Phosphorylation of the myosin phosphatase inhibitors, CPI-17 and PHI-1, by integrin-linked kinase.
Integrin-linked kinase (ILK) has been implicated in Ca(2+)- independent contraction of smooth muscle via its ability to phosphorylate myosin. We investigated the possibility that this kinase might also phosphorylate and regulate the myosin light-chain phosphatase inhibitor proteins CPI-17 [protein kinase C (PKC)-dependent phosphatase inhibitor of 17 kDa] and PHI-1 (phosphatase holoenzyme inhibitor-1), known substrates of PKC. Both phosphatase inhibitors were phosphorylated by ILK in an in-gel kinase assay and in solution. A Thr-->Ala mutation at Thr(38) of CPI-17 and Thr(57) of PHI-1 eliminated phosphorylation by ILK. Phosphopeptide mapping, phospho amino acid analysis and immunoblotting using phospho-specific antibodies indicated that ILK predominantly phosphorylated the site critical for potent inhibition, i.e. Thr(38) of CPI-17 or Thr(57) of PHI-1. CPI-17 and PHI-1 thiophosphorylated by ILK at Thr(38) or Thr(57) respectively inhibited myosin light-chain phosphatase (MLCP) activity bound to myosin, whereas the site-specific mutants CPI-17-Thr(38)Ala and PHI-1-Thr(57)Ala, treated with ILK under identical conditions, like the untreated wild-type proteins had no effect on the phosphatase. Consistent with these effects, both thiophospho-CPI-17 and -PHI-1 induced Ca(2+) sensitization of contraction of Triton X-100-demembranated rat-tail arterial smooth muscle, whereas CPI-17-Thr(38)Ala and PHI-1-Thr(57)Ala treated with ILK in the presence of adenosine 5'-[gamma-thio]triphosphate failed to evoke a contractile response. We conclude that ILK may activate smooth-muscle contraction both directly, via phosphorylation of myosin, and indirectly, via phosphorylation and activation of CPI-17 and PHI-1, leading to inhibition of MLCP.
Deng
Jing Ti
JT
Smooth Muscle Research Group and Canadian Institutes of Health Research Group in Regulation of Vascular Contractility, University of Calgary Faculty of Medicine, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada.
Sutherland
Cindy
C
Brautigan
David L
DL
Eto
Masumi
M
Walsh
Michael P
MP
D000818:Animals; D002118:Calcium / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D017830:Octoxynol / Q000494:pharmacology; D010449:Peptide Mapping; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D000099200:Scaffold Protein ILK
D002118:Calcium; D009124:Muscle Proteins; D043263:Myosin-Light-Chain Phosphatase; D017830:Octoxynol; D010749:Phosphoprotein Phosphatases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D000099200:Scaffold Protein ILK; C491519:Ppp1r14a protein, rat; C073882:phosphoprotein phosphatase inhibitor 1