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1.
Int J Mol Sci ; 22(22)2021 Nov 16.
Article in English | MEDLINE | ID: mdl-34830244

ABSTRACT

p130 Crk-associated substrate (p130Cas) is associated with poor prognosis and treatment resistance in breast and lung cancers. To elucidate p130Cas functional and clinical role in colorectal cancer (CRC) progression/therapy resistance, we performed cell culture experiments and bioinformatic/statistical analyses of clinical data sets. p130Cas expression was associated with poor survival in the cancer genome atlas (TCGA) data set. Knockdown/reconstitution experiments showed that p130Cas drives migration but, unexpectedly, inhibits proliferation in CRC cells. TCGA data analyses identified the growth factor epiregulin (EREG) as inversely correlated with p130Cas. p130Cas knockdown and simultaneous EREG treatment further enhanced proliferation. RNA interference and EREG treatment experiments suggested that p130Cas/EREG limit each other's expression/activity. Inverse p130Cas/EREG Spearman correlations were prominent in right-sided and earlier stage CRC. p130Cas was inducible by 5-fluorouracil (5-FU) and FOLFIRI (folinic acid, 5-FU, irinotecan), and p130Cas and EREG were upregulated in distant metastases (GSE121418). Positive p130Cas/EREG correlations were observed in metastases, preferentially in post-treatment samples (especially pulmonary metastases). p130Cas knockdown sensitized CRC cells to FOLFIRI independent of EREG treatment. RNA sequencing and gene ontology analyses revealed that p130Cas is involved in cytochrome P450 drug metabolism and epithelial-mesenchymal transition. p130Cas expression was associated with poor survival in right-sided, stage I/II, MSS (microsatellite stable), or BRAF-mutated CRC. In summary, p130Cas represents a prognostic factor and potential therapeutic target in CRC.


Subject(s)
Breast Neoplasms/diagnosis , Colorectal Neoplasms/diagnosis , Crk-Associated Substrate Protein/genetics , Epiregulin/genetics , Epithelial-Mesenchymal Transition/genetics , Adult , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Atlases as Topic , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/mortality , Camptothecin/analogs & derivatives , Camptothecin/therapeutic use , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/mortality , Crk-Associated Substrate Protein/antagonists & inhibitors , Crk-Associated Substrate Protein/metabolism , Epiregulin/metabolism , Female , Fluorouracil/therapeutic use , Gene Expression Regulation, Neoplastic , Gene Ontology , Humans , Leucovorin/therapeutic use , Male , Middle Aged , Molecular Sequence Annotation , Mutation , Neoplasm Metastasis , Neoplasm Staging , Prognosis , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Survival Analysis
2.
Cell Biochem Funct ; 38(3): 300-308, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31887784

ABSTRACT

Podosome formation in osteoclasts is an important initial step in osteoclastic bone resorption. Mice lacking c-Src (c-Src-/- ) exhibited osteopetrosis due to a lack of podosome formation in osteoclasts. We previously identified p130Cas (Crk-associated substrate [Cas]) as one of c-Src downstream molecule and osteoclast-specific p130Cas-deficient (p130CasΔOCL-/- ) mice also exhibited a similar phenotype to c-Src-/- mice, indicating that the c-Src/p130Cas plays an important role for bone resorption by osteoclasts. In this study, we performed a cDNA microarray and compared the gene profiles of osteoclasts from c-Src-/- or p130CasΔOCL-/- mice with wild-type (WT) osteoclasts to identify downstream molecules of c-Src/p130Cas involved in bone resorption. Among several genes that were commonly downregulated in both c-Src-/- and p130CasΔOCL-/- osteoclasts, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization. Reduced Kif1c expression was observed in both c-Src-/- and p130CasΔOCL-/- osteoclasts compared with WT osteoclasts. Kif1c exhibited a broad tissue distribution, including osteoclasts. Knockdown of Kif1c expression using shRNAs in WT osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in p130CasΔOCL-/- osteoclasts but not c-Src-/- osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas (191 words). SIGNIFICANCE OF THE STUDY: We previously showed that the c-Src/p130Cas (Cas) plays an important role for bone resorption by osteoclasts. In this study, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization, as a downstream molecule of c-Src/p130Cas axis, using cDNA microarray. Knockdown of Kif1c expression using shRNAs in wild-type osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in osteoclast-specific p130Cas-deficient (p130CasΔOCL-/- ) osteoclasts but not c-Src-/- osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas.


