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Syndecans are transmembrane proteoglycans that act as receptors for extracellular matrix molecules and co-receptors for growth factors, cytokines and morphogens. Syndecan-4 is a ubiquitously expressed member of the syndecan family and encompasses a unique PKCα-binding motif within its variable region. Engagement of syndecan-4 by fibronectin regulates Rho family guanosine triphosphatase (Rho GTPase) activity to promote focal adhesion formation and reorganisation of the actin cytoskeleton that is dependent on syndecan-4-mediated PKCα activity. Recent work in our laboratory has demonstrated a role for syndecan-4 in the dynamic recycling of other fibronectin receptors, α5β1 and αVβ3 integrins. Here, it is demonstrated that phosphorylation of syndecan-4 mediated by another PKC isoform, PKCδ, and a non-receptor tyrosine kinase, Src, regulates syndecan-4-dependent GTPase activity, heterodimer-specific integrin recycling and focal adhesion formation, providing a mechanism for spatiotemporal control of cell migration.Src is associated with processes regulating adhesion disassembly and cell migration, and aberrant activation of Src contributes to neoplastic progression. Using in vitro kinase assays, syndecan-4 was identified as a target for Src-mediated phosphorylation. Mutagenesis studies coupled with mass spectrometric analysis of phosphorylation indicated the existence of two novel Src phosphorylation sites within the syndecan-4 cytoplasmic domain, tyrosine180 (Y180) and tyrosine197 (Y197). Importantly, modulation of the phospho-competence of the PKCδ-phosphorylation site (Serine179 (S179)) within syndecan-4 cytoplasmic domain increased Src-mediated syndecan-4 phosphorylation by promoting preferential phosphorylation of Y180 over Y197. Thus, PKCδ primes syndecan-4 for Src-mediated phosphorylation, functioning as a molecular switch to regulate phosphorylation of alternative tyrosine residues. In cells, Src-mediated phosphorylation of Syn4Y180 suppressed activity of the small GTPase Arf6. Similarly, phosphomimetic mutation of the PKCδ-phosphorylation site within syndecan-4 inhibited Arf6 activation in response to fibronectin engagement. These results suggested that PKCδ-dependent phosphorylation of syndecan-4 regulated Arf6 activation by controlling Src-dependent Y180 phosphorylation. Furthermore, suppression of Arf6 activity or perturbation of the S179 residue promoted membrane delivery of internalised αVβ3 integrin but not α5β1. Thus, syndecan-4-dependent Arf6 activity regulates differential recycling of α5β1 and αVβ3 integrins and is controlled by phosphorylation by PKCδ and Src. As, the other identified Src kinase target, Y197, is located within the PDZ-binding motif of syndecan-4, it was hypothesised that phosphorylation of this residue may regulate the interaction of syndecan-4 with PDZ-domain-containing proteins. Protein binding to GST-syndecan-4 constructs was assessed in pull-down assays. Interestingly, binding of syntenin, a PDZ-domain containing syndecan-4 binding partner, was inhibited in the presence of a phosphomimetic Y197 residue. By contrast, syntenin binding was enhanced in PKCδ-phosphorylation site mutants of syndecan-4 that decrease Src-dependent Y197 phosphorylation. Therefore, the phosphorylation state of Y197 is a critical determinant of syndecan-4-PDZ-binding motif interactions. Intriguingly, cells expressing a syndecan-4 receptor defective for PDZ interactions exhibited constitutive activation of Arf6 and a severe defect in syndecan-4 and integrin receptor internalisation. These data suggest that syndecan-4 endocytic pathways may be intimately associated with integrin internalisation and that interactions at the PDZ-binding motif of syndecan-4 may be critical for regulating syndecan-4 internalisation.Finally, it was demonstrated that ECM engagement of syndecan-4 induces a transient wave of PKCα activity and that this is dependent on the integrity of the PKCδ-phosphorylation site. Furthermore, perturbation of S179 or siRNA-mediated knockdown of PKCδ resulted in suppressed Rac1 activity and enhanced focal adhesion formation. Thus, PKCδ-mediated phosphorylation of syndecan-4 appears to regulate several key processes involved in cell migration including cell-surface expression of integrin heterodimers, GTPase activity and focal adhesion formation. Consequently, cells expressing a syndecan-4 receptor with mutations in the PKCδ-phosphorylation site exhibited defects in migration speed and persistence on cell-derived matrices. Together, these data suggest that in migrating cells PKCδ activity is essential to coordinate differential phosphorylation of syndecan-4 by Src on two separate residues, to spatially and temporally restrict GTPase activity, heterodimer-specific integrin recycling and focal adhesion formation. |
