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Rho‐associated kinase and zipper‐interacting protein kinase: roles in rapid phosphorylation events in serum‐stimulated human arterial smooth muscle cells
Author(s) -
Walsh Michael P.,
Deng JingTi
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.1017.2
Subject(s) - phosphorylation , myosin light chain phosphatase , microbiology and biotechnology , myosin light chain kinase , rho associated protein kinase , biology , kinase , gene knockdown , rock1 , protein kinase a , biochemistry , apoptosis
Myosin regulatory light chain (LC 20 ) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Rho‐associated kinase (ROCK) and zipper‐interacting protein kinase (ZIPK) have been implicated in the regulation of LC 20 phosphorylation via direct phosphorylation of LC 20 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par‐4 (prostate‐apoptosis response‐4). Phosphorylation of MYPT1 at T696 and T850 inhibits MLCP activity whereas phosphorylation of Par‐4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of ROCK and ZIPK in these rapid serum‐induced phosphorylation events, we investigated the time‐courses of phosphorylation of LC 20 , MYPT1 and Par‐4 in serum‐stimulated human vascular smooth muscle cells, and examined the effects of siRNA‐mediated ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC 20 at T18 and S19, MYPT1 at T696 and T850, and Par‐4 at T163, peaking within 30–120 s. ROCK1 knockdown decreased the levels of phosphorylation of LC 20 , MYPT1 and Par‐4, whereas ZIPK knockdown decreased LC 20 phosphorylation, but increased phosphorylation of MYPT1 and Par‐4. Consistent with the effects of kinase knockdown, ROCK inhibition by GSK429286A and H1152 reduced serum‐induced LC 20 , MYPT1 and Par‐4 phosphorylation, while ZIPK inhibition by HS38 reduced only LC 20 phosphorylation. We conclude that (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC 20 , MYPT1 and Par‐4, (ii) both ROCK and ZIPK are involved in LC 20 phosphorylation, and (iii) only ROCK accounts for MYPT1 and Par‐4 phosphorylation. Support or Funding Information Supported by a grant from the Canadian Institutes of Health Research (MOP‐111262).