Calcium/calmodulin-dependent phosphorylation of vimentin in rat sertoli cells.

Abstract

Ca2+-dependent protein phosphorylation and the role of calmodulin in this process was investigated in subcellular fractions of primary cultures of rat Sertoli cells. Significant Ca2+/calmodulin-dependent protein phosphorylation in Sertoli cells was restricted to the cytosol fraction. The calmodulin dependence of these effects was confirmed by using the calmodulin inhibitor trifluoperazine. Purification of Calmodulin. Calmodulin was either obtained from Calbiochem (bovine brain) or purified from bovine brain or rat testis using filtration on Sepharose 4B and affinity chromatography on a W-7 affinity column (22). Calmodulin activity was determined by using the assay for calmodulin-sensitive phosphodiesterase. The purity ofthe preparations was analyzed by NaDodSO4/polyacrylamide gel electrophoresis.Purification and Assay of Phosphodiesterase. The procedure for the partial purification of calmodulin-sensitive phosphodiesterase from bovine brain was essentially that described by Cheung and Lin (23). Phosphodiesterase activity was assayed by the method of Thompson and Appleman (24).Preparation ofCell Fractions. Primary cultures ofrat Sertoli cells were prepared from testes of 20-to 22-day-old rats as described (25). Cells growing in culture flasks were collected in cold 50 mM Pipes, pH 7.0/10 mM MgCl2/50 mM NaCl with and without 0.1 mM phenylmethylsulfonyl fluoride and dispersed by sonication (2.5-3.5 W) for 15 sec. After centrifugation at 27,000 X g for 10 min, portions ofthe supernatant were used to assay for endogenous protein phosphorylation. When subcellular fractions were required, the cells were collected in 50 mM Pipes, pH 7.0/50 mM NaCl and then homogenized in a glass Dounce homogenizer (small clearance pestle). After homogenization, MgCl2 was added to a final concentration of 10 mM. Subcellular fractions (100,000 X g cytosol and particulate fractions) were prepared from homogenates as described by Li and Hsie (26). The crude nuclear fraction obtained after Triton extraction (26) was further purified by centrifugation through sucrose as described by Prashad et al (27). Nuclei were rinsed and resuspended in homogenization buffer prior to assay. Purity ofthe subcellular fractions was monitored by assaying for lactate dehydrogenase activity (28) and 5'-nucleotidase activity (29) in different fractions. Contamination of particulate and nuclear fractions with soluble proteins was <5% (n = 5). Contamination of the soluble fraction with particulate protein was <5% (n = 3) and of the nuclear fractions with particulate protein was <10% (n = 3).In Vitro Phosphorylation. Reaction mixtures (150 ,ul) 760The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Rat Sertoli cells acquire a beta-adrenergic response during primary culture.

“…2). Phosphoprotein of Mr/pI 58,000/5.1 has been identified as vimentin (6,21), and two of the additional phosphoproteins (Mr/pT 46,000/4.95 and 48,000/5.0) probably represent degradation products of vimentin (5,6,22). The observed changes were consistent with an earlier study showing potentiation of cAMP action in the presence of iBuMeXan (6) and correlated with the observation that only a slight increase in the phosphorylation of these four polypeptides was induced by FSH in the absence of iBuMeXan (not shown).…”

“…Aliquots of the sonicated cell and tissue samples were counted by spotting on filter paper squares as described (6,21). Samples containing the same number of counts were then analyzed by two-dimensional polyacrylamide gel electrophoresis and autoradiography as described (5,6,21).…”

Follicle-stimulating hormone-dependent phosphorylation of vimentin in cultures of rat Sertoli cells.

“…Reaction mixtures (150 ,ul) contained 20 mM Mops, pH 7.0, 10 mM MgCl2, 0.2mM MeiBuXan, 0.2 mM EGTA, 1-5 ,uM [y-32P]ATP (4 to 5 ,ACi per tube), 10-20 ,uM cAMP, and 25-50 ,ug ofprotein. Reactions were run for 10 min at 30°C and samples were spotted on filter disks as described (10). The remaining portion of each mixture was mixed with an equal volume of double-strength NaDodSO4 sample buffer (11) and boiled for 5 min.…”

“…One-and two-dimensional NaDodSO4/polyacrylamide gel electrophoresis, using a 5-15% acrylamide (linear gradient) slab gel, was carried out as described (11). Gels were processed as described (10).…”

“…(1.0 mg/ml), ,tM [y-32P]ATP (100-200 cpm/pmol), .and 20-30 ,ug of protein with or without 2-10 ,uM cAMP. Reactions were run for 10 min at 30°C and activity was assayed by spotting on filter disks as described (10).…”