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Identification of the regulatory site in smooth muscle calponin that is phosphorylated by protein kinase C
F Nakamura1, T Mino, J Yamamoto
1Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, Japan.
Abstract:
F-actin and tropomyosin inhibited the phosphorylation of calponin by protein kinase C, and the phosphorylation reduced the binding of calponin to F-actin and tropomyosin. Labeled phosphate from [gamma-32P]ATP was retained both on the chymotryptic NH2-terminal 22-kDa fragment, which contains the actin-, tropomyosin-, and calmodulin-binding regions, and on the COOH-terminal 12-kDa fragment. Fractionation of tryptic 32P-labeled peptides by high performance liquid chromatography allowed isolation of three phosphopeptides (designated T1, T2, and T3), each of which was located in three repeating amino acid motifs of calponin. Both the relative initial rates and extent of phosphorylation decreased in the order T2 > T3 > T1. Both serine and threonine residues were phosphorylated in T1 (GASQAGMTAPGTK), and only a threonine residue was phosphorylated in T2 (FASQQGMTAYGTR) and in T3 (GASQQGMTVYGLPR). As the 22-kDa fragment contained only T2, the phosphorylation site in T2 appeared to regulate the binding of calponin to F-actin and tropomyosin. The amino acid sequence of T2 indicates that protein kinase C phosphorylates Thr184. Thus Thr184 is the preferred site of phosphorylation and is functionally the most important of the sites phosphorylated by protein kinase C in smooth muscle calponin.
Insights
Protein kinase C phosphorylation of calponin reduces its binding to F-actin and tropomyosin. Thr184 is identified as the key phosphorylation site, regulating this interaction in smooth muscle.
Area of Science:
- Molecular and Cellular Biology
- Muscle Physiology
Background:
- Calponin is a key regulator of smooth muscle contraction.
- Its interaction with actin and tropomyosin is modulated by phosphorylation.
Purpose of the Study:
- To identify the specific sites of protein kinase C phosphorylation on calponin.
- To determine the functional significance of these phosphorylation sites on calponin's interaction with actin and tropomyosin.
Main Methods:
- In vitro phosphorylation of calponin using [gamma-32P]ATP and protein kinase C.
- Limited proteolysis (chymotrypsin and trypsin) to generate fragments.
- High-performance liquid chromatography (HPLC) for phosphopeptide isolation and analysis.
- Amino acid sequencing of phosphopeptides.
Main Results:
- Protein kinase C phosphorylates calponin at three distinct sites (T1, T2, T3) within repeating amino acid motifs.
- Phosphorylation rates varied, with T2 > T3 > T1.
- The NH2-terminal 22-kDa fragment, containing the actin- and tropomyosin-binding regions, was phosphorylated at site T2.
- Phosphorylation of calponin reduced its binding affinity to F-actin and tropomyosin.
- Thr184 was identified as the primary phosphorylation site within the T2 phosphopeptide.
Conclusions:
- Protein kinase C phosphorylation of smooth muscle calponin occurs at multiple sites, with Thr184 being the preferred and functionally most important site.
- Phosphorylation at Thr184 directly regulates the interaction between calponin, F-actin, and tropomyosin, impacting smooth muscle function.