Phosphorylation of Trask by Src Kinases Inhibits Integrin Clustering and Functions in Exclusion with Focal Adhesion Signaling

Department of Medicine, University of California, San Francisco, San Francisco, CA 94143-1387, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 02/2011; 31(4):766-82. DOI: 10.1128/MCB.00841-10
Source: PubMed


Trask is a recently described transmembrane substrate of Src kinases whose expression and phosphorylation has been correlated
with the biology of some cancers. Little is known about the molecular functions of Trask, although its phosphorylation has
been associated with cell adhesion. We have studied the effects of Trask phosphorylation on cell adhesion, integrin activation,
clustering, and focal adhesion signaling. The small hairpin RNA (shRNA) knockdown of Trask results in increased cell adhesiveness
and a failure to properly inactivate focal adhesion signaling, even in the unanchored state. On the contrary, the experimentally
induced phosphorylation of Trask results in the inhibition of cell adhesion and inhibition of focal adhesion signaling. This
is mediated through the inhibition of integrin clustering without affecting integrin affinity state or ligand binding activity.
Furthermore, Trask signaling and focal adhesion signaling inactivate each other and signal in exclusion with each other, constituting
a switch that underlies cell anchorage state. These data provide considerable insight into how Trask functions to regulate
cell adhesion and reveal a novel pathway through which Src kinases can oppose integrin-mediated cell adhesion.

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    • "The residual focus forming capacity of the single Y734 mutant supports its prominent role that has been described earlier [11], [30] but is not sufficient for efficient transformation and implicates additional pathways for CDCP1 signaling. Recently, Spassov et al. [31] showed that phosphorylation of the major tyrosine sites of CDCP1 inhibited cell adhesion and focal adhesion signaling by preventing integrin clustering. Also in their system, overexpression of a YΔF mutant lacking three intracellular Tyr residues was no longer able to inhibit cell adhesion. "
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    ABSTRACT: Cub domain containing protein 1 (CDCP1) is strongly expressed in tumors derived from lung, colon, ovary, or kidney. It is a membrane protein that is phosphorylated and then bound by Src family kinases. Although expression and phosphorylation of CDCP1 have been investigated in many tumor cell lines, the CDCP1 features responsible for transformation have not been fully evaluated. This is in part due to the lack of an experimental system in which cellular transformation depends on expression of exogenous CDCP1 and Src. Here we use retrovirus mediated co-overexpression of c-Src and CDCP1 to induce focus formation of NIH3T3 cells. Employing different mutants of CDCP1 we show that for a full transformation capacity, the intact amino- and carboxy-termini of CDCP1 are essential. Mutation of any of the core intracellular tyrosine residues (Y734, Y743, or Y762) abolished transformation, and mutation of a palmitoylation motif (C689,690G) strongly reduced it. Src kinase binding to CDCP1 was not required since Src with a defective SH2 domain generated even more CDCP1 dependent foci whereas Src myristoylation was necessary. Taken together, the focus formation assay allowed us to define structural requirements of CDCP1/Src dependent transformation and to characterize the interaction of CDCP1 and Src.
    PLoS ONE 12/2012; 7(12):e53050. DOI:10.1371/journal.pone.0053050 · 3.23 Impact Factor
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    • "They observed that integrin clusterings in the periphery of the cell occur within minutes, starts about after 3 minutes. There are also many recent studies on integrin clustering, such as by [19] [48] [39] [52] [55] [2] [25] [45]. "
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    ABSTRACT: When invading the tissue, malignant tumour cells (i.e. cancer cells) need to detach from neighbouring cells, degrade the basement membrane, and migrate through the extracellular matrix. These processes require loss of cell-cell adhesion and enhancement of cell-matrix adhesion. In this paper we present a mathematical model of an intracellular pathway for the interactions between a cancer cell and the extracellular matrix. Cancer cells use similar mechanisms as with normal cells for their interactions with the extracellular matrix. We develop a model of cell-matrix adhesion that accounts for reactions between the cell surface receptor integrins, the matrix glycoprotein fibronectin, and the actin filaments in the cytoskeleton. Each represents components for an intermediate compartment, the extracellular compartment, and the intracellular compartment, respectively. Binding of fibronectin with integrins triggers a clustering of protein complexes, which then activates and phosphorylates regulatory proteins that are involved in actin reorganisation causing actin polymerization and stress fibre assembly. Rearrangement of actin filaments with integrin/fibronectin complexes near adhesion sites and interaction with fibrillar fibronectin produces the force necessary for cell migration, accounting for cell-matrix adhesion.
    Mathematical Modelling of Natural Phenomena 01/2012; 7(1-1):29-48. DOI:10.1051/mmnp/20127103 · 0.81 Impact Factor
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    • "The mechanisms by which Trask may suppress tumor progression is unknown. We have shown that Trask, when phosphorylated, functions to inhibit integrin signaling, disrupt focal adhesions, and oppose cell adhesion (Spassov et al 2011b). Clearly integrin signaling and cell adhesion are functions that tumor cells require for processes of migration and metastases and this function of Trask may be tumor suppressive (Brakebusch et al 2002, Felding- Habermann 2003, Hood and Cheresh 2002, Ramsay et al 2007). "
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    ABSTRACT: Trask/CDCP1 is a transmembrane glycoprotein widely expressed in epithelial tissues whose functions are just beginning to be understood, but include a role as an anti-adhesive effector of Src kinases. Early studies looking at RNA transcript levels seemed to suggest overexpression in some cancers, but immunostaining studies are now providing more accurate analyses of its expression. In an immuno-histochemical survey of human cancer specimens, we find that Trask expression is retained, reduced or sometimes lost in some tumors compared with their normal epithelial tissue counterparts. A survey of human cancer cell lines also show a similar wide variation in the expression of Trask, including some cell types with the loss of Trask expression, and additional cell types that have lost the physiological detachment-induced phosphorylation of Trask. Three experimental models were established to interrogate the role of Trask in tumor progression, including two gain-of-function models with tet-inducible expression of Trask in tumor cells lacking Trask expression, and one loss-of-function model to suppress Trask expression in tumor cells with abundant Trask expression. The induction of Trask expression and phosphorylation in MCF-7 cells and in 3T3v-src cells was associated with a reduction in tumor metastases while the shRNA-induced knockdown of Trask in L3.6pl cancer cells was associated with increased tumor metastases. The results from these three models are consistent with a tumor-suppressing role for Trask. These data identify Trask as one of several potential candidates for functionally relevant tumor suppressors on the 3p21.3 region of the genome frequently lost in human cancers.
    Oncogene 06/2011; 31(4):419-31. DOI:10.1038/onc.2011.246 · 8.46 Impact Factor
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