Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function
Author(s)Travers, Timothy; Shao, Hanshuang; Joughin, Brian A.; Lauffenburger, Douglas A.; Wells, Alan; Camacho, Carlos J.; ... Show more Show less
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Phosphorylated residues occur preferentially in the intrinsically disordered regions of eukaryotic proteins. In the disordered amino-terminal region of human a-actinin-4 (ACTN4), Tyr[superscript 4] and Tyr[superscript 31] are phosphorylated in cells stimulated with epidermal growth factor (EGF), and a mutant with phosphorylation-mimicking mutations of both tyrosines exhibits reduced interaction with actin in vitro. Cleavage of ACTN4 by m-calpain, a protease that in motile cells is predominantly activated at the rear, removes the Tyr[superscript 4] site. We found that introducing a phosphomimetic mutation at only Tyr[superscript 31] was sufficient to inhibit the interaction with actin in vitro. However, molecular dynamics simulations predicted that Tyr[superscript 31] is mostly buried and that phosphorylation of Tyr[superscript 4] would increase the solvent exposure and thus kinase accessibility of Tyr[superscript 31]. In fibroblast cells, EGF stimulation increased tyrosine phosphorylation of a mutant form of ACTN4 with a phosphorylation-mimicking residue at Tyr[superscript 4], whereas a truncated mutant representing the product of m-calpain cleavage exhibited EGF-stimulated tyrosine phosphorylation at a background amount similar to that observed for a double phosphomimetic mutant of Tyr[superscript 4] and Tyr[superscript 31]. We also found that inhibition of the receptor tyrosine kinases of the TAM family, such as AXL, blocked EGF-stimulated tyrosine phosphorylation of ACTN4. Mathematical modeling predicted that the kinetics of phosphorylation at Tyr[superscript 31] can be dictated by the kinase affinity for Tyr[superscript 4]. This study suggests that tandem-site phosphorylation within intrinsically disordered regions provides a mechanism for a site to function as a switch to reveal a nearby function-regulating site.
DepartmentDavid H. Koch Institute for Integrative Cancer Research at MIT; Massachusetts Institute of Technology. Department of Biological Engineering
American Association for the Advancement of Science (AAAS)
Travers, T., H. Shao, B. A. Joughin, D. A. Lauffenburger, A. Wells, and C. J. Camacho. “Tandem Phosphorylation Within an Intrinsically Disordered Region Regulates ACTN4 Function.” Science Signaling 8, no. 378 (May 26, 2015): ra51–ra51.
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