK(-/-) cells is dramatically impaired and can be restored to control levels by expression of wild-type FAK. Surprisingly, reconstitution of bacterial internalization requires neither the kinase domain of FAK nor activation of c-Src, but does require a C-terminal PXXP motif through which FAK interacts with Cas. Infection of Cas(-/-) cells is also impaired, and reconstitution of invasiveness requires the central Cas YXXP repeat domain. The invasion defect in Cas(-/-) cells can be suppressed by overexpression of FAK, suggesting a functional link between FAK and Cas in the regulation of Salmonella invasion. Together, these findings reveal a novel role for focal adhesion proteins in the invasion of host cells by Salmonella.


Subject(s)
Crk-Associated Substrate Protein/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Salmonella typhimurium/physiology , Animals , Binding Sites , Crk-Associated Substrate Protein/chemistry , Crk-Associated Substrate Protein/deficiency , Dogs , Enzyme Activation , Fibroblasts/cytology , Fibroblasts/pathology , Focal Adhesion Protein-Tyrosine Kinases/deficiency , Focal Adhesions/metabolism , Gene Expression , HeLa Cells , Humans , Mice , Mice, Knockout , Paxillin/metabolism , Phagosomes/metabolism , Phosphotyrosine/metabolism , Protein Binding , Salmonella typhimurium/pathogenicity , rac1 GTP-Binding Protein/metabolism , rho GTP-Binding Proteins/metabolism
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