Subject(s)
Bone Resorption , Crk-Associated Substrate Protein/metabolism , Gene Expression Regulation , Kinesins/metabolism , Osteoclasts/metabolism , Actins/metabolism , Animals , Bone and Bones/metabolism , CSK Tyrosine-Protein Kinase/genetics , CSK Tyrosine-Protein Kinase/metabolism , HEK293 Cells , Heterozygote , Humans , Macrophage Colony-Stimulating Factor/metabolism , Mice , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Phenotype , Phosphorylation , RNA, Small Interfering/metabolism , Recombinant Proteins/metabolism , Signal Transduction , Zinc Fingers
3.
J Neurochem ; 150(2): 158-172, 2019 07.
Article in English | MEDLINE | ID: mdl-30589943

ABSTRACT

Oligodendrocytes (OLs) are myelinating cells of the central nervous system. Recent studies have shown that mechanical factors influence various cell properties. Mechanical stimulation can be transduced into intracellular biochemical signals through mechanosensors, such as integrin, p130Cas, talin and vinculin. However, the molecular mechanisms underlying the mechanical regulation of OLs by mechanosensors remain largely unknown. We found that morphology of OL was affected by knockdown of the mechanosensors p130Cas or talin1. Stretching of OL precursor cells induced the phosphorylation of p130Cas and talin-associated assembly of vinculin. Shear stress decreased the number of OL processes, whereas these effects were mechanically suppressed by dominant-negative (DN) p130Cas, but not by DN-talin1. To investigate the roles of p130Cas in post-natal OLs in vivo, we constructed a novel p130Cas knock-in mouse and found overexpression of p130Cas in vivo affected the number of mature OLs in the cortex. These results indicate that the mechanosensor p130Cas controls both OL morphogenesis and maturation.


Subject(s)
Crk-Associated Substrate Protein/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Oligodendroglia/cytology , Oligodendroglia/metabolism , Animals , Cell Differentiation/physiology , Cerebral Cortex/cytology , Cerebral Cortex/metabolism , Gene Knock-In Techniques , Mechanotransduction, Cellular/physiology , Mice , Mice, Inbred C57BL , Stress, Mechanical
4.
J Cell Sci ; 130(7): 1263-1273, 2017 04 01.
Article in English | MEDLINE | ID: mdl-28223315

ABSTRACT

Members of the Cas family of focal adhesion proteins contain a highly conserved C-terminal focal adhesion targeting (FAT) domain. To determine the role of the FAT domain in these proteins, we compared wild-type exogenous NEDD9 with a hybrid construct in which the NEDD9 FAT domain had been exchanged for the p130Cas (also known as BCAR1) FAT domain. Fluorescence recovery after photobleaching (FRAP) revealed significantly slowed exchange of the fusion protein at focal adhesions and significantly slower two-dimensional migration. No differences were detected in cell stiffness as measured using atomic force microscopy (AFM) and in cell adhesion forces measured with a magnetic tweezer device. Thus, the slowed migration was not due to changes in cell stiffness or adhesion strength. Analysis of cell migration on surfaces of increasing rigidity revealed a striking reduction of cell motility in cells expressing the p130Cas FAT domain. The p130Cas FAT domain induced rigidity-dependent phosphorylation of tyrosine residues within NEDD9. This in turn reduced post-translational cleavage of NEDD9, which we show inhibits NEDD9-induced migration. Collectively, our data therefore suggest that the p130Cas FAT domain uniquely confers a mechanosensing function.


Subject(s)
Crk-Associated Substrate Protein/chemistry , Crk-Associated Substrate Protein/metabolism , Focal Adhesions/metabolism , Mechanotransduction, Cellular , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Cell Line, Tumor , Cell Movement , Extracellular Matrix/metabolism , Focal Adhesions/drug effects , Gene Knockdown Techniques , Humans , Mechanotransduction, Cellular/drug effects , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Phosphorylation , Protein Domains , Protein Transport/drug effects , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Structure-Activity Relationship , Tetracycline/pharmacology
5.
J Virol ; 92(4)2018 02 15.
Article in English | MEDLINE | ID: mdl-29167336

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV) in vitro infection of dermal endothelial cells begins with its binding to host cell surface receptor molecules such as heparan sulfate (HS), integrins (α3ß1, αVß3, and αVß5), xCT, and EphA2 receptor tyrosine kinase (EphA2R). These initial events initiate dynamic host protein-protein interactions involving a multimolecular complex of receptors, signal molecules (focal adhesion kinase [FAK], Src, phosphatidylinositol 3-kinase [PI3-K], and RhoA-GTPase), adaptors (c-Cbl, CIB1, Crk, p130Cas, and GEF-C3G), actin, and myosin II light chain that lead to virus entry via macropinocytosis. Here we discuss how KSHV hijacks c-Cbl, an E3 ubiquitin ligase, to monoubiquitinate the receptors and actin, which acts like a marker for trafficking (similar to zip codes), resulting in the recruitment of the members of the host endosomal sorting complexes required for transport (ESCRT) Hrs, Tsg101, EAP45, and the CHMP5 and -6 proteins (zip code readers) recognizing the ubiquitinated protein and adaptor machinery to traffic through the different endosomal compartments in the cytoplasm to initiate the macropinocytic process and infection.


Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , Herpesviridae Infections/metabolism , Herpesvirus 8, Human/physiology , Proto-Oncogene Proteins c-cbl/metabolism , Virus Internalization , Endothelial Cells/virology , Humans , Pinocytosis , Signal Transduction , Ubiquitination
6.
J Biol Chem ; 292(44): 18281-18289, 2017 11 03.
Article in English | MEDLINE | ID: mdl-28860193

ABSTRACT

The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by which the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ∼4.2 µm) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.


Subject(s)
Avian Proteins/metabolism , Crk-Associated Substrate Protein/metabolism , Models, Molecular , Paxillin/metabolism , Amino Acid Motifs , Amino Acid Substitution , Animals , Avian Proteins/chemistry , Binding Sites , Chickens , Crk-Associated Substrate Protein/chemistry , Crk-Associated Substrate Protein/genetics , Crystallography, X-Ray , Hydrophobic and Hydrophilic Interactions , Kinetics , Leucine , Mice , Mutation , Paxillin/chemistry , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Conformation , Protein Interaction Domains and Motifs , Protein Interaction Mapping , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Structural Homology, Protein
7.
Biochim Biophys Acta ; 1860(8): 1753-63, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27068854

ABSTRACT

We previously demonstrated that focal adhesion kinase (FAK), p130Cas and paxillin are crucially involved in the enhanced malignant properties under expression of ganglioside GD3 in melanoma cells. Therefore, molecules existing in the GD3-mediated signaling pathway could be considered as suitable targets for therapeutic intervention in malignant melanoma. The aim of this study was to determine whether blockade of p130Cas and/or paxillin by RNAi suppresses melanoma growth. We found a suitable dose (40 µM siRNA, 25 µl/tumor) of the siRNA to suppress p130Cas in the xenografts generated in nu/nu mice. Based on these results, we performed intratumoral (i.t.) treatment with anti-p130Cas and/or anti-paxillin siRNAs mixed with atelocollagen as a drug delivery system in a xenograft tumor of a human melanoma cell line, SK-MEL-28. Mixture of atelocollagen (1.75%) and an siRNA (500 or 1000 pmol/tumor) was injected into the tumors every 3 days after the first injection. An siRNA against human p130Cas markedly suppressed tumor growth of the xenograft in a dose-dependent manner, whereas siRNA against human paxillin slightly inhibited the tumor growth. A control siRNA against firefly luciferase showed no effect. To our surprise, siRNA against human p130Cas (500 or 1000 pmol/tumor) combined with siRNA against human paxillin dramatically suppressed tumor growth. In agreement with the tumor suppression effects of the anti-p130Cas siRNA, reduction in Ki-67 positive cell number as well as in p130Cas expression was demonstrated by immunohistostaining. These results suggested that blockade of GD3-mediated growth signaling pathways by siRNAs might be a novel and promising therapeutic strategy against malignant melanomas, provided signaling molecules such as p130Cas and paxillin are significantly expressed in individual cases. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.


Subject(s)
Crk-Associated Substrate Protein , Gangliosides , Gene Expression Regulation, Neoplastic/drug effects , Melanoma , Neoplasm Proteins , Paxillin , RNA, Small Interfering , Animals , Cell Line, Tumor , Crk-Associated Substrate Protein/antagonists & inhibitors , Crk-Associated Substrate Protein/biosynthesis , Crk-Associated Substrate Protein/genetics , Gangliosides/biosynthesis , Gangliosides/genetics , Humans , Melanoma/drug therapy , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Mice , Mice, Nude , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Paxillin/antagonists & inhibitors , Paxillin/biosynthesis , Paxillin/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Signal Transduction , Xenograft Model Antitumor Assays
8.
J Biol Chem ; 290(19): 12247-55, 2015 May 08.
Article in English | MEDLINE | ID: mdl-25805500

