Yasuki Nonaka

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

Salt-inducible kinase (SIK), first cloned from the adrenal glands of rats fed a high salt diet, is a serine/threonine protein kinase belonging to an AMP-activated protein kinase family. Induced in Y1 cells at an early stage of ACTH stimulation, it regulated the initial steps of steroidogenesis. Here we report the identification of its isoform SIK2. When a green fluorescent protein-fused SIK2 was expressed in 3T3-L1 preadipocytes, it was mostly present in the cytoplasm. When coexpressed in cAMP-responsive element-reporter assay systems, SIK2 could repress the cAMP-responsive element-dependent transcription, although the degree of repression seemed weaker than that by SIK1. SIK2 was specifically expressed in adipose tissues. When 3T3-L1 cells were treated with the adipose differentiation mixture, SIK2 mRNA was induced within 1 h, the time of induction almost coinciding with that of c/EBPβ mRNA. Coexpressed with human insulin receptor substrate-1 (IRS-1) in COS cells, SIK2 could phosphorylate Ser794 of human IRS-1. Adenovirus-mediated overexpression of SIK2 in adipocytes elevated the level of phosphorylation at Ser789, the mouse equivalent of human Ser794. Moreover, the activity and content of SIK2 were elevated in white adipose tissues ofdb/db diabetic mice. These results suggest that highly expressed SIK2 in insulin-stimulated adipocytes phosphorylates Ser794 of IRS-1 and, as a result, might modulate the efficiency of insulin signal transduction, eventually causing the insulin resistance in diabetic animals.

Molecular cloning and expression of cDNAs encoding rat aldosterone synthase: Variants of cytochrome P-45011β

Abstract Two distinct forms of cDNA encoding rat aldosterone synthase were cloned from an adrenal capsular tissue cDNA library. The deduced amino acid sequences showed that one of the enzymes (P-45011β,aldo-1) had a long extension peptide composed of 34 amino acid residues while the other (P-45011β,aldo-2) had an extension peptide identical to that of rat P-45011β. Glu at the 320th position of P-45011β,aldo-1 was replaced with Lys in P-45011β,aldo-2. The amino acid sequence of the aldosterone synthase was highly homologous (81%) to rat P-45011β. Constructed expression vector containing the cDNA for extension peptide of P-45011β and the mature protein of P-45011β,aldo-1 was transfected into COS-7 cells. The cells converted 11-deoxycorticosterone into corticosterone, 18-hydroxycorticosterone, and aldosterone.

Cloning of a novel kinase (SIK) of the SNF1/AMPK family from high salt diet-treated rat adrenal1

PCR‐coupled cDNA subtraction hybridization was adapted to identify the genes expressed in the adrenocortical tissues from high salt diet‐treated rat. A novel cDNA clone, termed alt‐ nducible inase (SIK), encoding a polypeptide (776 amino acids) with significant similarity to protein serine/threonine kinases in the SNF1/AMPK family was isolated. An in vitro kinase assay demonstrated that SIK protein had autophosphorylation activity. Northern blot revealed that SIK mRNA levels were markedly augmented by ACTH treatment both in rat adrenal glands and in Y1 cells. SIK may play an important role in the regulation of adrenocortical functions in response to high plasma salt and ACTH stimulation.

Molecular cloning and sequence analysis of cDNA encoding rat adrenal cytochrome P-45011β

A cDNA clone encoding cytochrome P‐45011β of rat adrenal has been cloned and sequenced using a bovine P‐45011β cDNA insert (pcP‐450(11β)‐2; (1987) J. Biochem. 102, 559–568) as a probe. The nucleotide sequence contains an open reading frame sufficient to encode the entire amino acid sequence of a P‐45011β precursor protein consisting of 499 amino acids including an extension peptide of 24 amino acids at the NH2‐terminus. The cDNA contains 1247 nucleotides at the 3′‐noncoding region including 51 nucleotides of poly A, but lacks the 5′‐noncoding region. The deduced amino acid sequence shows 61% similarity to that of bovine P‐45011β. Putative binding sites for heme and steroid are highly conserved among steroidogenic P‐450s of known structure.

Molecular identification of adrenal inner zone antigen as a heme-binding protein

The adrenal inner zone antigen (IZA), which reacts specifically with a monoclonal antibody raised against the fasciculata and reticularis zones of the rat adrenal, was previously found to be identical with a protein variously named 25‐Dx and membrane‐associated progesterone receptor. IZA was purified as a glutathione S‐transferase‐fused or His6‐fused protein, and its molecular properties were studied. The UV‐visible absorption and EPR spectra of the purified protein showed that IZA bound a heme chromophore in high‐spin type. Analysis of the heme indicated that it is of the b type. Site‐directed mutagenesis studies were performed to identify the amino‐acid residues that bind the heme to the protein. The results suggest that two Tyr residues, Tyr107 and Tyr113, and a peptide stretch, D99–K102, were important for anchoring the heme into a hydrophobic pocket. The effect of IZA on the steroid 21‐hydroxylation reaction was investigated in COS‐7 cell expression systems. The results suggest that the coexistence of IZA with CYP21 enhances 21‐hydroxylase activity.

