Phosphorylation on threonine 11 of β-dystrobrevin alters its interaction with kinesin heavy chain

Abstract

Dystrobrevin family members (α and β) are cytoplasmic components of the dystrophin-associated glycoprotein complex, a multimeric protein complex first isolated from skeletal muscle, which links the extracellular matrix to the actin cytoskeleton. Dystrobrevin shares high homology with the cysteine-rich and C-terminal domains of dystrophin and a common domain organization. The β-dystrobrevin isoform is restricted to nonmuscle tissues, serves as a scaffold for signaling complexes, and may participate in intracellular transport through its interaction with kinesin heavy chain. We have previously characterized the molecular determinants affecting the β-dystrobrevin- kinesin heavy chain interaction, among which is cAMP-dependent protein kinase [protein kinase A (PKA)] phosphorylation of β-dystrobrevin. In this study, we have identified β-dystrobrevin residues phosphorylated in vitro by PKA with pull-down assays, surface plasmon resonance measurements, and MS analysis. Among the identified phosphorylated residues, we demonstrated, by site-directed mutagenesis, that Thr11 is the regulatory site for the β-dystrobrevin- kinesin interaction. As dystrobrevin may function as a signaling scaffold for kinases/phosphatases, we also investigated whether β-dystrobrevin is phosphorylated in vitro by kinases other than PKA. Thr11 was phosphorylated by protein kinase C, suggesting that this represents a key residue modified by the activation of different signaling pathways. Structured digital abstract Kif5a binds to β-DB by surface plasmon resonance (View interaction) β-DB physically interacts with Kinesin and syntrophin. by pull down (View interaction) β-dystrobrevin, a component of the dystrophin complex, can be phosphorylated by PKA and PKC. By mass spectrometry analysis, we have identified β-dystrobrevin residues phosphorylated in vitro by both the kinases and, using pull-down assays and surface plasmon resonance measurements, demonstrated that threonine 11 is a key residue for β-dystrobrevin-kinesin interaction. © 2012 The Authors Journal compilation © 2012 FEBS.