Integrin-linked kinase (ILK) has been implicated in Ca2+- 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 17kDa] 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 Thr38 of CPI-17 and Thr57 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. Thr38 of CPI-17 or Thr57 of PHI-1. CPI-17 and PHI-1 thiophosphorylated by ILK at Thr38 or Thr57 respectively inhibited myosin light-chain phosphatase (MLCP) activity bound to myosin, whereas the site-specific mutants CPI-17-Thr38Ala and PHI-1-Thr57Ala, 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 Ca2+ sensitization of contraction of Triton X-100-demembranated rat-tail arterial smooth muscle, whereas CPI-17-Thr38Ala and PHI-1-Thr57Ala treated with ILK in the presence of adenosine 5′-[γ-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.
- Ca2+ sensitization
- muscle contraction
- smooth muscle
Abbreviations used: CPI-17, protein kinase C-dependent phosphatase inhibitor of 17 kDa; DTT, dithiothreitol; H-T buffer, Hepes-Tyrode's buffer; ILK, integrin-linked kinase; LC20, 20kDa light chain of myosin; MLCK, myosin light-chain kinase; MLCP, myosin light-chain phosphatase; MYPT1, myosin-targeting subunit of MLCP; PHI-1, phosphatase holoenzyme inhibitor-1; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PKN, protein kinase N; PP1, type 1 protein serine/threonine phosphatase; ROCK, Rho-associated kinase; ZIP, zipper-interacting protein.
- The Biochemical Society, London ©2002