Format

Send to

Choose Destination
Sci Signal. 2015 May 26;8(378):ra51. doi: 10.1126/scisignal.aaa1977.

Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function.

Author information

1
Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA.
2
Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15260, USA.
3
Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
4
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
5
Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA. ccamacho@pitt.edu.

Abstract

Phosphorylated residues occur preferentially in the intrinsically disordered regions of eukaryotic proteins. In the disordered amino-terminal region of human α-actinin-4 (ACTN4), Tyr(4) and Tyr(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(4) site. We found that introducing a phosphomimetic mutation at only Tyr(31) was sufficient to inhibit the interaction with actin in vitro. However, molecular dynamics simulations predicted that Tyr(31) is mostly buried and that phosphorylation of Tyr(4) would increase the solvent exposure and thus kinase accessibility of Tyr(31). In fibroblast cells, EGF stimulation increased tyrosine phosphorylation of a mutant form of ACTN4 with a phosphorylation-mimicking residue at Tyr(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(4) and Tyr(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(31) can be dictated by the kinase affinity for Tyr(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.

PMID:
26012634
PMCID:
PMC4522051
DOI:
10.1126/scisignal.aaa1977
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center