Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function
| Autor: | Brian A. Joughin, Hanshuang Shao, Carlos J. Camacho, Timothy Travers, Douglas A. Lauffenburger, Alan Wells |
|---|---|
| Přispěvatelé: | Massachusetts Institute of Technology. Department of Biological Engineering, Koch Institute for Integrative Cancer Research at MIT, Joughin, Brian A., Lauffenburger, Douglas A. |
| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
Mutant
Mutation Missense macromolecular substances Molecular Dynamics Simulation Models Biological Biochemistry Article Receptor tyrosine kinase Cell Line Phosphorylation cascade Structure-Activity Relationship chemistry.chemical_compound Epidermal growth factor Humans Actinin Protein phosphorylation Phosphorylation Molecular Biology Epidermal Growth Factor biology Calpain Kinase Tyrosine phosphorylation Cell Biology Molecular biology chemistry biology.protein Biophysics |
| Zdroj: | PMC |
| Popis: | 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. National Institutes of Health (U.S.) (Grant R01 GM69668) |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|