Calponin and SM 22 isoforms in avian and mammalian smooth muscle. Absence of phosphorylation in vivo.
Calponin is a basic smooth-muscle-specific protein capable of binding to F-actin, tropomyosin and calmodulin in vitro. Using two-dimensional gel electrophoresis, we show that calponin exists as multiple isoelectric variants in avian and mammalian tissues. During chick embryogenesis, one isoform is expressed in gizzard that shows a pI identical to the most basic adult alpha variant; around 10 d after hatching multiple isoforms then appear. SM 22 [Pearlstone, J. R., Weber, M., Lees-Miller, J. P., Carpenter, M. R. & Smillie, L. B. (1987) J. Biol. Chem. 262, 5985-5991], which has sequence-motifs related to calponin, displays a similar isoform pattern during development; one isoform (alpha) is present in the embryo and three in the adult. In living smooth-muscle strips from chicken gizzard and guinea pig taenia coli, labelled with 32PO4, no phosphate incorporation could be detected in any of the calponin or SM 22 isoforms during either contraction or relaxation. From the additional observation that antibodies against phosphoserine also failed to label calponin and SM 22 in two-dimensional gel immunoblots, we conclude that the multiple isoforms do not arise via differential phosphorylation. These results support the claim [Barany, M., Rokolya, A. & Barany, K. (1991) FEBS Lett. 279, 65-68] that calponin phosphorylation is not involved in smooth muscle regulation in vivo, as has been suggested from in vitro studies [Winder, S. J. & Walsh, M. J. (1990) J. Biol. Chem. 265, 10148-10155]. In vitro translation of porcine and chicken smooth-muscle mRNA produced only a single (alpha) isoform of calponin, suggesting that the adult isoforms do not derive from multiple gene products; in the same assay two polypeptides appeared in the position of SM 22, one corresponding to the alpha isoform and a second more basic spot, not observed in tissue samples. Whereas calponin and SM 22 appear synchronously during smooth muscle differentiation in vivo, SM 22 is not fully down-regulated like calponin, metavinculin and heavy-caldesmon in smooth muscle cells in culture, pointing to a differential regulation of expression of the alpha SM 22 isoform during smooth-muscle phenotype modulation in vitro.
Gimona
M
M
Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg.
Sparrow
M P
MP
Strasser
P
P
Herzog
M
M
Small
J V
JV
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002642:Chick Embryo; D002645:Chickens; D015180:Electrophoresis, Gel, Two-Dimensional; D066298:In Vitro Techniques; D008840:Microfilament Proteins; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000378:metabolism*; D009126:Muscle Relaxation; D009130:Muscle, Smooth / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D013552:Swine; D000096985:Calponins
D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D000096985:Calponins; C058992:Tagln protein, mouse; C051615:transgelin