We recently demonstrated that cyclic GMP (cGMP)-dependent protein kinase (G-kinase) activates the human fos promoter in a strictly cGMP-dependent manner (T. Gudi et al., J. Biol. Chem. 271: [4597][4598][4599][4600] 1996). Here, we demonstrate that G-kinase translocates to the nucleus by an active transport mechanism which requires a nuclear localization signal (NLS) and is regulated by cGMP. Immunofluorescent staining of G-kinase was predominantly cytoplasmic in untreated cells, but intense nuclear staining appeared in 8-bromo (Br)-cGMP-treated cells. We identified a putative NLS in the G-kinase ATP binding domain which resembles the NLS of the interleukin-1␣ precursor. Fusion of the G-kinase NLS to the N terminus of ␤-galactosidase produced a chimeric protein which localized to the nucleus. Mutation of a single amino acid residue (K 407 3E) within the G-kinase NLS produced an enzyme with normal cGMP-dependent activity in vitro which did not translocate to the nucleus and did not transactivate the fos promoter in the presence of 8-Br-cGMP in vivo. In contrast, N-terminally truncated versions of G-kinase with constitutive, cGMP-independent activity in vitro localized to the nucleus and transactivated the fos promoter in the absence of 8-Br-cGMP. These results indicate that nuclear localization of G-kinase is required for transcriptional activation of the fos promoter and suggest that a conformational change of the kinase, induced by cGMP binding or by removal of the N-terminal autoinhibitory domain, functionally activates an otherwise cryptic NLS.The NO/cyclic GMP (cGMP) signal transduction pathway is present in many mammalian cells and involved in the regulation of important physiological functions such as neurotransmission, cell differentiation and proliferation, changes in vascular smooth muscle tone, endothelial cell permeability, and platelet aggregation (13,36,37,39,40,44,45). Whereas the cyclic AMP (cAMP) signal transduction pathway is well known to regulate gene transcription, regulation of gene expression by the NO/cGMP signal transduction pathway has been demonstrated only recently in different cell types (2,5,16,18,19,24,38,41,47). In rat embryo fibroblasts and thyroid epithelial cells, we showed that NO-releasing agents and membranepermeable cGMP analogs increase c-fos and junB mRNA expression, AP-1 DNA binding, and the transcriptional activity of promoters containing phorbol ester response elements (41). We subsequently demonstrated that transfection of G-kinase I␤ into G-kinase-deficient baby hamster kidney (BHK) cells causes induction of endogenous c-fos mRNA and activation of a cotransfected human fos promoter construct in a strictly cGMP-dependent manner (18, 18a). The effect of G-kinase is mediated by several sequence elements in the fos promoter, most notably the cAMP response element, the AP-1 binding site, and the serum response element with adjacent C/EBP-␤ binding site (18). The magnitude of G-kinase transactivation of the fos promoter was similar to that of cAMP-dependent protein kinase (A...

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Many of nitric oxide's biological effects are mediated via NO binding to the iron in heme-containing proteins. Cobalamin (vitamin B 12 ) is structurally similar to heme and is a cofactor for methionine synthase, a key enzyme in folate metabolism. NO inhibits methionine synthase activity in vitro, but data concerning NO binding to cobalamin are controversial. We now show spectroscopically that NO reacts with all three valency states of cobalamin and that NO's inhibition of methionine synthase activity most likely involves its reaction with monovalent cobalamin. By following incorporation of the methyl moiety of [ 14 C]methyltetrahydrofolic acid into protein, we show that NO inhibits methionine synthase activity in vivo, in cultured mammalian cells. The inhibition of methionine synthase activity disrupted carbon flow through the folate pathway as measured by decreased incorporation of [ 14 C]formate into methionine, serine, and purine nucleotides. Homocysteine, but not cysteine, attenuated NO's inhibition of purine synthesis, providing further evidence that NO was acting through methionine synthase inhibition. NO's effect was observed both when NO donors were added to cells and when NO was produced physiologically in co-culture experiments. Treating cells with an NO synthase inhibitor increased formate incorporation into methionine, serine, and purines and methyl-tetrahydrofolate incorporation into protein. Thus, physiological concentrations of NO appear to regulate carbon flow through the folate pathway.Vitamin B 12 deficiency leads to pernicious anemia and subacute combined degeneration of the spinal cord (1). Pernicious anemia is characterized by megaloblastic erythropoiesis and is secondary to decreased activity of methionine synthase, one of two mammalian enzymes that requires vitamin B 12 (cobalamin) as a cofactor (2, 3). Methionine synthase catalyzes the transfer of the methyl group of 5-methyltetrahydrofolate to homocysteine via a methylcobalamin intermediate with cycling of cobalamin between the ϩ1 valency state (i.e. cbl(I)) 1 and the ϩ3 valency state (i.e. cbl(III)) (2, 3). Methyltetrahydrofolate is the major intracellular storage form of folates, and its synthesis from 5,10-methylene tetrahydrofolate is essentially irreversible in vivo (2, 4) (Fig. 1). Thus, decreased methionine synthase activity leads to trapping of intracellular folates as 5-methyltetrahydrofolate, and the megaloblastic anemia of vitamin B 12 deficiency is virtually indistinguishable from the megaloblastosis of folate deficiency (1).Nitric oxide (NO) is produced by most cell types and regulates a diverse array of biological functions (5, 6). NO has been reported to inhibit methionine synthase activity in vitro (7-9), and it might be expected to bind to the cobalt in cobalamin because (i) NO binds tightly to the iron in heme (10); (ii) ferrous heme and cbl(III) are isoelectronic; and (iii) in both heme and cobalamin, the metal ion is coordinated to four in-plane nitrogen atoms of a tetrapyrrole ring and has two out-of-plane ligands (3...

We have shown that nitric oxide (NO) regulates c-fos gene expression via cGMP-dependent protein kinase (G-kinase), but NO's precise mechanism of action is unclear. We now demonstrate that: (1) NO targets two transcriptional elements in the fos promoter, i.e., the fos AP-1 binding site and the cAMP-response element (CRE); (2) NO activation of these two enhancer elements requires the CRE binding protein CREB because a dominant negative CREB fully inhibits NO transactivation of reporter genes whereas dominant negative Fos or CCAAT enhancer binding proteins have no effect; (3) CREB is phosphorylated by G-kinase in vitro and its phosphorylation increases in vivo when G-kinase is activated either directly by cGMP or indirectly by NO via soluble guanylate cyclase; (4) NO activation of fos promoter elements requires nuclear translocation of G-kinase but not activation of mitogen-activated protein kinases.