Analysis of phosphorylation of the myosin targeting subunit of smooth muscle myosin light chain phosphatase by Phos‐tag SDS‐PAGE

Abstract

Phosphorylation of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase plays an important role in the regulation of smooth muscle contraction, and several sites of phosphorylation by different protein Ser/Thr kinases have been identified. Furthermore, in some instances, phosphorylation at specific sites affects phosphorylation at neighboring sites, with functional consequences. Characterization of the complex phosphorylation of MYPT1 in tissue samples at rest and in response to contractile and relaxant stimuli is, therefore, challenging. We have exploited Phos‐tag SDS‐PAGE in combination with western blotting using antibodies to MYPT1 to separate multiple phosphorylated MYPT1 species and quantify MYPT1 phosphorylation stoichiometry using purified, full‐length recombinant MYPT1 phosphorylated by Rho‐associated coiled‐coil kinase (ROCK) and cyclic AMP‐dependent protein kinase (PKA). This approach confirmed that phosphorylation of MYPT1 by ROCK occurs at Thr697 and Thr855, PKA phosphorylates these two sites and the neighboring Ser696 and Ser854, and prior phosphorylation at Thr697 and Thr855 by ROCK precludes phosphorylation at Ser696 and Ser854 by PKA. Furthermore, phosphorylation at Thr697 and Thr855 by ROCK exposes two other sites of phosphorylation by PKA. Treatment of Triton‐skinned rat caudal arterial smooth muscle strips with the membrane‐impermeant phosphatase inhibitor microcystin, or of intact tissue with the membrane‐permeant calyculin A, induced slow, sustained contractions that correlated with phosphorylation of MYPT1 at 7 to ≥10 sites. Phos‐tag SDS‐PAGE thus provides a suitable and convenient method for analysis of the complex, multi‐site MYPT1 phosphorylation events involved in the regulation of myosin light chain phosphatase activity and smooth muscle contraction.Support or Funding InformationFunded by the Canadian Institutes of Health Research (MOP‐111262)