Aldosterone biosynthesis by a reconstituted cytochrome P-45011β system

Abstract [ 3 H]Corticosterone was incubated with cytochrome P-450 11β purified to electrophoretic homogeneity from bovine adrenocortical mitochondria, and the reaction products were analyzed by high performance liquid chromatography. The production of aldosterone (21.2 pmol/nmol P-450/min) and 18-hydroxycorticosterone (1.17 nmol/nmol P-450/min) was observed. When lipidic extracts from mitochondria of bovine adrenocortical zona glomerulosa were added to the reaction mixture, the rate of production of aldosterone was increased 28-fold. When [ 3 H]18-hydroxycorticosterone was incubated with cytochrome P-450 11β , the amount of aldosterone produced was 55.7 pmol/nmol P-450/min in the absence of the lipidic extracts and the enhancing effect of the lipidic extracts was 4-fold.

Characterization of the adrenal-specific antigen IZA (inner zone antigen) and its role in the steroidogenesis

Inner zone antigen (IZA) is a protein specifically expressed in the zona fasciculata and reticularis of the adrenal cortex. The cDNA encoding IZA was found to be identical to that encoding the previously reported putative membrane-associated progesterone receptor (MPR) and the TCDD-induced 25kDa protein (25-Dx). From its structure, MPR was classed as a member of a protein family containing a haem-binding domain, and progesterone was proposed to be a ligand of this domain. Indeed, when GST-tagged IZA was expressed in Escherichia coli and purified, the purified GST-IZA had a brown colour with maximum absorbance at 400 nm. The addition of dithionate shifted the absorbance peak to 420 nm, suggesting a haem-binding function. The possible role of IZA in steroidogenesis has been addressed, and the inhibition of adrenal steroidogenesis by the addition of an anti-IZA monoclonal antibody has been reported. When COS-7 cells were transformed with plasmids for appropriate steroidogenic enzymes in the presence or absence of an IZA expression plasmid and tested for their steroidogenic activities, 21-hydroxylation of progesterone was found to be specifically activated by IZA overexpression, suggesting the involvement of IZA in progesterone metabolism. Taken together, the available evidence suggests that IZA may have an important role in the functions of the adrenal zona fasciculata and reticularis.

Fish testicular 11β-hydroxylase : cDNA cloning and mRNA expression during spermatogenesis

We isolated and characterized a cDNA encoding testicular 11β‐hydroxylase, cytochrome P450(11β) from the Japanese eel (Anguilla japonica) testis. The cDNA contains an open reading frame that encodes a protein of 511 amino acids. The predicted amino acid sequence shares 38–48% homology with those of adrenal P450(11β) from mammals and frog. Transient expression in COS 1 cells confirmed that the protein encoded by this cDNA had P450(11β) activity. Northern blotting revealed a single 1.8 kb long transcript of P450(11β). This transcript was not found in immature eel testes prior to an injection with human chorionic gonadotropin (hCG), but it was present in eel testes after hCG injection.

Implication of zog protein (zona glomerulosa-specific protein) in zone development of the adrenal cortex

The three zones of adrenal cortex are thought to arise from a single multipotential stem cell. Immunohistochemical studies of fetal and adult adrenals using an antibody against a previously-cloned ZOG protein, a rat homolog of Pref-1, were conducted to explore its roles in the differentiation of cortical tissues. At the early embryonic stage, ZOG was already expressed in adrenogonadal primordial cells. The ZOG-positive cells gradually formed the adrenal primordium by E14.5. By E17.5 the expression was repressed in the inner part of the aggregate and these cells began to express CYP11B1. The ZOG-positive cells at this stage existed at the periphery of the aggregate but they did not express CYP11B2 yet. Not until E20.5 did the aldosteronogenic cells appear among the ZOG-positive cells at the outermost part of the gland. Based on these and the other findings the zonal development of the adrenal cortex is discussed.

Molecular biology of rat steroid 11ß-hydroxylase [P450(11ß)] and aldosterone synthase [P450(11ß, Aldo)]

The molecular features of rat steroid 11 beta-hydroxylase [P450(11 beta)] and aldosterone synthase [P450(11 beta, aldo)] are discussed. P450(11 beta) is biosynthesized as a precursor form composed of 499 amino acids, having a 24-amino acid extension peptide. Two species of P450(11 beta, aldo) were identified; a precursor form of P450(11 beta, aldo)-1 is 510 amino acids long and has a 34-amino acid extension peptide, while that of P450(11 beta, aldo)-2 is 500 amino acids long and has a 24-amino acid extension peptide. The 286th amino acid of P450(11 beta, aldo)-1 is Glu, while that of P450(11 beta, aldo)-2 is Lys. The cDNA-expression studies showed that P450(11 beta, aldo)-1 had the aldosterone producing activity whereas P450(11 beta, aldo)-2 had no activity, suggesting that Glu286 of P450(11 beta, aldo) plays an important role in the catalysis. The amino acid sequence of a region in P450(11 beta) from Leu337 through Pro352 is highly conserved among the steroidogenic P450s. Functional expression studies on the cDNAs for two P450(11 beta)s showed that P450(11 beta) catalyzes the 11 beta-, 18- and 19-hydroxylations of 11-deoxycorticosterone, but not the aldosterone synthesis. P450(11 beta, aldo), on the other hand, catalyzes the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. The two P450(11 beta)s were also shown to catalyze the conversion of 11-deoxycortisol to cortisol, 18-hydroxycortisol and cortisone.