Salt-Inducible Kinase Is Involved in the ACTH/cAMP-Dependent Protein Kinase Signaling in Y1 Mouse Adrenocortical Tumor Cells

Abstract

The involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1-2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase's inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.

Adipose-specific Expression, Phosphorylation of Ser794 in Insulin Receptor Substrate-1, and Activation in Diabetic Animals of Salt-inducible Kinase-2

“…It was induced in adrenocortical cells at a very early phase of ACTH stimulation, and its molecular properties have been investigated in detail (27)(28)(29). Its presence in tissues other than the adrenal cortex was also reported (27). Examining the genomic data base, we noticed the presence of an isoform of SIK.…”

“…The mixture was centrifuged at 15,000 rpm for 15 min, and the resulting infranatant was recovered, and the protein concentration was determined with Protein Assay (Bio-Rad). The protein solutions, 3 mg obtained from white adipose, 3 mg from brown adipose, 18 mg from liver, or 18 mg from skeletal muscles, were incubated with 10 l of anti-SIK2-specific antiserum and 50 l of protein-GSepharose at 4°C for 3 h. The SIK2-IgG-protein-G-Sepharose complex was precipitated by centrifugation at 3000 ϫ g for 5 s, washed three times with 1 ml of lysis buffer, and washed once with 1 ml of SIK-reaction buffer (27). The final precipitate was suspended in the SIK-reaction buffer with a final volume of 50 l. Two aliquots of the SIK2-IgG suspension were used for the immunoblot analysis and in vitro kinase assay.…”

“…Other Procedures-Expression of GST-tagged proteins in E. coli and COS-7 cells and their purification was described before (27,29). Procedures for Northern blot analysis, in vitro kinase assays, and reporter assays were described in Ref.…”

“…Procedures for Northern blot analysis, in vitro kinase assays, and reporter assays were described in Ref. 27.…”

“…It is a serine/ threonine protein kinase that belongs to a sucrose-nonfermenting-1 protein kinase/AMP-activated protein kinase (AMPK) family. It was induced in adrenocortical cells at a very early phase of ACTH stimulation, and its molecular properties have been investigated in detail (27)(28)(29). Its presence in tissues other than the adrenal cortex was also reported (27).…”