The effect of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) on the activation and differentiation of normal B cells was investigated. B cells of transgenic mice expressing LMP1 under the control of immunoglobulin promoter/enhancer displayed enhanced expression of activation antigens and spontaneously proliferated and produced antibody. Humoral immune responses of LMP1 transgenic mice in CD40-deficient or normal backgrounds revealed that LMP1 mimics CD40 signals to induce extrafollicular B cell differentiation but, unlike CD40, blocks germinal center formation. Thus, these specific properties of LMP1 may determine the site of primary B cell infection and the state of infection in the natural course of EBV infection, whereas subsequent loss of LMP1 expression may affect the site of persistent latent infection.

Cyclic AMP-responsive element (CRE)-binding protein (CREB) is a transcription factor that plays an important role in numerous physiological events, such as cell proliferation, survival, tumorigenesis, glucose metabolism and memory, in a phosphorylation-dependent manner [1,2] Cyclic AMP responsive element (CRE)-binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. Two independent investigations have suggested the presence of Ser133-independent activation. One study identified a kinase, salt-inducible kinase (SIK), which repressed CREB; the other isolated a novel CREB-specific coactivator, transducer of regulated CREB activity (TORC), which upregulated CREB activity. These two opposing signals are connected by the fact that SIK phosphorylates TORC and induces its nuclear export. Because LKB1 has been reported to be an upstream kinase of SIK, we used LKB1-defective HeLa cells to further elucidate TORC-dependent CREB activation. In the absence of LKB1, SIK was unable to phosphorylate TORC, which led to constitutive activation of CRE activity. Overexpression of LKB1 in HeLa cells improved the CRE-dependent transcription in a regulated manner. The inactivation of kinase cascades by 10 nm staurosporine in LKB1-positive HEK293 cells also induced unregulated, constitutively activated, CRE activity. Treatment with staurosporine completely inhibited SIK kinase activity without any significant effect on the phosphorylation level at the LKB1-phosphorylatable site in SIK or the activity of AMPK, another target of LKB1. Constitutive activation of CREB in LKB1-defective cells or in staurosporine-treated cells was not accompanied by CREB phosphorylation at Ser133. The results suggest that LKB1 and its downstream SIK play an important role in silencing CREB activity via the phosphorylation of TORC, and such silencing may be indispensable for the regulated activation of CREB.Abbreviations A-loop, activation loop; AMPK, AMP-activated protein kinase; bZIP, basic leucine zipper domain; CRE, cAMP-response element; CREB, CRE-binding protein; DAPI, 4¢,6-diamidino-2-phenylindole; GFP, green fluorescent protein; GST, glutathione-S-transferase; HA, hemagglutinin; KID, kinase-inducible domain; moi, multiplicities of infection; PKA, protein kinase A; RT, reverse transcription; SIK, salt-inducible kinase; TORC, transducer of regulated CREB activity.

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/EBP␤ mRNA. Coexpressed with human insulin receptor substrate-1 (IRS-1) in COS cells, SIK2 could phosphorylate Ser 794 of human IRS-1. Adenovirus-mediated overexpression of SIK2 in adipocytes elevated the level of phosphorylation at Ser 789 , the mouse equivalent of human Ser 794 . Moreover, the activity and content of SIK2 were elevated in white adipose tissues of db/db diabetic mice. These results suggest that highly expressed SIK2 in insulin-stimulated adipocytes phosphorylates Ser 794 of IRS-1 and, as a result, might modulate the efficiency of insulin signal transduction, eventually causing the insulin resistance in diabetic animals.The lipid metabolism in adipose tissues is under the control of two hormonal signaling pathways; insulin stimulates glucose uptake and lipogenesis, whereas cAMP, generated by exogenous stimuli like adrenalin and glucagon, stimulates lipolysis. If the balance between the two signaling systems becomes lost and the adipose tissues are exposed to hyperinsulinemia for a prolonged time, they gradually become resistant to insulin stimulation (1, 2). The insulin resistance occurring in tissues involved in biological fuel metabolism, such as adipose tissues, liver, and skeletal muscles, would finally cause disorders in energy metabolism of the whole body, such as obesity and type 2 diabetes (3, 4). Insulin receptor substrate (IRS) 1 proteins are key molecules of the insulin-signaling cascade (5); they are phosphorylated on tyrosine residues by the action of insulindependently activated insulin receptor kinase, and the tyrosine-phosphorylated IRS proteins trigger further intracellular cascades. Several investigators recently reported (6, 7) that IRS proteins, under certain non-physiological conditions, were phosphorylated on serine residues. The serine phosphorylation of IRS proteins would modulate the efficiency of the insulinsignaling cascade (8, 9) and eventually render the animals resistant to insulin stimulation (10, 11). Molecular identification of several protein kinases responsible for the serine phosphorylation of IRS proteins has been reported (12-24).Salt-inducible kinase (SIK) was first cloned from ...

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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 ÔRK-rich regionÕ 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 CREBrepressing 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 hormoneinduced 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.