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Tensin 2 interactomics reveals interaction with GAPDH and a phosphorylation-mediated regulatory role in glycolysis

ABSTRACT: Integrin adaptor proteins, like tensin-2, are crucial for cell adhesion and signaling. However, the function of tensin-2 beyond localizing to focal adhesions remain poorly understood. We utilized proximity-dependent biotinylation and strep-tag affinity proteomics to identify interaction partners of tensin-2 in HEK293 cells. Interactomics linked tensin-2 to known focal adhesion proteins and the dystrophin glycoprotein complex, while also uncovering novel interaction with the glycolytic enzyme GAPDH. We demonstrated that Y483-phosphorylation of tensin-2 regulates the glycolytic rate in HEK293 and MEF cells and found that pY483 tensin-2 is enriched in adhesions in MEF cells. Our study unveils novel interaction partners for tensin-2 and further solidifies its speculated role in cell energy metabolism. These findings shed fresh insight on the functions of tensin-2, highlighting its potential as a therapeutic target for diseases associated with impaired cell adhesion and metabolism.

ORGANISM(S): Homo sapiens

PROVIDER: GSE231782 | GEO | 2024/07/20

REPOSITORIES: GEO

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Tensin 2 interactomics reveals interaction with GAPDH and a phosphorylation-mediated regulatory role in glycolysis

Project description:Integrin adaptor proteins, like tensin-2, are crucial for cell adhesion and signalling. However, its functions beyond localizing to focal adhesions remain poorly understood. To identify tensin-2's interaction partners in HEK293 cells, we utilized proximity-dependent biotinylation and strep-tag affinity proteomics. Results linked tensin-2 to known focal adhesion proteins and the dystrophin-glycoprotein complex, while also uncovering novel interaction with the glycolytic enzyme GAPDH. We demonstrated that Y483-phosphorylation of tensin-2 regulates the glycolytic rate in HEK293 and MEF cells, as well as gene expression in HEK293 cells. Of note, Y483-phosphorylation directs tensin-2 to adhesion sites in MEF cells. Our study unveils numerous novel interaction partners for tensin-2, further solidifies its speculated role in cell energy metabolism and identifies Y483-phosphorylation as a crucial regulator for tensin-2 localization. These findings shed fresh insight into the functions of tensin-2, highlighting its potential as a therapeutic target for diseases associated with impaired cell adhesion and metabolism.

2023-05-02 | MSV000091836 | MassIVE

Ambra1 spatially regulates Src activity and Src/FAK-mediated cancer cell invasion via trafficking networks

Project description:Here, using mouse squamous cell carcinoma cells, we report a completely new function for the autophagy protein Ambra1 as the first described ‘spatial rheostat’ controlling the Src/FAK pathway. Ambra1 regulates the targeting of active phospho-Src away from focal adhesions into autophagic structures that cancer cells use to survive adhesion stress. Ambra1 binds to both FAK and Src in cancer cells. When FAK is present, Ambra1 is recruited to focal adhesions, promoting FAK-regulated cancer cell direction-sensing and invasion. However, when Ambra1 cannot bind to FAK, abnormally high levels of phospho-Src and phospho-FAK accumulate at focal adhesions, positively regulating adhesion and invasive migration. Spatial control of active Src requires the trafficking proteins Dynactin 1 and IFITM3, which we identified as Ambra1 binding partners by interaction proteomics. We conclude that Ambra1 is a core component of an intracellular trafficking network linked to tight spatial control of active Src and FAK levels, and so crucially regulates their cancer-associated biological outputs.

2017-04-04 | PXD006002 | Pride

U2OS Adhesion Isolations +/- CytochalasinD and Neomycin

Project description:U2OS cells preferentially utilise integrin alphaV beta5 in adhesion complexes when grown under standard cell culture conditions for 3 days. AlphaV beta5 can be found in both classical 'Focal' adhesions and in novel 'Reticular' adhesions that do not associate with F-actin or other known adhesion protein components. In order to identify components of reticular adhesions, cells were treated with cytochalasinD in order to disrupt F-actin and promote loss of focal adhesions. Remaining reticular adhesion complexes were stabilised with DTBP crosslinking reagent before adhesion complexes were isolated. Preliminary data suggested that depletion of PIP2 by Neomycin treatment would lead to disruption of reticular adhesions preferentially over focal adhesions and so this manipulation was included in these experiments.

