Abstract
ABSTRACTCell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration.
Highlights
IntroductionProtein tyrosine phosphatases, including PTP1B, have been established as important regulators of integrin-mediated signal transduction implied in cytoskeletal rearrangements and cell migration (Larsen et al, 2003; Burridge et al, 2006; Arregui et al, 2013).PTP1B is an endoplasmic reticulum (ER)-anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics (Frangioni et al, 1992; Hernández et al, 2006; Anderie et al, 2007; Fuentes and Arregui, 2009; Nievergall et al, 2010; Haj et al, 2012; Monteleone et al, 2012; Burdisso et al, 2013)
We hypothesized that inefficient activation of the Src/ FAK signaling pathway in KO cells impairs lamellipodium and adhesion formation due to enhanced myosin activity. To test this we examined whether myosin inhibition by blebbistatin restores integrin-dependent signaling, lamellipodium and adhesion assembly at the periphery of KO cells plated for 10 min on fibronectin
Our results suggest that early after cell contact with the matrix, PTP1B cooperates with β3 integrins to activate a Src/FAK signaling pathway leading to the transient repression of RhoA and myosindependent contractility, allowing adhesion and lamellipodium assembly
Summary
Protein tyrosine phosphatases, including PTP1B, have been established as important regulators of integrin-mediated signal transduction implied in cytoskeletal rearrangements and cell migration (Larsen et al, 2003; Burridge et al, 2006; Arregui et al, 2013).PTP1B is an endoplasmic reticulum (ER)-anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics (Frangioni et al, 1992; Hernández et al, 2006; Anderie et al, 2007; Fuentes and Arregui, 2009; Nievergall et al, 2010; Haj et al, 2012; Monteleone et al, 2012; Burdisso et al, 2013). PTP1B dephosphorylates the autoinhibitory tyrosine of Src (Tyr 529 in mouse), contributing to its activation (Arregui et al, 1998; Bjorge et al, 2000). BiFC (bimolecular fluorescence complementation) analysis demonstrated that ER-bound PTP1B targets Src associated with the plasma membrane in contact with the substrate (Monteleone et al, 2012).
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