Molecular and Cellular Endocrinology 265–266 (2007) 196–204 Dephosphorylation of TORC initiates expression of the StAR gene Hiroshi Takemori a,∗ , Mariko Kanematsu a,b , Junko Kajimura a , Osamu Hatano c , Yoshiko Katoh a , Xing-zi Lin a , Li Min a , Takeshi Yamazaki d , Junko Doi b , Mitsuhiro Okamoto e a Laboratory of Cell Signaling and Metabolism, National Institute of Biomedical Innovation, Osaka 567-0085, Japan b Faculty of Food and Nutrition, Senri Kinran University, Osaka 565-0873, Japan c Department of Anatomy, Nara Medical University, Kashihara, Nara 634-8521, Japan d Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima 739-8524, Japan e Faculty of Contemporary Human Life Science, Tezukayama University, Nara 631-8585, Japan Abstract Cyclic AMP responsive element (CRE) binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. However, transducer of regulated CREB activity (TORC), a CREB specific co-activator, upregulates CREB activity in a phospho-Ser133-independent manner. Interestingly, TORC is also regulated by phosphorylation; the phospho-form is inactive, and the dephospho-form active. When PKA phosphorylates CREB, it inhibits TORC kinases simultaneously and accelerates dephosphorylation of TORC. We show in this report that staurosporine, a kinase inhibitor, induces the expression of the StAR gene in Y1 adrenocortical cells, possibly a result of an increase in the population of dephospho-TORC. The expression of the StAR gene is known to be regulated by SF-1 and CREB, and the co-activators CBP/p300 may mediate the actions of both factors. Our experiments using KG501, a disruptor of the interaction between phospho-CREB and CBP/p300, also support the importance of TORC in the regulation of StAR gene expression. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: CREB; SIK; TORC; StAR; SF-1; Adrenal gland 1. Introduction Cyclic AMP responsive element (CRE) binding protein (CREB) is a transcription factor that plays numerous roles in a variety of physiological events, such as cell proliferation, survival, tumorigenesis, glucose metabolism, and memory (Impey et al., 2004). CREB is activated in the adrenocortical cells by kinase cascades, e.g., the adrenocorticotropic hormone (ACTH)-protein kinase A (PKA) (Schimmer et al., 2006) pathway and the angiotensin II-Ca 2+ /calmodulin-dependent kinase pathway (Condon et al., 2002). Active CREB induces Abbreviations: CRE, cAMP responsive element; CREB, CRE-binding protein; ACTH, adrenocorticotropic hormone; CBP, CREB binding protein; SIK, salt-inducible kinase; TORC, transducer of regulated CREB activity; PKA, protein kinase A; SF-1, steroidogenic factor 1; StAR, steroidogenic acute regulatory protein; CYP11A1, side chain cleavage cytochrome P450; CYP11B1, 11-hydroxylase P450; CYP11B2, aldosterone synthase P450; GST, glutathione-S-transferase; PCR, polymerase chain reaction; MOI, multiplicity of infection ∗ Corresponding author at: Laboratory of Cell Signaling and Metabolism, National Institute of Biomedical Innovation, 7-6-8, Asagi, Saito, Ibaraki, Osaka 567-0085, Japan. Tel.: +81 72 641 9834; fax: +81 72 641 9836. E-mail address: takemori@nibio.go.jp (H. Takemori). gene expression of steroidogenic enzymes in cooperation with a nuclear factor SF-1/Ad4BP (steroidogenic factor-1/Ad4- binding protein) (Morohashi and Omura, 1996; Parker and Schimmer, 1997). The following genes for the steroidogenic enzymes/proteins are reportedly regulated by CREB. Steroido- genic acute regulatory protein (the StAR protein, which transfers cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane) (Clark et al., 1994; Manna et al., 2002), side chain cleavage cytochrome P450 (the enzyme CYP11A1, which catalyzes the conversion of cholesterol to pregnenolone) (Rice et al., 1990; Morohashi et al., 1993), 11-hyrodxylase P450 (the enzyme CYP11B1, which catalyzes the final step of glucocorticoid synthesis) (Wang et al., 2000) and aldosterone synthase P450 (the enzyme CYP11B2, which catalyzes the final step of aldosterone synthesis) (Clyne et al., 1997). The CREB-dependent transcription is believed to be acti- vated, when Ser133 of CREB is phosphorylated (Johannessen et al., 2004). This phosphorylation alters the affinity of the transactivation domain of CREB to the acceptor domain of the CREB-binding protein (CBP) and p300, and eventually results in enhancing transcriptions of CRE-dependent genes. The small compound KG501 that disrupts the interaction between 0303-7207/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2006.12.020