ABSTRACT

Elevated levels of p130(Cas) (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1 gene) are associated with aggressiveness of breast tumors. Following phosphorylation of its substrate domain, p130(Cas) promotes the integration of protein complexes involved in multiple signaling pathways and mediates cell proliferation, adhesion, and migration. In addition to the known BCAR1-1A (wild-type) and 1C variants, we identified four novel BCAR1 mRNA variants, generated by alternative first exon usage (1B, 1B1, 1D, and 1E). Exons 1A and 1C encode for four amino acids (aa), whereas 1D and 1E encode for 22 aa and 1B1 encodes for 50 aa. Exon 1B is non-coding, resulting in a truncated p130(Cas) protein (Cas1B). BCAR1-1A, 1B1, and variant 1C mRNAs were ubiquitously expressed in cell lines and a survey of human tissues, whereas 1B, 1D, and 1E expression was more restricted. Reconstitution of all isoforms except for 1B in p130(Cas)-deficient murine fibroblasts induced lamellipodia formation and membrane ruffling, which was unrelated to the substrate domain phosphorylation status. The longer isoforms exhibited increased binding to focal adhesion kinase (FAK), a molecule important for migration and adhesion. The shorter 1B isoform exhibited diminished FAK binding activity and significantly reduced migration and invasion. In contrast, the longest variant 1B1 established the most efficient FAK binding and greatly enhanced migration. Our results indicate that the p130(Cas) exon 1 variants display altered functional properties. The truncated variant 1B and the longer isoform 1B1 may contribute to the diverse effects of p130(Cas) on cell biology and therefore will be the target of future studies.


Subject(s)
Crk-Associated Substrate Protein/genetics , Crk-Associated Substrate Protein/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Gene Expression Regulation, Enzymologic , Alternative Splicing , Animals , Base Sequence , Cell Adhesion , Cell Movement , Exons , Fibroblasts/metabolism , Humans , Mice , Molecular Sequence Data , Neoplasm Invasiveness , Protein Isoforms , Protein Structure, Tertiary , RNA, Messenger/metabolism , Sequence Homology, Nucleic Acid , Signal Transduction , src Homology Domains
9.
J Cell Sci ; 127(Pt 12): 2647-58, 2014 Jun 15.
Article in English | MEDLINE | ID: mdl-24762811

ABSTRACT

DOK1 regulates platelet-derived growth factor (PDGF)-BB-stimulated glioma cell motility. Mechanisms regulating tumour cell motility are essential for invasion and metastasis. We report here that PDGF-BB-mediated glioma cell invasion and migration are dependent on the adaptor protein downstream of kinase 1 (DOK1). DOK1 is expressed in several glioma cell lines and in tumour biopsies from high-grade gliomas. DOK1 becomes tyrosine phosphorylated upon PDGF-BB stimulation of human glioma cells. Knockdown of DOK1 or expression of a DOK1 mutant (DOK1FF) containing Phe in place of Tyr at residues 362 and 398, resulted in inhibition of both the PDGF-BB-induced tyrosine phosphorylation of p130Cas (also known as BCAR1) and the activation of Rap1. DOK1 colocalises with tyrosine phosphorylated p130Cas at the cell membrane of PDGF-BB-treated cells. Expression of a non-tyrosine-phosphorylatable substrate domain mutant of p130Cas (p130Cas15F) inhibited PDGF-BB-mediated Rap1 activation. Knockdown of DOK1 and Rap1 inhibited PDGF-BB-induced chemotactic cell migration, and knockdown of DOK1 and Rap1 and expression of DOK1FF inhibited PDGF-mediated three-dimensional (3D) spheroid invasion. These data show a crucial role for DOK1 in the regulation of PDGF-BB-mediated tumour cell motility through a p130Cas-Rap1 signalling pathway. [Corrected]


Subject(s)
Brain Neoplasms/metabolism , Crk-Associated Substrate Protein/metabolism , DNA-Binding Proteins/physiology , Glioblastoma/metabolism , Phosphoproteins/physiology , Proto-Oncogene Proteins c-sis/physiology , RNA-Binding Proteins/physiology , Telomere-Binding Proteins/metabolism , Becaplermin , Brain Neoplasms/pathology , Cell Line, Tumor , Chemotaxis , Glioblastoma/pathology , Humans , Neoplasm Invasiveness , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Processing, Post-Translational , Shelterin Complex , Signal Transduction , src-Family Kinases/metabolism
10.
J Cell Sci ; 127(Pt 16): 3440-50, 2014 Aug 15.
Article in English | MEDLINE | ID: mdl-24928898