2019-06-03 | PXD008680 | Pride

Potential interactors of alpha-tubulin acetyltransferase 1 (αTAT1/MEC17)

Project description:Cell adhesion to the extracellular matrix occurs through integrin-mediated focal adhesions, which sense the mechanical properties of the substrate and impact cellular functions such as cell migration. Mechanotransduction at focal adhesions affects the actomyosin network and contributes to cell migration. Despite being key players in cell adhesion and migration, the role of microtubules in mechanotransduction has been overlooked. Here, we show that substrate rigidity increases microtubule acetylation through β1 integrin signalling in primary rat astrocytes. Moreover, αTAT1, the enzyme responsible for microtubule acetylation, interacts with a major mechanosensing focal adhesion protein, Talin, and is able to tune the distribution of focal adhesions depending on the matrix rigidity. αTAT1 also reorganises the actomyosin network, increases traction force generation and cell migration speed on stiff substrates. Mechanistically, acetylation of microtubules promotes the release of microtubule-associated RhoGEF, GEF-H1 into the cytoplasm, which then leads to RhoA activation and high actomyosin contractility. Thus, we propose a novel feedback loop involving a crosstalk between microtubules and actin in mechanotransduction at focal adhesions whereby, cells sense the rigidity of the substrate through integrin-mediated adhesions, modulate their levels of microtubule acetylation, which then controls the actomyosin cytoskeleton and force transmission on the substrate to promote mechanosensitive cell migration.

2021-11-02 | PXD015871 | Pride

Proteomics exploiting modular baits with different subcellular locations reveal hydrogen peroxide-inducible clone 5 protein (HIC-5) and a glucocorticoid receptor variant as novel testin partners

Project description:The LIM domain and tumour suppressor protein Testin (Tes) is downregulated in a variety of human tumours and tumour cell lines. Depending on its conformation, Tes localises to stress fibres and focal adhesions where it forms protein complexes with other members of the actin cytoskeleton, such as zyxin and VASP, and thereby influences cellular processes like cell migration, adhesion or spreading. Tes is a modular protein and Tes variants lacking specific domains, localize to different locations in cells. To better understand the molecular basis of its function, we utilized an interaction proteomics approach combined with pathway analysis. This revealed proteins present in complexes in which Tes participates as a function of its modular structure as well as novel Tes interaction partners. We demonstrate that Tes interacts with a short isoform of the glucocorticoid receptor (GR), independently of the conventional full length GRα, a transcription factor important in regulating cellular metabolism and immune function. Tes also interacts with the focal adhesion protein and GR-coactivator Hic-5. In addition, we found that upon overexpression, Tes and Hic-5 induce opposite effects on cell spreading on a fibronectin matrix.

2018-10-26 | PXD005058 | Pride

Remodeling of the focal adhesion complex by hydrogen-peroxide- induced senescence

Project description:Cellular senescence is a phenotype characterized by cessation of cell division, which can be caused by exhaustive replication or environmental stress. It is involved in age-related pathophysiological conditions and affects both the cellular cytoskeleton and the prime cellular mechanosensors, focal adhesion complexes. While the size of focal adhesions increases during senescence, it is unknown if and how this is accompanied by a remodeling of the internal focal adhesion structure. Our study uses metal-induced energy transfer to study the axial dimension of focal adhesion proteins from oxidative-stress-induced senescent cells with nanometer precision, and compares these to unstressed cells. We influenced cytoskeletal tension and the functioning of mechanosensitive ion channels using drugs and studied the combined effect of senescence and drug intervention on the focal adhesion structure. We find that H 2 O 2 induced restructuring of the focal adhesion complex indicates a loss of tension and altered talin complexation. Mass spectroscopy-based proteomics confirmed the differential regulation of several cytoskeletal proteins induced by H 2 O 2 treatment.

2023-07-06 | PXD041766 | Pride

Transcription profiling of mouse fibroblasts derived from p130Cas exon 2-specific knockout mice.

Project description:p130Cas (Cas, Crk-associated substrate) is an adaptor molecule composed of an N-terminal Src homology 3 (SH3) domain, a substrate domain (SD), and a C-terminal Src binding domain (SBD). The SH3 domain of Cas has been shown to associate with various signaling molecules, including focal adhesion kinase (FAK), but its role in cellular function remains unclear. To address this issue, we established and analyzed primary fibroblasts derived from mice expressing a truncated Cas lacking the exon 2 encoding the SH3 domain (Cas exon 2â/â). In comparison to wild-type (Cas exon 2+/+) cells, Cas exon 2â/â primary fibroblasts showed delayed migration in wound healing and reduced spreading on fibronectin (FN), which would be due to reduced complex formation of Cas exon 2â/â with FAK and CrkII and also to impaired localization of Cas exon 2â/â to focal adhesions on FN. In addition, to analyze downstream signaling pathway regulated by Cas exon 2, we compared gene expression profiles between Cas exon 2+/+ and Cas exon 2â/â cells by a microarray analysis. Interestingly, we found that Cas exon 2-deficiency upregulated expression of CXC Chemokine Receptor-4 (CXCR4), CC Chemokine Receptor-5 (CCR5), and thrombospondin 4, which are implicated in cell motility and adhesion. These results define the role of Cas SH3-encoding exon in cell motility, FAK/Cas/CrkII complex formation, recruitment of Cas to focal adhesions, and regulation of cell adhesion-associated gene expression in primary fibroblasts. Experiment Overall Design: RNA samples extracted from Cas exon 2+/+, Cas exon 2+/â, and Cas exon 2â/â fibroblasts (12.5dpc, two embryos for each genotype) were labeled and hybridized on Affymetrix microarray. Gene expression patterns of Cas exon 2â/â fibroblasts were compared with those of Cas exon 2+/+ and Cas exon 2+/â cells.

