2ϩsensitization in rat ileal longitudinal smooth muscle. Am J Physiol Gastrointest Liver Physiol 293: G699-G710, 2007. First published July 26, 2007; doi:10.1152/ajpgi.00214.2007.-We investigated the protein kinases responsible for myosin regulatory light chain (LC20) phosphorylation and regulation of myosin light chain phosphatase (MLCP) activity during microcystin (phosphatase inhibitor)-induced contraction at low Ca 2ϩ concentrations of rat ileal smooth muscle stretched in the longitudinal axis. Application of 1 M microcystin induced LC20 diphosphorylation and contraction of ␤-escin-permeabilized rat ileal smooth muscle at pCa 9. The PKC inhibitor GF109203x, the MEK inhibitor PD-98059, and the p38 MAPK inhibitor SB-203580 significantly reduced this contraction. These inhibitory effects were abolished when the microcystin concentration was increased to 10 M, indicating that application of these kinase inhibitors generated an increase in MLCP activity. GF-109203x and PD-98059, but not SB-203580, significantly decreased the phosphorylation level of the myosin-targeting subunit of MLCP, MYPT1, at Thr-697 (rat sequence) during microcystin-induced contraction at pCa 9. On the other hand, SB-203580, but not GF-109203x or PD-98059, significantly reduced the phosphorylation level of the PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17). A zipper-interacting protein kinase (ZIPK) inhibitor (SM1 peptide) and a Rho-associated kinase inhibitor (Y-27632) had little effect on microcystin-induced contraction at pCa 9. In conclusion, PKC, ERK1/2, and p38 MAPK pathways facilitate microcystin-induced contraction at low Ca 2ϩ concentrations by contributing to the inhibition of MLCP activity either through phosphorylation of MYPT1 or CPI-17 [probably mediated by integrin-linked kinase (ILK)]. ILK and not ZIPK is likely to be the protein kinase responsible for LC20 diphosphorylation during microcystin-induced contraction of rat ileal smooth muscle at pCa 9, similar to its recently described role in vascular smooth muscle. The negative regulation of MLCP by PKC and MAPKs during microcystin-induced contraction at pCa 9, which is not observed in vascular smooth muscle, may be unique to phasic smooth muscle. ileum; protein kinase C; myosin phosphatase; mitogen-activated protein kinase; protein kinase C-potentiated phosphatase inhibitor protein of 17 kDa; myosin-targeting subunit of myosin light chain phosphatase SMOOTH MUSCLE CONTRACTION is a dynamic and highly regulated process. The contractile state of smooth muscle is mainly regulated by phosphorylation of the 20-kDa myosin regulatory light chain (LC 20 ] i )} is the primary determinant of smooth muscle contraction. Force can be further increased through signaling pathways that modulate MLCK and/or MLCP activities. The Ca 2ϩ sensitivity of contraction can be affected by any change in the ratio of MLCK:MLCP activity. A decrease in MLCP activity will shift the balance in favor of MLCK, resulting in a greater degree of LC 20 phosphorylation and contraction. The phenomenon of ...

The SMTNL1 protein contains a single type-2 calponin homology (CH) domain at its C terminus that shares sequence identity with the smoothelin family of smooth muscle-specific proteins. In contrast to the smoothelins, SMTNL1 does not associate with F-actin in vitro, and its specific role in smooth muscle remains unclear. In addition, the biological function of the C-terminal CH-domains found in the smoothelin proteins is also poorly understood. In this work, we have therefore determined the solution structure of the CH-domain of mouse SMTNL1 (SMTNL1-CH; residues 346 -459). The secondary structure and the overall fold for the C-terminal type-2 CHdomain is very similar to that of other CH-domains. However, two clusters of basic residues form a unique surface structure that is characteristic of SMTNL1-CH. Moreover, the protein has an extended C-terminal ␣-helix, which contains a calmodulin

No abstract