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Abstract
Smooth muscle caldesmon was phosphorylated in vitro by sea star p44mpk up to 2.0 mol of phosphate/mol of protein at both Ser and Thr residues. The phosphorylation sites were contained mainly in the COOH-terminal 10-kDa cyanogen bromide fragment which houses the binding sites for calmodulin, tropomyosin, and F-actin. Tryptic peptide maps of 32P-labeled caldesmon by p44mpk and p34cdc2 showed that while both enzymes recognized similar sites of phosphorylation, they have different preferred sites. Phosphorylation of caldesmon attenuated slightly its interaction with actin and had no effect on its binding to calmodulin and tropomyosin. Smooth muscle cell extracts from chicken gizzard and rat aorta contained 42- and 44-kDa proteins, respectively, which were cross-reactive with an antibody to sea star p44mpk. Immunoprecipitates from gizzard and aorta cell extracts, generated with the p44mpk antibody, possessed kinase activities toward myelin basic protein as well as caldesmon. These results suggest that MAP kinase may have functions in the differentiated smooth muscle cells distinct from those involved in the cell cycle.
Highlights
Tro by sea star ~ 4 4 u"p t~o 2~.0 mol of phosphate/mol Yamashiro et al [14] have demonstrated that 1-caldesmon ofproteinatboth Ser andThrresidues.Thephosin fibroblasts is phosphorylated during mitosis and becomes phorylation sites were contained mainlyin the COOH- detached from the actinfilaments
The smooth muscle caldesmon has a M,of 87,000 (h-caldesmon) and consists of three domains: the NH, domain contains thebinding sites for myosin [4]and calmodulin [5]; a helical central segment consists of repetitive motifs; and a COOH domain contains binding sites for F-actin [6],tropomyosin [7,8], andcalmodulin [6].The smaller non-muscle caldesmon
Activation of MAP kinases as a result of dual phosphorylation at neighboring Thr and Tyrresidues is believed to play a pivotal role in the GO/G1 transition of somatic cells and theG2/M transition in the oocyte cell cycle
Summary
MATERIALS ANDMETHODS pl) was subjected to two-dimensional mapping on Kodak thin layer. The membrane was incubated in TBS containing incorporation of 32Pin caldesmon was assayed by the Whatman P81 0.05% Tween (TTBS), 1%skim milk, and antibodies against MAP filter paper method and SDS-polyacrylamide gel electrophoresis fol- kinase (1:lOOO dilution) overnight at room temperature. Immunoprecipitation and Kinase Assay-Chicken gizzard and rat Tryptic Peptide Mapping-"P-Labeled caldesmon (0.25 mg) was aorta smooth muscle tissue was homogenized in 20 mM MOPS, pH digestedwith trypsin (L-1-tosylamido-2-phenylethychlloromethyl ke- 7.2, 60 mM P-glycerophosphate, 5 mM EGTA, 1 mM EDTA, 5 mM tone-treated from Sigma) at anenzyme:substrate weight ratio of 1:50 NaF, 5 mM Na3V04,1 mM phenylmethylsulfonyl fluoride, 1 d m 1 for 20 h at 37 "C in 50 mM NH4C03,pH 8.0. The digested sample was leupeptin, 1 mM DTT and clarified at 13,000 rpm for 5 min.One lyophilizedand suspended in 20 pl of pH 1.8thin layer electrophoresis hundred-pl aliquots were incubated with 5 g1 of rabbit preimmune buffer (water:acetic acidformic acid, 9008020). Reactions were stopped after 40 min for MBP andafter 1h for caldesmon by the addition of Laemmli sample buffer, and the samples were analyzed on 12% SDSpolyacrylamide gels followed byautoradiography
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