AMP-activated protein kinase (AMPK) activation reportedly suppresses transcriptional activity of the cAMP-responsive element (CRE) in the phosphoenolpyruvate carboxykinase C (PEPCK-C) promoter and reduces hepatic PEPCK-C expression. Although a previous study found TORC2 phosphorylation to be involved in the suppression of AMPK-mediated CRE transcriptional activity, we herein present evidence that glycogen synthase kinase 3beta (GSK3beta) phosphorylation induced by AMPK also plays an important role. We initially found that injecting fasted mice with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) markedly increased Ser-9 phosphorylation of hepatic GSK3beta within 15 min. Stimulation with AICAR or the GSK3beta inhibitor SB-415286 strongly inhibited CRE-containing promoter activity in HepG2 cells. Using the Gal4-based transactivation assay system, the transcriptional activity of cAMP-response element-binding protein (CREB) was suppressed by both AICAR and SB415286, whereas that of TORC2 was repressed significantly by AICAR but very slightly by SB415286. These results show inactivation of GSK3beta to directly inhibit CREB but not TORC2. Importantly, the AICAR-induced suppression of PEPCK-C expression was shown to be blunted by overexpression of GSK3beta(S9G) but not wild-type GSK3beta. In addition, AICAR stimulation decreased, whereas Compound C (AMPK inhibitor) increased CREB phosphorylation (Ser-129) in HepG2 cells. The time-courses of decreased CREB phosphorylation (Ser-129) and increased GSK3beta phosphorylation were very similar. Furthermore, AMPK-mediated GSK3beta phosphorylation was inhibited by an Akt-specific inhibitor in HepG2 cells, suggesting involvement of the Akt pathway. In summary, phosphorylation (Ser-9) of GSK3beta is very likely to be critical for AMPK-mediated PEPCK-C gene suppression. Reduced CREB phosphorylation (Ser-129) associated with inactivation of GSK3beta by Ser-9 phosphorylation may be the major mechanism underlying PEPCK-C gene suppression by AMPK-activating agents such as biguanide.
Protein kinase B (PKB)/Akt reportedly plays a role in the survival and/or proliferation of cells. We identified a novel protein, which binds to PKB, using a yeast two-hybrid screening system. This association was demonstrated not only in vivo by overexpressing both proteins or by coimmunoprecipitation of the endogenous proteins, but also in vitro using glutathione S-transferase fusion proteins. Importantly, this protein specifically associates with the C terminus of PKB but not with other AGC kinases and enhances PKB phosphorylation and kinase activation without growth factor stimulation. Thus, we termed this Akt-specific binding protein APE (Akt-phosphorylation enhancer). Since APE-induced phosphorylation of PKB did not occur in cells treated with wortmannin or LY294002, APE itself is not a kinase but seems to enhance or prolong the phosphoinositide 3-kinase-dependent phosphorylation of PKB. In cells in which APE was suppressed by small interfering RNA, DNA synthesis was significantly reduced with suppression of PKB phosphorylation, suggesting a synergistic role of APE in PKB-induced proliferation. On the other hand, in cells overexpressing both PKB and APE, despite markedly increased basal phosphorylation of PKB, both DNA rereplication and subsequent Chk2 phosphorylation and apoptosis were seen, suggesting the involvement of APE in the regulation of cell cycling replication licensing. Taking these observations together, APE appears to be a novel regulator of PKB phosphorylation. Furthermore, the interaction between APE and PKB, possibly dependent on the expression levels of both proteins, may be a novel molecular mechanism leading to proliferation and/or apoptosis.
