Adrenocorticotropin (ACTH) induces phosphorylation of a cytoplasmic protein in intact isolated adrenocortical cells.

Abstract

In 32p incorporation experiments with intact adrenocortical cells, adrenocorticotropin (ACTH) or adenosine 3',5'-cyclic monophosphate (cAMP) induced a rapid and transient increase of approximately 300-500% in the phosphorylation of a 32P-containing cytoplasmic protein of about 150,000 daltons (APS15o). Half-maximal stimulation of APS150 phosphorylation was observed with about 3 pM ACTH. Receptorbound cAMP, corticosterone production, and the appearance of phosphorylated APS150 increased in parallel with respect to both time and ACTH concentration. All three responses were dependent on extracellular calcium. Inhibition of protein synthesis with cycloheximide suggested a half-life of APS150 of about 10 min. The time course of 32p incorporation into ACTH-induced APS150 in the absence and presence of nonradioactive phosphate shows that the phosphorylation of APS150 is under simultaneous control of cAMP-dependent protein kinase and of phosphatase activity. Thus a rapid ACTI-dependent and cAMP-dependent protein phosphorylation in intact adrenocortical cells within steroidogenic ACTH concentrations has now been demonstrated.It is well recognized that steroidogenesis in adrenal cortex and similar steroid-elaborating tissue is under complex hormonal control. In the current model of adrenocorticotropin (ACTH) action, it is proposed that the hormone binds to a specific receptor on plasma membrane and stimulates adenylate cyclase activity, with accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) and activation of a cAMP-dependent protein kinase (1). Until recently, the intermediate role of cAMP in the acute steroidogenic response to physiological concentrations of ACTH has been questioned (1). However, it has now been shown that hormonal stimulation of adrenocortical cells by ACTH in the steroidogenic concentration range is accompanied by a simultaneous increase in endogenous cAMP bound to the regulatory subunit of the protein kinase (2). Furthermore, studies with mutant clones of Y1 adrenocortical tumor cells (adenylate cyclase and cAMP-dependent protein kinase deficiency) provide additional evidence that cAMP and cAMPdependent protein kinase are obligatory components of the ACTH-stimulated steroidogenesis pathway (3). In this context, the characterization of an endogenous specific substrate(s) for cAMP-dependent protein kinase(s) would substantially enlarge our understanding of the mechanism of acute ACTH action.Several laboratories have reported phosphorylation of subcellular proteins in broken cells of adrenocortical tissue (4-8). Ribosomal protein phosphorylation stimulated by ACTH and cAMP in cultured mouse tumor cells was also described (9). However, this stimulation was achieved only by high ACTH concentrations. It has been shown that the activity of enzymes involved in steroidogenesis is increased by phosphorylation. Evidence has been presented that in adrenal cortex the phos- [32Plphosphate (400 MCi/ml; 1 Ci = 3.7 X 10l°becquerels) for different times at 37°C or 0°C in a total volume of 0.7 ml; this...

Compartmentalization of corticotropin-dependent steroidogenic factors in adrenal cortex: evidence for a post-translational cascade in stimulation of the cholesterol side-chain split.

“…Nevertheless the operation of a cascade seems very likely. It has been shown that in intact adrenocortical cells steroidogenic concentrations of ACTH were accompanied by a cAMP-dependent phosphorylation of at least one cytosolic protein (8). The present results indicate that one of the factors participating in the cascade mentioned above could be the post-translational link in the immediate stimulation ofsteroidogenesis after the ACTH-and cAMP-dependent phosphorylation pathway.…”

“…The question still remains as to how a peptide hormone such as corticotropin (ACTH, adrenocorticotropic hormone, adrenocorticotropin) binding on a cell surface receptor can transmit a specific signal to its mitochondrial site ofaction. The current ideas on the major mechanism ofthe immediate action ofACTH on adrenocortical zona fasciculata-reticularis cells suggest the following steps: activation of adenylate cyclase in the presence of extracellular Ca2+, production of cAMP, activation of a cAMP-dependent protein kinase, and phosphorylation of specific cycloheximidesensitive proteins that may be involved somehow in activating the rate-limiting step (2,(6)(7)(8)(9), possibly mediated by phospholipids (10). So far, no direct link between these events has been established because of the lack of an appropriate cell-free system.…”

The Hypothyroidism in an Inbred Kindred with Congenital Thyroid Hormone and Glucocorticoid Deficiency is Due to a Mutation Producing a Truncated Thyrotropin Receptor

“…Thus, as described in other children with RACTH (25,26,28,41), the hypoadrenocortisolism in this family was not caused by a defective ACTHR. Given that the phenotype is identical to that observed in children with ACTHR defects, it is tempting to speculate that the defect may involve some of the protein cofactors that regulate ACTHR gene expression (42) or defects in the protein kinase-mediated steroidogenesis involving adrenocortical-specific proteins that are phosphorylated by cAMP (43,44). The apparent ACTH-mediated skin hyperpigmentation is not mediated through the action of ACTH on the ACTHR but that of ACTH acting on the cutaneous MSH receptor, the product of another gene.…”

Corticotropin increases protein tyrosine phosphatase activity by a cAMP‐dependent mechanism in rat adrenal gland

“…Male Wistar rats (90‐day‐old) were used throughout. Adrenal glands or ZF cells were obtained from animals supplied with dexamethasone (10 µg·mL −1 , ad libitum) in the drinking water for 16 h before killing [19].…”