2008-06-16 | E-GEOD-8357 | biostudies-arrayexpress

The ShcA adapter protein cooperates with LPP to mediate adhesion dynamics and invadopodia formation

Project description:ShcA and Lipoma Preferred Partner (LPP) are mediators of TGFβ-induced breast cancer cell migration and invasion. Reduced expression of either protein results in diminished breast cancer lung metastasis. Total internal reflection fluorescence (TIRF) microscopy reveals that TGFβ enhances the assembly and disassembly rates of paxillincontaining adhesions in a ShcA-dependent manner through the phosphorylation of specific ShcA tyrosine residues (Y239/Y240/Y313). Using a BioID proximity labeling approach, we show that ShcA exists in a complex with a variety of actin cytoskeletal proteins, including paxillin and LPP. Consistent with a functional interaction between ShcA and LPP, TGFβ-induced LPP localization to adhesions depends on ShcA. Once localized to cellular adhesions, LPP is required to mediate TGFβ-induced cell migration and adhesion dynamics. Mutations that impair LPP localization to adhesions (mLIM1) or interaction with the actin cytoskeleton via α-actinin (ΔABD) abrogate migratory responses to TGFβ. Live-cell TIRF microscopy reveals that ShcA clustering at the cell membrane precedes LPP recruitment. We hypothesize that ShcA promotes the formation of small, dynamic adhesions in the presence of TGFβ by acting as a nucleator of focal complex formation. Finally, we define a previously unknown function for ShcA in the formation of invadopodia, a process that also requires LPP. Our data reveals that ShcA controls the formation and function of two key cellular structures required for breast cancer metastasis.

2020-04-27 | PXD018265 | Pride

MaTAR25 lncRNA regulates the Tensin1 gene to impact breast cancer progression

Project description:Misregulation of long non-coding RNA genes has been linked to a wide variety of cancer types. Here we report on Mammary Tumor Associated RNA 25 (MaTAR25), a nuclear enriched and chromatin associated lncRNA that plays a role in mammary tumor cell proliferation, migration, and invasion, both in vitro and in vivo. MaTAR25 functions by interacting with purine rich element binding protein B, and associating with a major downstream target gene Tensin 1 (Tns1) to regulate its expression in trans. Knockout of MaTAR25 results in down-regulation of Tns1 leading to a reorganization of the actin cytoskeleton, and a reduction of focal adhesions and microvilli. The human ortholog of MaTAR25, LINC01271, is upregulated with human breast cancer stage and metastasis.

2020-10-19 | GSE142169 | GEO

Functional analysis of exon 2 of p130Cas in fibroblasts derived from exon 2-specific knockout mice.

Project description:p130Cas (Cas, Crk-associated substrate) is an adaptor molecule composed of an N-terminal Src homology 3 (SH3) domain, a substrate domain (SD), and a C-terminal Src binding domain (SBD). The SH3 domain of Cas has been shown to associate with various signaling molecules, including focal adhesion kinase (FAK), but its role in cellular function remains unclear. To address this issue, we established and analyzed primary fibroblasts derived from mice expressing a truncated Cas lacking the exon 2 encoding the SH3 domain (Cas exon 2∆/∆). In comparison to wild-type (Cas exon 2+/+) cells, Cas exon 2∆/∆ primary fibroblasts showed delayed migration in wound healing and reduced spreading on fibronectin (FN), which would be due to reduced complex formation of Cas exon 2∆/∆ with FAK and CrkII and also to impaired localization of Cas exon 2∆/∆ to focal adhesions on FN. In addition, to analyze downstream signaling pathway regulated by Cas exon 2, we compared gene expression profiles between Cas exon 2+/+ and Cas exon 2∆/∆ cells by a microarray analysis. Interestingly, we found that Cas exon 2-deficiency upregulated expression of CXC Chemokine Receptor-4 (CXCR4), CC Chemokine Receptor-5 (CCR5), and thrombospondin 4, which are implicated in cell motility and adhesion. These results define the role of Cas SH3-encoding exon in cell motility, FAK/Cas/CrkII complex formation, recruitment of Cas to focal adhesions, and regulation of cell adhesion-associated gene expression in primary fibroblasts. Keywords: SH3-encoding exon, p130Cas

2007-12-31 | GSE8357 | GEO

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