Phosphorylation of tyrosine 285 of PAK1 facilitates βPIX/GIT1 binding and adhesion turnover

Abstract

The p21-activated serine-threonine kinase (PAK1) regulates cell motility and adhesion. We have previously shown that the prolactin (PRL)-activated tyrosine kinase JAK2 phosphorylates PAK1 in vivo and in vitro and identified tyrosines 153, 201, and 285 in PAK1 as sites of JAK2 tyrosyl phosphorylation. Here, we further investigate the role of the tyrosyl phosphorylated PAK1 (pTyr-PAK1) in regulation of cell adhesion. We use human breast cancer T47D cell lines that stably overexpress PAK1 wild type or PAK1 Y3F mutant in which these 3 JAK2 phosphorylation sites were mutated to phenylalanine. We demonstrate that PRL/JAK2-dependent phosphorylation of these tyrosines promotes a motile phenotype in the cells upon adhesion, participates in regulation of cell adhesion on collagen IV, and is required for maximal PAK1 kinase activity. Down-regulation of PAK1 abolishes the effect of PAK1 on cell adhesion. We show that the tyrosyl phosphorylation of PAK1 promotes PAK1 binding to β-PAK1-interacting guanine-nucleotide exchange factor (βPIX) and G protein-coupled receptor kinase-interacting target 1 (GIT1), phosphorylation of paxillin on Ser273, and formation and distribution of adhesion complexes. Using phosphospecific antibodies (Abs) directed to single phosphorylated tyrosines on PAK1, we identified Tyr285 as a site of PRL-dependent phosphorylation of PAK1 by JAK2. Furthermore, using PAK1 Y285F mutant, we provide evidence for a role of pTyr285 in cell adhesion, enhanced βPIX/GIT1 binding, and adhesion turnover. Our immunohistochemistry analysis demonstrates that pTyr285- PAK1 may modulate PAK1 signaling during tumor progression.

Synergistic Activation of ERα by Estrogen and Prolactin in Breast Cancer Cells Requires Tyrosyl Phosphorylation of PAK1

“…Immunoprecipitation, PAK1 in vitro kinase assay and gene silencing were performed as described in [21]. Briefly, IP’d PAK1 was subjected to an in vitro kinase assay in the presence of 10 μCi of [γ- 32 P]ATP and histone H4 (substrate of PAK1).…”

“…Tyrosyl phosphorylation of PAK1 (pTyr-PAK1) enhances PAK1 kinase activity and its ability to form protein/protein interactions. Both PAK1 activities are important for PRL-dependent breast cancer cell adhesion, motility, and invasion [19,20,21]. pTyr-PAK1 also regulates PRL-induced cyclin D1 promoter activity [22].…”

The serum/PDGF-dependent "melanogenic" role of the minute level of the oncogenic kinase PAK1 in melanoma cells proven by the highly sensitive kinase assay

“…It has been shown that α-MSH activates the Tyrkinase JAK2 (18), which in turn activates PAK1 by the phosphorylation at Tyr 285, (instead of Thr 423), leading to the PIX-PAK1 interaction (19). This could explain why neither α-MSH-dependent activation of PAK1 nor melanogenesis involves the autophosphorylation of PAK1 at Thr 423.…”

PAK1 translocates into nucleus in response to prolactin but not to estrogen

“…Tyrosyl phosphorylation of PAK1 (pTyr-PAK1) enhances such important PAK1 functions as kinase activity and the ability to form protein/protein interactions that are important for adhesion, motility, and invasion of breast cancer cells in response to PRL ([14,15,16]; reviewed in [17]). We have also previously demonstrated that the three tyrosines on PAK1 molecules and PAK1-Nck interaction play a critical role in PAK1-dependent regulation of cyclin D1 promoter activity in response to PRL [18].…”