Regulation of Gene Expression by Cyclic GMP-Dependent Protein Kinase Requires Nuclear Translocation of the Kinase: Identification of a Nuclear Localization Signal

Abstract

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...

Nitric Oxide Activation of p38 Mitogen-activated Protein Kinase in 293T Fibroblasts Requires cGMP-dependent Protein Kinase

“…The cyclic nucleotide-dependent kinases have also been shown directly to mediate some gene expression, although emphasis has been on the ability to activate the transcription factor CREB by direct phosphorylation. These studies have lead to some debate as to how the cytosolically located PKG can activate the predominantly nuclear CREB as a substrate (31,43,44). The activation of MAPKs by PKG can therefore provide an alternative mechanism by which PKG can regulate transcription without nuclear translocation.…”

Nitric Oxide–Induced Increase in p21 Sdi1/Cip1/Waf1 Expression During the Cell Cycle in Aortic Adventitial Fibroblasts

“…31 Previous studies have shown that colocalization of p53, protein kinase A, and protein kinase G in the nucleus provide access for these 2 kinases to phosphorylate p53. 32,33 Because NO can induce p53 activation and nuclear localization and can activate protein kinase G through the ability to increase guanylate cyclase activity, it is reasonable to propose that the NOinduced activation of p53 is related to increased cGMP levels. At high levels of NO, it is presumed that protein kinase A can be activated by the inordinately high concentrations of cGMP produced, 6 and because the catalytic subunit of protein kinase A can translocate to the nucleus, the possibility exists that either or both protein kinases might mediate an effect of cGMP by nuclear localization and subsequent p53 activation.…”

“…At high levels of NO, it is presumed that protein kinase A can be activated by the inordinately high concentrations of cGMP produced, 6 and because the catalytic subunit of protein kinase A can translocate to the nucleus, the possibility exists that either or both protein kinases might mediate an effect of cGMP by nuclear localization and subsequent p53 activation. Furthermore, because protein kinases A and G can phosphorylate transcriptional factors related to cAMP response element binding protein, 32 other diverse effects could promote either p53-dependent or -independent increases in p21 transcription.…”

Nuclear entry of a cGMP-dependent kinase converts transient into long-lasting olfactory adaptation

“…In other PKGs, cGMP binding has been shown to relieve an inhibitory interaction between an amino terminal pseudosubstrate region and the kinase domain (31). cGMP binding has been shown to both activate the kinase and promote nuclear translocation of PKG 1α in mammalian cells (32). Altering key residues within either or both cGMP-binding domains (T276A and T398A) of GFP-EGL-4 led to exclusively cytosolic GFP, even after prolonged odor exposure (Fig.…”

“…3B). The NLS of the kinasedefective mutant should be exposed by cGMP binding (32), and yet, it is defective for nuclear entry. Since the NLS of the kinase defective mutant should be exposed by cGMP binding, the fact that we observe a partial dependence on the kinase domain indicates that nuclear translocation may be enhanced by a kinase function.…”