Paxillin nuclear-cytoplasmic localization is regulated by phosphorylation of the LD4 motif: evidence that nuclear paxillin promotes cell proliferation

Paxillin, a major focal-adhesion complex component belongs to the subfamily of LIM domain proteins and participates in cell adhesion-mediated signal transduction. It is implicated in cell-motility responses upon activation of cell-surface receptors and can recruit, among others, the GIT1 [GRK (G-protein-coupled-receptor kinase)-interacting ARF (ADP-ribosylation factor) GAP (GTPase-activating protein)]–PIX [PAK (p21-activated kinase)-interacting exchange factor]–PAK1 complex. Several adhesion proteins including zyxin, Hic5 and Trip6 are also nuclear and can exert transcriptional effects. In the present study we show that endogenous paxillin shuttles between the cytoplasm and nucleus, and we have used a variety of tagged paxillin constructs to map the nuclear export signal. This region overlaps an important LD4 motif that binds GIT1 and FAK1 (focal-adhesion kinase 1). We provide evidence that phosphorylation of Ser272 within LD4 blocks nuclear export, and we show that this modification also reduces GIT1, but not FAK1, binding; however, Ser272 phosphorylation does not appear to be mediated by PAK1 as previously suggested. Expression of nuclear-localized paxillin LIM domains stimulate DNA synthesis and cell proliferation. By real-time PCR analysis we have established that overexpression of either full-length paxillin or a truncated nuclear form suppresses expression of the parental imprinted gene H19, and modulation of this locus probably affects the rate of NIH-3T3 cell proliferation. Abbreviations: BrdU, bromodeoxyuridine; CRM1, chromosome region maintenance 1; CRP, cysteine-rich protein; DMEM, Dulbecco's modified Eagle's medium; FAK, focal-adhesion kinase; FCS, foetal calf serum; GFP, green fluorescent protein; GIT1, GRK (G-protein-coupled-receptor kinase)-interacting ARF (ADP-ribosylation factor) GAP (GTPase-activating protein); GIT1-C, the C-terminal of GIT-1; GST, glutathione transferase; Igf2, insulin-like growth factor 2; IMP1, Igf2 mRNA binding protein 1; LHX, LIM homeodomain protein; mGFP, monomeric GFP; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NES, nuclear export signal; NLS, nuclear localization signal(s); PABP1, polyA-binding protein 1; PAK1, p21-activated kinase 1; PFA, paraformaldeyhde; β-PIX, β PAK-interacting exchange factor