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The role of tyrosine phosphorylated caveolin-1 in regulating focal adhesion dynamics and cancer cell migration Strugnell, Scott Stuart

Abstract

Caveolin-1 (Cav1) is a conditional tumor suppressor whose expression is associated with a poor prognosis for many human cancer types. Cavi is a Src kinase substrate phosphorylated on tyrosine-14 (pYl4Cavl) that interacts with integrin and has been localized to focal adhesions (FAs). We undertook to study the role of pYl4Cavl in FA dynamics and tumor cell migration. Using FRAP analysis, we showed that pYl4Cavl phosphorylation and stabilization of FA kinase (FAK)-GFP in FAs occurs via a process that requires Rho/ROCK and Src signalling. In Cavi expressing MDA-231 breast carcinoma cells, pYl4Cavl was enriched in purified pseudopodial fractions while in low Cav1-expressing MDA-435 tumor cells, transfected Cav1, but not the Y1 4F mutant of Cay 1 -mRFP, was recruited to actin-rich protrusions. In MDA-435 tumor cells, transfected Cav1 and the phosphomimetic Cav1 mutant, Cav1Y14D, were found in close proximity to FA-associated proteins. The recruitment of either Cavl or Cav1Y14D to FAs was associated with increased cell migration. The positively charged Cav1Y14R mutant was not as closly associated with FA associated proteins, and increased cell spreading and stress fiber formation. The spatial association of pYl4Cavl with FAs may therefore regulate FA signaling and dynamics. The Mgat5 gene encodes the Golgi N-acetylglucosaminyltransferase-V that generates B1,6 branched N-glycans that bind galectin-3 at the cell surface forming a lattice domain. We found that the Mgat5/galectin lattice acts together with pYl4Cavl to stabilize FAK in FAs, thereby enhancing FA disassembly and de novo formation leading to the activation of directional tumor cell migration. Our data therefore argue that local interactions between the Mgat5/galectin lattice, Rho/ROCK and Src signalling and downstream phosphorylation of pY14Cav1 at FA sites promote tumor cell migration by regulating local FA dynamics in protrusive domains of tumor cells.

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