The causal relationship between mutations in cAMP-dependent protein kinase and the loss of adrenocorticotropin-regulated adrenocortical functions.

Abstract

The Y1 adrenocortical tumor cell mutants, Kin-7 and Kin-8, harbor point mutations in the regulatory subunit (RI) of the type 1 cAMP-dependent protein kinase (cAMPdPK) that render the enzyme resistant to activation by cAMP. These mutants also are resistant to many of the regulatory effects of ACTH and cAMP. In order to examine the causal relationships between the mutations in cAMPdPK and the resistance to ACTH and cAMP, the Kin mutants were transfected with expression vectors encoding wild type subunits of cAMPdPK in order to restore cAMP-responsive protein kinase activity. The transformants then were screened for the concomitant recovery of cellular responsiveness to ACTH and cAMP. In the mutant Kin-7, cAMP-responsive protein kinase activity was recovered after transfection with an expression vector encoding wild type mouse RI. Protein kinase activity in the mutant Kin-8 remained largely cAMP-resistant after transfection with the RI expression vector but could be rendered cAMP-responsive by transfection with an expression vector encoding the wild type catalytic subunit. The recovery of cAMP-responsive protein kinase activity was accompanied by the recovery of steroidogenic and morphological responses to ACTH and cAMP, suggesting that the cAMP-dependent signaling cascade plays an obligatory role in these actions of ACTH. The growth-regulatory effects of cAMP were not reversed with the recovery of cAMP-responsive protein kinase activity, suggesting that cAMP-resistant growth regulation results from second-site, adaptive mutations either in the original Kin mutant population or in the transformants. Studies on the conversion of 22(R)-hydroxycholesterol into steroid products in parent and mutant cells indicate that the Kin mutations reduce the steroidogenic capacity of the cell as well as inhibit the hormone- and cyclic nucleotide-dependent mobilization of substrate cholesterol.

De Novo Biosynthesis and Radiolabeling of Mammalian Digitalis-Like Factors

“…3, 2004 crease the synthesis of progesterone and its derivatives in response to adrenocorticotropin hormone (ACTH) (33 ). ACTH binds a specific G-protein-coupled 7-transmembrane receptor that activates adenylate cyclase and produces the increase in intracellular cAMP (34,35 ). When we treated Y-1 cells with 8-bromoadenosine 3Ј:5Ј-cAMP, a cAMP analog, we observed a fivefold increase in steroids, as expected (36 ), but no increase in DLIF was detected ( Table 1).…”

Adrenocorticotropin Induction of Stress-activated Protein Kinase in the Adrenal Cortex in Vivo

“…The intracellular Ca ϩ2 chelating agent BAPTA also reduced angiotensin II-in- duced SAPK activity (19) and ACTH-induced SAPK activity by 40%. 2 Calcium plays an important role in several aspects of normal adrenal function. ACTH-induced steroid secretion was inhibited by calcium channel blockade in cultured bovine adrenal cells (56,57).…”

“…In many circumstances, induction of ERK activity is associated with enhanced cellular proliferation (12, 60), S-phase progression, and DNA synthesis (25). ACTH inhibits cellular proliferation in fetal adrenal cells (61) and inhibits DNA synthesis in Y1 cells (2). The inhibition of ERK activity by ACTH may contribute to the inhibition of cellular proliferation and DNA synthesis induced by ACTH (2).…”

“…The stimulatory guanine nucleotide-bound Gs couples to adenylate cyclase, leading to a series of signaling cascades. The acute ACTH response is associated with a rapid increase in steroid secretion and is mediated by cAMP and cAMP-dependent protein kinase A (PKA) 1 (1,2). Although ACTH stimulates an increase in cAMP formation (3)(4)(5), other secondary messengers including protein kinase C (PKC) (6), Ca ϩ2 -calmodulin-dependent protein kinase (7) and the phosphoinositol pathway (8,9) are also induced by ACTH.…”

ACTH-induced Nucleocytoplasmic Translocation of Salt-inducible Kinase

“…When GFP tag alone was introduced into Y1 cells, the fluorescence signal did not localize at any specific subcellular area, and its distribution did not change after the ACTH addition (data not shown). To answer whether the cAMP/PKA signaling was involved in the intracellular redistribution of SIK, Y1 cells and PKA-less mutant Y1, Kin-7, cells (38) were treated with 8-Br-cAMP (Fig. 1D).…”