ABSTRACT

Cell adhesion complexes provide platforms where cell-generated forces are transmitted to the extracellular matrix (ECM). Tyrosine phosphorylation of focal adhesion proteins is crucial for cells to communicate with the extracellular environment. However, the mechanisms that transmit actin cytoskeletal motion to the extracellular environment to drive cell migration are poorly understood. We find that the movement of p130Cas (Cas, also known as BCAR1), a mechanosensor at focal adhesions, correlates with actin retrograde flow and depends upon actomyosin contraction and phosphorylation of the Cas substrate domain (CasSD). This indicates that CasSD phosphorylation underpins the physical link between Cas and the actin cytoskeleton. Fluorescence recovery after photobleaching (FRAP) experiments reveal that CasSD phosphorylation, as opposed to the association of Cas with Src, facilitates Cas displacement from adhesion complexes in migrating cells. Furthermore, the stabilization of Src-Cas binding and inhibition of myosin II, both of which sustain CasSD phosphorylation but mitigate Cas displacement from adhesion sites, retard cell migration. These results indicate that Cas promotes cell migration by linking actomyosin contractions to the adhesion complexes through a dynamic interaction with Src as well as through the phosphorylation-dependent association with the actin cytoskeleton.


Subject(s)
Actomyosin/physiology , Cell Movement , Crk-Associated Substrate Protein/metabolism , Focal Adhesions/metabolism , Actins/metabolism , Crk-Associated Substrate Protein/genetics , Cytoskeleton/genetics , Cytoskeleton/metabolism , Focal Adhesions/genetics , HEK293 Cells , Humans , Phosphorylation , Proto-Oncogene Proteins pp60(c-src)/genetics , Proto-Oncogene Proteins pp60(c-src)/metabolism
11.
J Cell Sci ; 127(Pt 7): 1394-405, 2014 Apr 01.
Article in English | MEDLINE | ID: mdl-24481817

ABSTRACT

Tyrosine phosphorylation of the substrate domain of Cas (CasSD) correlates with increased cell migration in healthy and diseased cells. Here, we address the mechanism leading to the phosphorylation of CasSD in the context of fibronectin-induced early spreading of fibroblasts. We have previously demonstrated that mechanical stretching of CasSD exposes phosphorylation sites for Src family kinases (SFKs). Surprisingly, phosphorylation of CasSD was independent of myosin contractile activity but dependent on actin polymerization. Furthermore, we found that CasSD phosphorylation in the early stages of cell spreading required: (1) integrin anchorage and integrin-mediated activation of SFKs, (2) association of Cas with focal adhesion kinase (FAK), and (3) N-WASP-driven actin-assembly activity. These findings, and analyses of the interactions of the Cas domains, indicate that the N-terminus of Cas associates with the FAK-N-WASP complex at the protrusive edge of the cell and that the C-terminus of Cas associates with the immobilized integrin-SFK cluster. Thus, extension of the leading edge mediated by actin polymerization could stretch Cas during early cell spreading, priming it for phosphorylation.


Subject(s)
Actins/metabolism , Crk-Associated Substrate Protein/metabolism , Pseudopodia/metabolism , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , Actin Cytoskeleton/metabolism , Animals , Cell Adhesion/physiology , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Humans , Mice , Phosphorylation , Polymerization , Protein Structure, Tertiary , Signal Transduction
12.
Tumour Biol ; 37(8): 10665-73, 2016 Aug.
Article in English | MEDLINE | ID: mdl-26867768

ABSTRACT

Elevated p130Cas (Crk-associated substrate) levels are found in aggressive breast tumors and are associated with poor prognosis and resistance to standard therapeutics in patients. p130Cas signals majorly through its phosphorylated substrate domain (SD) that contains 15 tyrosine motifs (YxxP) which recruit effector molecules. Tyrosine phosphorylation of p130Cas is important for mediating migration, invasion, tumor promotion, and metastasis. We previously developed a Src*/SD fusion molecule approach, where the SD is constitutively phosphorylated. In a polyoma middle T-antigen (PyMT)/Src*/SD double-transgenic mouse model, Src*/SD accelerates PyMT-induced tumor growth and promotes a more aggressive phenotype. To test whether Src*/SD also drives metastasis and which of the YxxP motifs are involved in this process, full-length and truncated SD molecules fused to Src* were expressed in breast cancer cells. The functionality of the Src*/SD fragments was analyzed in vitro, and the active proteins were tested in vivo in an orthotopic mouse model. Breast cancer cells expressing the full-length SD and the functional smaller SD fragment (spanning SD motifs 6-10) were injected into the mammary fat pads of mice. The tumor progression was monitored by bioluminescence imaging and caliper measurements. Compared with control animals, the complete SD promoted primary tumor growth and an earlier onset of metastases. Importantly, both the complete and truncated SD significantly increased the occurrence of metastases to multiple organs. These studies provide strong evidence that the phosphorylated p130Cas SD motifs 6-10 (Y236, Y249, Y267, Y287, and Y306) are important for driving mammary carcinoma progression.


