Tyro3-mediated phosphorylation of ACTN4 at tyrosines is FAK-dependent and decreases susceptibility to cleavage by m-Calpain.

In this study, we sought the molecular mechanism of Tyro3 effects avoiding endogenous background by overexpression of Tyro3 in fibroblasts that have negligible levels of Tyro3. This introduction triggers the tyrosyl-phosphorylation of ACTN4, a member of actin binding protein family involved in motility, a behavior critical for invasive progression, as shown by siRNA to Tyro3 limiting melanoma cell migration and invasion. Tyro3-mediated phosphorylation of ACTN4 required FAK activation at tyrosine 397 and the EGF receptor cascade, but not EGFR ligand binding. Using PCR-based mutagenesis, the sites of Tyro3-mediated ACTN4 phosphorylation were mapped to ACTN4 tyrosine 11 and 13, and this occurs in conjunction with EGF-mediated phosphorylation on Y4 and Y31. Interestingly, Tyro3-mediated phosphorylation only slightly decreases the actin binding activity of ACTN4. However, this rendered the phosphorylated ACTN4 resistant to the m-calpain cleavage between Y13 and G14, a limited proteolysis that prevents growth factor regulation of ACTN4 interaction with F-actin. Overexpression of both WT ACTN4 and ACTN4Y11/13E, a mimic of ACTN4 phosphorylated at tyrosine 11 and 13, in melanoma WM983b cells resulted in a likely mesenchymal to amoeboidal transition. ACTN4Y11/13E-expressing cells were more amoeboidal, less migratory on collagen I gel coated surface but more invasive through collagen networks. In parallel, expression of ACTN4Y11/13E, in ACTN4 knockdown melanoma WM1158 cells resulted in ...

Source: The International Journal of Biochemistry and Cell Biology - December 20, 2017 Category: Biochemistry Authors: Shao H, Wang A, Lauffenburger D, Wells A Tags: Int J Biochem Cell Biol Source Type: research