Salt‐inducible kinase‐1 (SIK1) is phosphorylated at Ser577 by protein kinase A in adrenocorticotropic hormone‐stimulated Y1 cells, and the phospho‐SIK1 translocates from the nucleus to the cytoplasm. The phospho‐SIK1 is dephosphorylated in the cytoplasm and re‐enters the nucleus several hours later. By using green‐fluorescent protein‐tagged SIK1 fragments, we found that a peptide region (586–612) was responsible for the nuclear localization of SIK1. The region was named the ‘RK‐rich region’ because of its Arg‐ and Lys‐rich nature. SIK1s mutated in the RK‐rich region were localized mainly in the cytoplasm. Because SIK1 represses cAMP‐response element (CRE)‐mediated transcription of steroidogenic genes, the mutants were examined for their effect on transcription. To our surprise, the cytoplasmic mutants strongly repressed the CRE‐binding protein (CREB) activity, the extent of repression being similar to that of SIK1(S577A), a mutant localized exclusively in the nucleus. Several chimeras were constructed from SIK1 and from its isoform SIK2, which was localized mainly in the cytoplasm, and they were examined for intracellular localization as well as CREB‐repression activity. A SIK1‐derived chimera, where the RK‐rich region had been replaced with the corresponding region of SIK2, was found in the cytoplasm, its CREB‐modulating activity being similar to that of wild‐type SIK1. On the other hand, a SIK2‐derived chimera with the RK‐rich region of SIK1 was localized in both the nucleus and the cytoplasm, and had a CREB‐repressing activity similar to that of the wild‐type SIK2. Green fluorescent protein‐fused transducer of regulated CREB activity 2 (TORC2), a CREB‐specific co‐activator, was localized in the cytoplasm and nucleus of Y1 cells, and, after treatment with adrenocorticotropic hormone, cytoplasmic TORC2 entered the nucleus, activating CREB. The SIK1 mutants, having a strong CRE‐repressing activity, completely inhibited the adrenocorticotropic hormone‐induced nuclear entry of green fluorescent protein‐fused TORC2. This suggests that SIK1 may regulate the intracellular movement of TORC2, and as a result modulates the CREB‐dependent transcription activity. Together, these results indicate that the RK‐rich region of SIK1 is important for determining the nuclear localization and attenuating CREB‐repressing activity, but the degree of the nuclear localization of SIK1 itself does not necessarily reflect the degree of SIK1‐mediated CREB repression.
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.
The CREB-specific coactivator TORC2 (also known as CRTC2) upregulates gluconeogenic gene expression in the liver. Salt-inducible kinase (SIK) family enzymes inactivate TORC2 through phosphorylation and localize it in the cytoplasm. Ser 171 and Ser 275 were found to be phosphorylated in pancreatic β-cells. Calcineurin (Cn) is proposed as the Ser 275 phosphatase, because its inhibitor cyclosporin A (CsA) stabilizes phospho-Ser 275 and retains TORC2 in the cytoplasm. Because the regulation of dephosphorylation at Ser 171 has not been fully clarified, we performed experiments with a range of doses of okadaic acid (OA), an inhibitor of PP2A/PP1, and with overexpression of various phosphatases and found that PP1 functions as an activator for TORC2, whereas PP2A acts as an inhibitor. In further studies using TORC2 mutants, we detected a disassociation between the intracellular distribution and the transcription activity of TORC2. Additional mutant analyses suggested the presence of a third phosphorylation site, Ser 307 . The Ser 307 -disrupted TORC2 was constitutively localized in the nucleus, but its coactivator activity was normally suppressed by SIK1 in COS-7 cells. CsA, but not OA, stabilized the phosphogroup at Ser 307 , suggesting that differential dephosphorylation at Ser 171 and Ser 307 cooperatively regulate TORC2 activity and that the nuclear localization of TORC2 is insufficient to function as a coactivator. Because the COS-7 cell line may not possess signaling cascades for gluconeogenic programs, we next examined the importance of Ser 307 and Ser 171 for TORC2's function in mouse liver. Levels of phosphorylation at Ser 171 and Ser 307 changed in response to fasting or fed conditions and insulin resistance of the mouse liver, which were modified by treatment with CsA/OA and by overexpression of PP1/PP2A/Cn. These results suggest that multiple phosphorylation sites and their phosphatases may play important roles in regulating TORC2/CREB-mediated gluconeogenic programs in the liver.