Subject(s)
Breast Neoplasms/pathology , Crk-Associated Substrate Protein/physiology , Neoplasm Proteins/physiology , Amino Acid Motifs , Animals , Breast Neoplasms/genetics , CSK Tyrosine-Protein Kinase , Cell Line, Tumor , Crk-Associated Substrate Protein/chemistry , Crk-Associated Substrate Protein/genetics , Disease Progression , Female , Genes, Reporter , Heterografts , Humans , MCF-7 Cells , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Neoplasm Metastasis , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Peptide Fragments/genetics , Phosphorylation , Phosphotyrosine/metabolism , Protein Domains , Protein Processing, Post-Translational , Recombinant Fusion Proteins/metabolism , src-Family Kinases/genetics , src-Family Kinases/metabolism
13.
Cell Biol Int ; 40(3): 241-56, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26909547

ABSTRACT

Cell-matrix adhesion and cell-cell contacts are essential for the metabolism, protein synthesis, survival, and cancer metastasis of cells. Major transmembrane receptors are the integrins, which are responsible for cell-matrix adhesions, and the cadherins, which are important for cell-cell adhesions. Adherent cells anchor via focal adhesion proteins to the extracellular matrix, whereas cell-cell contacts connect via focal adherens junction proteins. The temporal formation of these connections is greatly strengthened either through externally applied stresses on the cell or by myosin-driven cell contractility. The mechanism by which protein(s) within these connections sense, transmit, and respond to mechanochemical signaling is currently strongly debated and various candidates have been named. Vinculin has been described as one of the key players in cell-matrix and cell-cell adhesions that build a strong physical connection for transmitting forces between the cytoskeleton, the extracellular matrix, and cell-cell connections.


Subject(s)
Mechanotransduction, Cellular/physiology , Vinculin/metabolism , Actins/metabolism , Adherens Junctions/metabolism , Cell Adhesion , Crk-Associated Substrate Protein/metabolism , Extracellular Matrix/metabolism , Humans , Integrins/metabolism , Protein Binding , Vinculin/chemistry
14.
J Korean Med Sci ; 31(4): 535-41, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27051236

ABSTRACT

Angiotensin II (Ang II) induces the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. In kidneys, Ang II plays an important role in the development of proteinuria by the modification of podocyte molecules. We have previously found that Ang II suppressed podocyte AMP-activated protein kinase (AMPK) via Ang II type 1 receptor and MAPK signaling pathway. In the present study, we investigated the roles of AMPK on the changes of p130Cas of podocyte by Ang II. We cultured mouse podocytes and treated them with various concentrations of Ang II and AMPK-modulating agents and analyzed the changes of p130Cas by confocal imaging and western blotting. In immunofluorescence study, Ang II decreased the intensity of p130Cas and changed its localization from peripheral cytoplasm into peri-nuclear areas in a concentrated pattern in podocytes. Ang II also reduced the amount of p130Cas in time and dose-sensitive manners. AMPK activators, metformin and AICAR, restored the suppressed and mal-localized p130Cas significantly, whereas, compound C, an AMPK inhibitor, further aggravated the changes of p130Cas. Losartan, an Ang II type 1 receptor antagonist, recovered the abnormal changes of p130Cas suppressed by Ang II. These results suggest that Ang II induces the relocalization and suppression of podocyte p130Cas by the suppression of AMPK via Ang II type 1 receptor, which would contribute to Ang II-induced podocyte injury.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Angiotensin II/pharmacology , Crk-Associated Substrate Protein/metabolism , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/chemistry , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Angiotensin II Type 1 Receptor Blockers/pharmacology , Animals , Blotting, Western , Cell Line , Cell Nucleus/metabolism , Cytoplasm/metabolism , Focal Adhesion Kinase 1/metabolism , Losartan/pharmacology , Metformin/pharmacology , Mice , Microscopy, Confocal , Podocytes/cytology , Podocytes/drug effects , Podocytes/metabolism , Ribonucleotides/pharmacology
15.
Biochem Biophys Res Commun ; 444(4): 509-13, 2014 Feb 21.
Article in English | MEDLINE | ID: mdl-24472550

