Targeting of FAK Ser(910) by ERK5 and PP1 delta in non-stimulated and phorbol ester-stimulated cells

Set(910) of FAK (focal adhesion kinase) was phosphorylated in fibroblasts treated with the phorbol ester PMA and dephosphorylated by PP1 delta (protein phosphatase 18), as indicated by shRNA (small-hairpin RNA) gene silencing. Ser(910) of FAK was reported previously to be an ERK (extracellular-signal-regulated kinase) 1/2 target in cells treated with phorbol esters. In contrast, various approaches, including the use of the MEK (mitogen-activated protein kinase/ERK kinase) inhibitors UO126 and CI-1040 to inhibit ERK 1/2 pointed to the involvement of ERK5. This hypothesis was confirmed by: (i) shRNA ERK5 gene silencing, which resulted in complete pSer(910) loss in non-stimulated and PMA-stimulated cells; (ii) direct phosphorylation of recombinant FAK by ERK5; and (iii) ERK5 activation by PMA. PMA stimulation and ERK5 silencing in MDA-MB 231 and MDA-MB 361 breast cancer cells indicated Ser(910) targeting by ERK5 also in these cells. Given the proximity of Set(910) to the FAT (focal adhesion targeting) regulatory domain of FAK, cell proliferation and morphology were investigated in FAK(-/-) cells expressing S910A mutant FAK. The cell growth rate decreased and exposure to PMA induced peculiar morphological changes in cells expressing S910A, with respect to wild-type FAK, suggesting a role for Ser(910) in these processes. The present study indicates, for the first time, the phosphorylation of Ser(910) of FAK by ERK5 and its dephosphorylation by PP1 delta, and suggested a role for Ser(910) in the control of cell shape and proliferation.