WDR1 is a novel EYA3 substrate and its dephosphorylation induces modifications of the cellular actin cytoskeleton

Abstract

Eyes absent (EYA) proteins are unusual proteins combining in a single polypeptide chain transactivation, threonine phosphatase, and tyrosine phosphatase activities. They play pivotal roles in organogenesis and are involved in a variety of physiological and pathological processes including innate immunity, DNA damage repair or cancer metastasis. The molecular targets of EYA tyrosine phosphatase activity are still elusive. Therefore, we sought to identify novel EYA substrates and also to obtain further insight into the tyrosine-dephosphorylating role of EYA proteins in various cellular processes. We show here that Src kinase phosphorylates tyrosine residues in two human EYA family members, EYA1 and EYA3. Both can autodephosphorylate these residues and their nuclear and cytoskeletal localization seems to be controlled by Src phosphorylation. Next, using a microarray of phosphotyrosine-containing peptides, we identified a phosphopeptide derived from WD-repeat-containing protein 1 (WDR1) that is dephosphorylated by EYA3. We further demonstrated that several tyrosine residues on WDR1 are phosphorylated by Src kinase, and are efficiently dephosphorylated by EYA3, but not by EYA1. The lack of phosphorylation generates major changes to the cellular actin cytoskeleton. We, therefore, conclude that WDR1 is an EYA3-specific substrate, which implies that EYA3 is a key modulator of the cytoskeletal reorganization.

Analysis of EYA3 Phosphorylation by Src Kinase Identifies Residues Involved in Cell Proliferation

“…Recently, we have demonstrated that c-Src phosphorylates tyrosine residues of human EYA1 and EYA3 to control their nuclear and cytoskeletal localization [30]. We have also found that EYA1 and EYA3 are capable of autodephosphorylation [30]. These data indicate a potential implication of EYA tyrosine phosphorylation and autodephosphorylation in regulating physiological processes and contributing to pathological conditions.…”

“…For example, c-Src activation has been reported to generate more than 50% of tumors in liver, colon, breast, lung, and pancreas [35]. Recently, we have demonstrated that c-Src phosphorylates tyrosine residues of human EYA1 and EYA3 to control their nuclear and cytoskeletal localization [30]. We have also found that EYA1 and EYA3 are capable of autodephosphorylation [30].…”

“…The interaction between two EYA3 molecules is mediated by the ED and depends on Src-induced phosphorylation [30]. However, it is still unclear how many tyrosine residues of EYA3 are phosphorylated by Src and, subsequently, autodephosphorylated.…”

“…Incubation of EYA3 WT with Src and ATP for 2 h generated molecules with only one phosphorylated residue (Figure 1c, Figure S3). However, molecules with up to three phosphorylated residues were observed after only 5 min when using a phosphatase-deficient mutant of EYA3 (EYA3 D311N) [23,27,30] (Figure S5). After 2 h, up to 12 phosphorylation sites were observed on EYA3 D311N (Figure 1b versus Figure 1d, when compared to Figure S5, the intensities of the signals corresponding to the unphosphorylated molecules decreased and those of the phosphorylated molecules increased) highlighting the importance of the ED in controlling the phosphorylation status of EYA3.…”

“…Despite the large number of reports implicating EYA proteins in pathological conditions, limited information is available regarding their substrates. So far, three physiological substrates for EYA's PTP activity have been identified: histone H2A.X (phosphotyrosine-pY-142) [27,28], estrogen receptor β (pY36) [29], which both have nuclear localization, and WD repeat-containing protein 1 (WDR1), which is a cytoskeletal protein [30].…”