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Am J Physiol Gastrointest Liver Physiol (October 14, 2004). doi:10.1152/ajpgi.00330.2004
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Submitted on July 27, 2004
Accepted on October 7, 2004

Agonist-Induced Association of Tropomyosin with Protein Kinase C{alpha} in Colonic Smooth Muscle

Sita Somara1, Haiyan Pang1, and Khalil N. Bitar1*

1 Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan, USA

* To whom correspondence should be addressed. E-mail: bitar{at}umich.edu.

Smooth muscle contraction regulated by myosin light chain phosphorylation, is also regulated at the thin-filament level. Tropomyosin, a thin filament regulatory protein, regulates contraction by modulating actin-myosin interactions. Present investigation shows that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of tropomyosin in colonic smooth muscle cells. Our data also shows that acetylcholine induces a significant and sustained increase in PKC-mediated association of tropomyosin with PKC{alpha} in the particulate fraction of colonic smooth muscle cells. Immunoblotting studies revealed that in colonic smooth muscle cells, there is no significant change in the amount of tropomyosin or actin in particulate fraction in response to acetylcholine indicating that the increased association of tropomyosin with PKC{alpha} in the particulate fraction may be due to acetylcholine-induced translocation of PKC{alpha} to the particulate fraction. To investigate whether the association of PKC{alpha} with tropomyosin was due to a direct interaction, we performed in vitro direct binding assay. Tropomyosin cDNA amplified from colonic smooth muscle mRNA was expressed as GST-tropomyosin fusion protein. In vitro binding experiments using GST-tropomyosin and recombinant PKC{alpha} indicated direct interaction of tropomyosin with PKC{alpha}. PKC-mediated phosphorylation of tropomyosin and direct interaction of PKC{alpha} with tropomyosin suggest that tropomyosin could be a substrate for PKC. Phosphorylation of tropomyosin may aid in holding the slided tropomyosin away from myosin binding sites on actin resulting in actomyosin interaction and sustained contraction.




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