ABSTRACT

Myogenesis is regulated by the basic helix-loop-helix (bHLH) myogenic regulatory factor MyoD, which induces muscle-specific gene expression by binding to the E-box sequence as a heterodimer with ubiquitous bHLH E2A (E12/E47) proteins. Here, we report that a 31-kDa caspase-generated cleavage product of Crk-associated substrate (p130Cas), herein called 31-kDa, is downregulated during muscle cell differentiation. 31-kDa contains a helix-loop-helix (HLH) domain that shows greater sequence homology with Id (inhibitor of DNA binding) proteins than with bHLH proteins. This HLH domain, lacking the basic region required for DNA binding, mediated the direct interaction of 31-kDa with MyoD. Overexpression of 31-kDa in C3H10T1/2 cells inhibited not only the transcriptional activation of p21(Waf1/Cip1) and E-box-dependent muscle-specific genes by MyoD and/or E2A but also MyoD-induced myosin heavy chain expression and myogenic conversion. In sum, our results suggest a role for 31-kDa as a negative regulator of MyoD in the muscle differentiation program.


Subject(s)
Caspases/metabolism , Crk-Associated Substrate Protein/metabolism , Muscle Development , MyoD Protein/metabolism , Animals , Caspases/chemistry , Caspases/genetics , Cell Line , Down-Regulation , Gene Expression Regulation, Developmental , Helix-Loop-Helix Motifs , Mice
16.
Biochem Biophys Res Commun ; 447(2): 352-7, 2014 May 02.
Article in English | MEDLINE | ID: mdl-24726648

ABSTRACT

Although osteoblast lineage cells, especially osteocytes, are thought to be a primary mechanosensory cell in bone, the identity of the mechano-receptor and downstream mechano-signaling pathways remain largely unknown. Here we show using osteoblastic cell model of mechanical stimulation with fluid shear stress that in the absence of integrin αv, phosphorylation of the Src substrate p130Cas and JNK was impaired, culminating in an inhibition of nuclear translocation of YAP/TAZ and subsequent transcriptional activation of target genes. Targeted deletion of the integrin αv in osteoblast lineage cells results in an attenuated response to mechanical loading in terms of Sost gene expression, indicative of a role for integrin αv in mechanoreception in vivo. Thus, integrin αv may be integral to a mechanosensing machinery in osteoblastic cells and involved in activation of a Src-JNK-YAP/TAZ pathway in response to mechanical stimulation.


Subject(s)
Integrin alphaV/physiology , Mechanotransduction, Cellular/physiology , Osteoblasts/physiology , Shear Strength/physiology , Stress, Mechanical , Acyltransferases , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Cycle Proteins , Cell Lineage , Cells, Cultured , Integrin alphaV/genetics , MAP Kinase Kinase 4/metabolism , Mechanotransduction, Cellular/genetics , Mice , Phosphoproteins/metabolism , Transcription Factors/metabolism , YAP-Signaling Proteins , src-Family Kinases/metabolism
17.
Chembiochem ; 15(8): 1161-70, 2014 May 26.
Article in English | MEDLINE | ID: mdl-24771685

ABSTRACT

The interaction between VEGF-A and its neuropilin (NRP) receptors mediates a number of important biological effects. NRP1 and the related molecule NRP2 are widely expressed on multiple tumour types and throughout the tumour vasculature, and are emerging as critical molecules required for the progression of angiogenic diseases. Given the increasing evidence supporting a role for NRP1 in tumour development, there is growing interest in developing inhibitors of NRP1 interactions with VEGF and its other ligands. In order to probe the interaction we synthesised a number of exon 7- and 8-derived bicyclic peptides with N-terminal lipophilic groups and found a simple N-octanoyl derivative (EG00086) to be the most potent and functionally active. Detailed modelling studies indicated that new intramolecular hydrogen bonds were formed, stabilising the structure and possibly contributing to the potency. Removal of a salt bridge between D142 and R164 implicated in VEGF-A binding to neuropilin-1 had a minor effect on potency. Isothermal calorimetry was used to assess binding of EG00086 to NRP1 and NRP2, and the stability of the peptide in serum and in vivo was investigated. EG00086 is a potent blocker of VEGF-promoted cellular adhesion to extracellular matrices, and phosphorylation of p130Cas contributes to this effect.


