Lutropin-dependent protein phosphorylation and steroidogenesis in rat tumour Leydig cells

Abstract

Tumour Leydig cells have been incubated in the presence or absence of lutropin (luteinizing hormone, ;LH'). Stimulation of cells with lutropin (1000ng/ml) in the presence of 1-methyl-3-isobutylxanthine (0.25mm) resulted in increased steroid production and increased protein phosphorylation. When pregnenolone metabolism was inhibited, basal pregnenolone production was 26.9+/-7.4ng/60min per 10(6) cells; stimulated production was 156.1+/-39.5ng/60min per 10(6) cells (means+/-s.d., n=4). Lutropin-dependent phosphorylated proteins of molecular mass 17000, 22000, 24000, 33000 and 57000Da were detected. A significant increase of [(32)P]P(i) incorporation into these phosphorylated proteins was observed concomitant with the increased pregnenolone production. The occurrence of the phosphoproteins in nuclei, mitochondria and postmitochondrial-supernatant was investigated. The 17000Da phosphoprotein was found in the nuclear fraction, whereas the 22000, 24000, 33000 and 57000Da phosphoproteins were localized in the postmitochondrial-supernatant fraction. Of the cholesterol-side-chain-cleavage activity, 80.3+/-6.1% (mean+/-s.d., n=5) was present in the mitochondrial fraction isolated from tumour Leydig cells, and this activity was 2.5-fold increased when cells had been preincubated with lutropin/1-methyl-3-isobutylxanthine (basal production: 194.6+/-28.6ng/30min per mg of protein; lutropinstimulated production: 498.8+/-91.5ng/30min per mg of protein; means+/-s.d., n=3). The similarities in the kinetics of the phosphorylation of proteins and the pregnenolone production after addition of lutropin/1-methyl-3-isobutylxanthine indicate that the phosphoproteins could be involved in the lutropin-dependent increase in steroidogenesis in tumour Leydig cells. It remains to be demonstrated, however, to what extent the phosphoproteins outside the mitochondria can influence the cholesterol-side-chain-cleavage activity inside the mitochondria.

Lutropin increases phosphorylation of a 33 000-dalton ribosomal protein in rat tumour Leydig cells

“…Details of chemicals and methods used for isolation and incubation of tumour Leydig cells have been described previously (Bakker et al, 1981).…”

“…Isolation of testis Leydig cells from immature (21-23 days) and mature (90-100 days) Wistar rats (substrain R -Amsterdam) was performed as described by Janszen et al (1976), except that no dextran-centrifugation step was used. Incubation of cells was essentially as described by Bakker et al (1981). In brief, cells were preincubated for 60 min and incubated with or without lutropin/3-isobutyl-1methylxanthine as indicated (lutropin concentrations: lOOng/ml for immature and mature testis Leydig cells, 10OOng/ml for tumour Leydig cells; 3-isobutyll-methylxanthine: 0.25mM).…”

“…The action of lutropin (luteinizing hormone, 'LH') on steroidogenesis in rat tumour Leydig cells is accompanied by activation of protein kinase and phosphorylation of proteins, and increased synthesis of specific proteins (Janszen et al, 1978;Cooke et al, 1979;Bakker et al, 1981; for a review, see Rommerts & Brinkmann, 1981). It is not known whether, and if so how, phosphorylated proteins or newly synthesized proteins are related to the rate-limiting step in steroid production, i.e.…”

“…the cholesterol-side-chain-cleavage activity in mitochondria. Lutropin-dependent phosphorylation of five proteins (17000, 22000, 24000, 33000 and 57000 Da) has been demonstrated in rat tumour Leydig cells, and phosphorylation of these proteins coincided with increased steroid production (Bakker et al, 1981). The 17000-Da protein was isolated from the nuclear fraction, and the other four proteins were isolated from the postmitochondrial supernatant.…”

Localization and Hormonal Regulation of the Non-Specific Lipid Transfer Protein (Sterol Carrier Protein2) in the Rat Testis

“…Molenaar & van der,Conk, Grootegoed & van der Molen (1985) and Grootegoed, Krïger-Sewnarain,Jutte, Rommerts & van der Molen (1982) respectively.Tissue or isolated cells were disrupted by sonication (5 10 sec, amplitude 16 microns, frequence 60 Hz) and a 105,000 g supernatant fraction was prepared as described previously(Bakker, Hoogerbrugge, Rommerts & van der Molen, 1981). Levels of nsLTP were determined in supernatants after acid (pH 5.1) and heat treatment (5 min, 90°C) using an…”