Abstract
FAK is a non-receptor tyrosine kinase contributing to migration and proliferation downstream of integrin and/or growth factor receptor signaling of normal and malignant cells. In addition to well-characterized tyrosine phosphorylations, FAK is phosphorylated on several serines, whose role is not yet clarified. We observed that phosphorylated FAK on serine 732 (P-FAKSer732) is present at variable levels in vitro, in several melanoma, ovarian and thyroid tumor cell lines and in vivo, in tumor cells present in fresh ovarian cancer ascites. In vitro P-FAKSer732 was barely detectable during interphase while its levels strongly increased in mitotic cells upon activation of the EGFR/MEK/ERK axis in an integrin-independent manner. P-FAKSer732 presence was crucial for the maintenance of the proliferation rate and its levels were inversely related to the levels of acetylated α-tubulin. P-FAKSer732 localized at the microtubules (MTs) of the spindle, biochemically associated with MTs and contributed to MT depolymerization. The lack of the phosphorylation on Ser732 as well as the inhibition of CDK5 activity by roscovitine impaired mitotic spindle assembly and correct chromosome alignment during mitosis. We also identified, for the first time, that the EGF-dependent EGFR activation led to increased P-FAKSer732 and polymerized MTs. Our data shed light on the multifunctional roles of FAK in neoplastic cells, being involved not only in integrin-dependent migratory signaling but also in integrin-independent MT dynamics and mitosis control. These findings provide a new potential target for inhibiting the growth of tumor cells in which the EGFR/MEK/ERK/CDK5 pathway is active.
Highlights
Can influence cell movement regulating cytoskeleton remodeling, focal adhesions (FAs) turnover and membrane protrusions.[1]
In all cell lines the expression of focal adhesion kinase (FAK) was associated with the presence of P-FAKSer[732] and -Tyr[576], at variable levels (Figure 1a)
Western blot analysis of Me#28 samples following immunoprecipitation (IP) with anti-P-FAKSer[732] antibody (Ab) indicated that the principal form in the immunoprecipitate consisted of FAK phosphorylated on Ser[732] (Figure 1e), whereas the dominant form in the unbound fraction contained mainly P-FAKTyr[576]. These results demonstrate that FAK exists as two distinct pools: the first associated with FAs and the second, phosphorylated on Ser[732], localized in the cytoplasm in a perinuclear region
Summary
Can influence cell movement regulating cytoskeleton remodeling, FA turnover and membrane protrusions.[1]. FRNK inhibits both integrinand growth factor-induced FAK activation and blocks FAK-mediated cell migration and proliferation.[9]. The COOH-terminal domain of FAK undergoes several serine (Ser) phosphorylation events whose role is not well understood. Phosphorylation of FAK at Ser[732] (P-FAKSer732), by the post-mitotic stability and migration by pulling the proximal region of the nucleus into the leading process, likely in the direction of the centrosome.[10,11] In endothelial cells (ECs), P-FAKSer[732], phosphorylated by Rho-associated protein kinase (ROCK),[12] has been found co-localized with g-tubulin at the centrosomes; deletion of FAK in primary ECs causes an increase in centrosome numbers, and multipolar and disorganized spindles.[13]. P-FAKSer[732] has been characterized mainly in normal cells.[10,12,13] Here, we aimed to analyze in detail the presence and the role of P-FAKSer[732] in tumor cells
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