REGULATION OF GENE-EXPRESSION BY CYCLIC GMP-DEPENDENT PROTEIN-KINASE REQUIRES NUCLEAR TRANSLOCATION OF THE KINASE - IDENTIFICATION OF A NUCLEAR-LOCALIZATION SIGNAL

We recently demonstrated that cyclic GMP (cGMP)-dependent protein kina se (G-kinase) activates the human fos promoter in a strictly cGMP-depe ndent manner (T. Gudi et al., J. Biol. Chem, 271:4597-4600, 1996). Her e, we demonstrate that G-kinase translocates to the nucleus by an acti ve transport mechanism which requires a nuclear localization signal (N LS) and is regulated by cGMP. Immunofluorescent staining of G-kinase w as predominantly cytoplasmic in untreated cells, but intense nuclear s taining appeared in S-bromo (Br)-cCMP-treated cells. We identified a p utative NLS in the G-kinase ATP binding domain which resembles the NLS of the interleukin-1 alpha precursor. Fusion of the G-kinase NLS to t he N terminus of beta-galactosidase produced a chimeric protein which localized to the nucleus. Mutation of a single amino acid residue (K-4 07-->E) within the C-kinase NLS produced an enzyme with normal cGMP-de pendent activity in vitro which did not translocate to tile nucleus an d 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 nuc leus and transactivated the Sos promoter in the absence of 8-Br-cGMP. These results indicate that nuclear localization of G-kinase is requir ed 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 activate s an otherwise cryptic NLS.