Expression cloning of a cDNA encoding the type II regulatory subunit of the cAMP-dependent protein kinase

We report here the isolation and sequence of a cDNA for the type II regulatory subunit of the cAMP-dependent protein kinase (cAMP-PK) from a lambda gt-11 cDNA library derived from a porcine epithelial cell line (LLC-PK1). The cDNA was detected by immunological screening using an affinity purified polyclonal antibody for bovine RII. DNA sequence analysis of the 467 bp EcoRI insert confirmed the identity of the clone, because the deduced amino acid sequence corresponded to the published sequence for the bovine RII protein. Northern analysis of total RNA from the LLC-PK1 cells indicated a single mRNA species of about 6.0 kb, probably derived from a single copy gene.     

cAMP mediated proteolysis of the catalytic subunit of cAMP-dependent protein kinase

The cAMP-dependent protein kinase from LLC-PK1 cells can be activated in vivo by calcitonin and vasopressin, or forskolin. Continuous treatment of cells with these agents results in a decrease of total cAMP-PK activity. The loss of kinase activity was enhanced when either of these three agents was incubated in the presence of isobutylmethylxanthine. Results obtained using affinity purified antibodies to the catalytic subunit show that the loss of kinase was due to specific proteolysis of this subunit.     

Unilateral ovariectomy restores ovulatory cyclicity in rats with a polycystic ovarian condition

An acyclic polycystic ovarian condition can be induced in adult rats with a single injection of estradiol valerate (EV). The ovaries are small and contain multiple cystic follicles and no new corpora lutea. In the early stages of the condition, both basal plasma luteinizing hormone (LH) and LH responses to luteinizing hormone-releasing hormone (LHRH) are attenuated. Plasma androgens are indistinguishable from normal controls. The present study examines the effect of unilateral ovariectomy (ULO) on this condition. Removal of one cystic ovary results in almost immediate resumption of vaginal cyclicity that persists for at least 3 wk. At 1 or 3 wk after ULO the remaining ovary contains fresh corpora lutea, appears histologically normal, and is significantly heavier than the cystic ovary removed at ULO, indicative of compensatory hypertrophy. Despite the resumption of apparently normal cyclic function, basal plasma LH concentrations and LH responses to LHRH are not significantly better than those in intact animals with polycystic ovaries. Thus, the previously polycystic ovary is fully capable of normal ovulatory function despite obvious impairments in the hypothalamo-pituitary axis. Since ovulatory function resumes on a background of continued poor pituitary responsiveness, the primary defect, which ULO corrects, is probably at the hypothalamic level. Finally, the cystic ovary clearly contributes to the hypothalamic aberration to which it subsequently responds.     

DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated neuronal phosphoprotein. I. Amino acid sequence around the phosphorylated threonine

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) is a major endogenous cytosolic substrate for dopamine- and cyclic AMP-stimulated protein phosphorylation in neurons of the basal ganglia of mammalian brain. It shares many properties with phosphatase inhibitor 1, a substrate for cyclic AMP-dependent protein kinase, and with G-substrate, a substrate for cyclic GMP-dependent protein kinase. We have, therefore, undertaken an analysis of the amino acid sequence around the site at which purified DARPP-32 is phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. The results indicate that DARPP-32 is phosphorylated at a single threonine residue contained in the sequence Arg-Arg-Arg-Pro-Thr(P)-Pro-Ala-Met-Leu-Phe-Arg. This sequence was obtained by automated solid phase sequencing of two overlapping tryptic phosphopeptides and one overlapping chymotryptic phosphopeptide which were purified by reverse-phase high-performance liquid chromatography. A 9-amino acid sequence containing the phosphorylatable threonine residue in DARPP-32 shares 8 identical residues with a sequence containing the phosphorylatable threonine residue in phosphatase inhibitor 1, and shares 5 identical residues with the two identical sequences surrounding the 2 phosphorylatable threonine residues in G-substrate. These observations support the view that DARPP-32, inhibitor 1, and G-substrate are members of a family of regulatory proteins which are involved in the control of protein phosphatase activity by both cyclic AMP and cyclic GMP, but which differ in their cellular and tissue distributions.     

The impact of type I diabetes on rat liver γ-glutamyltranspeptidase

The impact of type 1 diabetes mellitus on liver -glutamyltranspeptidase, a premalignant marker, was studied. Diabetes was induced in male Sprague Dawley and Fischer 344 rats by administration of Streptozotocin, which produced a stable and moderately severe diabetic state. In liver homogenates, -glutamyltranspeptidase was increased over control levels: 1.2, 8.1 and 13,2 fold in Strague-Dawley rats; 4.8, 58.4 and 84.7 fold in Fischer 344 rats; at 1, 3 and 6 weeks following Streptozotocin treatment. In plasma membranes isolated from the livers of Fischer 344 rats, -glutamyltranspeptidase was increased over control levels: 5.6, 75 and 127 fold at weeks 1, 3 and 6 following Streptozotocin treatment. The relative specific activity of 5-nuleohdase was found to be similar: 9–14, indicating comparable degrees of plasma membrane purity. Plasma glutamate-pyruvate transaminase levels were minimally and similarly affected at all time points indicating lack of association of increasing -glutamyltranspeptidase activity with overt liver damage. Thyroid hormone replacement, with both T₃ (0.6 g/Kg) once a day and T₄ (6.0 g/kg) twice a day for three days elicited a further 30% increment in enzyme activity. Insulin replacement (20–40 units/200 g body weight) twice a day for five days reduced enzyme activity 51% at week 6. This was associated with an increase in -glutamyltranspeptidase in the plasma from 14 fold over control levels in the diabetic state at week 6 to 53 fold ever control levels after insulin replacement at week 6. It is proposed that the diabetes-induced increase in -glutamyltranspeptidase is reduced by an insulin-directed shedding of the enzyme into the plasma.     

Protein phosphatase 2A--a 'ménage à trois'

Protein phosphorylation is probably the major regulatory mechanism employed by eukaryotic cells. Much work has been devoted to the role of protein kinases and their modulation by hormones, growth factors and neurotransmitters. It is now appreciated that protein phosphatases are also key players in actively regulating many cellular processes. In this article we discuss recent advances in our understanding of the function of protein phosphatase 2A, one of the major serine/threonine-specific protein phosphatases.     

cAMP-dependent protein kinase activation affects vasopressin V2-receptor number and internalization in LLC-PK1 renal epithelial cells.

The relationship between activation of the cAMP-dependent protein kinase (cAMP-PK) and ligand binding and internalization by the vasopressin renal (V2-type) receptor of LLC-PK1 renal epithelial cells was examined. Upon cAMP-PK activation through 1 h treatment with the cAMP analogue 8-bromo-cAMP (BrcA), a marked reduction in V2-receptor steady state number and internalization in LLC-PK1 cells was effected. In cells treated for 17 h with BrcA and hence down-regulated for cAMP-PK, the V2-receptor number was normal but internalization was markedly reduced. Cells of the LLC-PK1 mutant FIB4, which possesses about 10% parental cAMP-PK catalytic subunit activity, exhibited lower V2-receptor steady state number and internalization in comparison to untreated LLC-PK1 cells. A negative correlation was thus evident between cAMP-PK activation and V2-receptor number, and internalization. Phosphorylation by cAMP-PK may effect ligand-independent removal of receptor from the plasma membrane.