Subject(s)
Neuropilin-1/metabolism , Peptides, Cyclic/chemistry , Peptides, Cyclic/metabolism , Vascular Endothelial Growth Factor A/chemistry , Vascular Endothelial Growth Factor A/metabolism , Binding Sites , Cell Adhesion/drug effects , Cell Survival/drug effects , Cells, Cultured , Crk-Associated Substrate Protein/metabolism , Exons/genetics , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Humans , Lipopeptides/chemistry , Lipopeptides/metabolism , Lipopeptides/pharmacology , Molecular Dynamics Simulation , Neuropilin-1/chemistry , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Phosphorylation/drug effects , Protein Binding , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/genetics
18.
Stem Cells ; 31(7): 1422-33, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23592522

ABSTRACT

It has recently been proposed that defective differentiation of mammary luminal progenitors predisposes to basal-like breast cancer. However, the molecular and cellular mechanisms involved are still unclear. Here, we describe that the adaptor protein p130Cas is a crucial regulator of mouse mammary epithelial cell (MMEC) differentiation. Using a transgenic mouse model, we show that forced p130Cas overexpression in the luminal progenitor cell compartment results in the expansion of luminal cells, which aberrantly display basal cell features and reduced differentiation in response to lactogenic stimuli. Interestingly, MMECs overexpressing p130Cas exhibit hyperactivation of the tyrosine kinase receptor c-Kit. In addition, we demonstrate that the constitutive c-Kit activation alone mimics p130Cas overexpression, whereas c-Kit downregulation is sufficient to re-establish proper differentiation of p130Cas overexpressing cells. Overall, our data indicate that high levels of p130Cas, via abnormal c-Kit activation, promote mammary luminal cell plasticity, thus providing the conditions for the development of basal-like breast cancer. Consistently, p130Cas is overexpressed in human triple-negative breast cancer, further suggesting that p130Cas upregulation may be a priming event for the onset of basal-like breast cancer.


Subject(s)
Crk-Associated Substrate Protein/metabolism , Mammary Glands, Animal/cytology , Mammary Neoplasms, Experimental/pathology , Proto-Oncogene Proteins c-kit/metabolism , Triple Negative Breast Neoplasms/pathology , Animals , Cell Differentiation/physiology , Cell Growth Processes/physiology , Cells, Cultured , Crk-Associated Substrate Protein/biosynthesis , Crk-Associated Substrate Protein/genetics , Female , Humans , Immunohistochemistry , Mammary Glands, Animal/metabolism , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/metabolism , Mice , Mice, Transgenic , Pregnancy , Proto-Oncogene Proteins c-kit/genetics , Signal Transduction , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/metabolism
19.
Cell Biol Int ; 38(3): 283-6, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24497348

ABSTRACT

Adherent cells, when mechanically stressed, show a wide range of responses including large-scale changes in their mechanical behaviour and gene expression pattern. This is in part facilitated by activating the focal adhesion (FA) protein p130Cas through force-induced conformational changes that lead to the phosphorylation by src family kinases. Janostiak et al. [Janostiak et al. Cell Mol Life Sci (2013) DOI 10.1007/s00018-013-1450-x] have reported that the phosphorylation site Y12 on the SH3 domain of p130Cas modulates the binding with vinculin, a prominent mechano-coupling protein in FAs. Tension changes in FAs (due to the anchorage of the SH3 domain and C-terminal) bring about an extension of the substrate domain of p130Cas by unmasking the phosphorylation sites. These observations demonstrate that vinculin is an important modulator of the p130Cas-mediated mechano-transduction pathway in cells. The central aim should be now to test that vinculin is critical for p130Cas incorporation into the focal adhesion complex and for transmitting forces to the p130Cas molecule.


Subject(s)
Crk-Associated Substrate Protein/metabolism , Focal Adhesions/metabolism , Mechanotransduction, Cellular/physiology , Vinculin/metabolism , Cell Adhesion/physiology , Humans , Phosphorylation/physiology
20.
Am Heart J Plus ; 44: 100416, 2024 Aug.
Article in English | MEDLINE | ID: mdl-39036012

ABSTRACT

Breast cancer anti-estrogen resistance-1 (p130Cas/BCAR1) is an adaptor protein of the cas(Cas) family. This protein regulates multiple complex pathways in different organs including bones, pancreas, and immune and cardiovascular systems. Although previous research well demonstrated the role of p130Cas/BCAR1 in different diseases especially cancers, a precise review study on the various effects of p130Cas/BCAR1 on cardiovascular diseases is missing. In this study, we reviewed mechanisms of action for p130Cas/BCAR1 impact, on cardiac embryonic development defects, hypertrophy and remodeling, pulmonary artery hypertension (PAH), and atherosclerosis. Also, we suggest feature direction for research and potential therapeutic implications. This study showed that p130Cas/BCAR1 can affect cardiovascular diseases in various mechanisms including actin stress fiber formation, attachment to focal adhesion kinase (FAK) and angiotensin II (Ang II), generation of reactive oxygen species (ROS), and growth factor signaling through amplifying receptor tyrosine kinase (RTKs).

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