Targeting of FAK Ser⁹¹⁰ by ERK5 and PP1δ in non-stimulated and phorbol ester-stimulated cells

Ser⁹¹⁰ of FAK (focal adhesion kinase) was phosphorylated in fibroblasts treated with the phorbol ester PMA and dephosphorylated by PP1δ (protein phosphatase 1δ), as indicated by shRNA (small-hairpin RNA) gene silencing. Ser⁹¹⁰ 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 ERK1/2 pointed to the involvement of ERK5. This hypothesis was confirmed by: (i) shRNA ERK5 gene silencing, which resulted in complete pSer⁹¹⁰ 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 Ser⁹¹⁰ targeting by ERK5 also in these cells. Given the proximity of Ser⁹¹⁰ 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 Ser⁹¹⁰ in these processes. The present study indicates, for the first time, the phosphorylation of Ser⁹¹⁰ of FAK by ERK5 and its dephosphorylation by PP1δ, and suggested a role for Ser⁹¹⁰ in the control of cell shape and proliferation.