We recently obtained evidence that treatment of human colon cancer cells with exisulind (sulindac sulfone) and related compounds induces apoptosis by activation of protein kinase G (PKG) and c-Jun kinase (JNK1). The present study further explores this mechanism. We demonstrate that in NIH3T3 cells a constitutively active mutant of PKG causes a dose-dependent activation of JNK1 and thereby transactivates c-Jun and stimulates transcription from the AP-1 enhancer element. The activation of JNK1

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... the transactivation of c-Jun by this mutant of PKG were inhibited by a dominant negative MEKK1. In vitro assays showed that a purified PKG directly phosphorylated the N-terminal domain of MEKK1. PKG also directly phosphorylated a full-length MEKK1, and this was associated with enhanced MEKK1 phosphorylation. Thus, it appears that PKG activates JNK1 through a novel PKG-MEKK1-SEK1-JNK1 pathway, by directly phosphorylating and activating MEKK1. Cyclic GMP (cGMP) is an important second messenger that mediates several signal transduction pathways in mammalian cells (1). It is involved in the regulation of various physiological functions, including neurotransmission, cell differentiation, proliferation, and platelet aggregation (2). Cyclic GMP also modulates intracellular calcium levels in vascular smooth muscle cells and thereby modulates smooth muscle tone (3). Intracellular levels of cGMP are tightly regulated through synthesis by guanylate cyclases and hydrolysis by specific phosphodiesterases (PDEs) 1 (4, 5). cGMP has several intracellular targets, including gated ion channels, cGMP-dependent protein kinases (PKG), cGMP-activated phosphodiesterases, and cGMP-inhibited phosphodiesterases (6, 7). PDE2 and PDE5 are cGMP phosphodiesterases (5). In recent studies we obtained evidence that in human colon cancer cell lines, novel PDE2/5 inhibitors, including exisulind (sulindac sulfone) and the high affinity derivatives CP248 and CP461, increase cellular levels of cGMP and that this leads to activation of PKG and c-Jun kinase (JNK1) and induction of apoptosis (8, 9) . However, the biochemical details by which cGMP leads to activation of JNK1 were not definitely established. The present study presents evidence that the activation of PKG by cGMP leads to direct phosphorylation and activation of MEKK1. This then leads to activation of SEK1 and subsequently JNK1. This PKG-MEKK1-SEK1-JNK1 pathway represents a novel signal transduction pathway that may be important in understanding the roles of cGMP and PKG in cell proliferation and apoptosis. EXPERIMENTAL PROCEDURES Cell Cultures and Transfection and Reporter Assays-NIH3T3 mouse fibroblasts were routinely grown in Dulbecco's minimal essential medium (DMEM) containing 10% calf serum. For reporter assays, triplicate samples of 1 ϫ 10 5 cells in 35-mm plates were transfected using Lipofectin (Life Technologies, Inc.) with 1 g of the reporter plasmid, 0.05-5 g of various expression vectors, and 1 g of the control plasmid pCMV-␤-gal. The pcDNA3 plasmid DNA was added to the transfections, as needed, to achieve the same total amount of plasmid DNA per transfection. Twenty-four hours after transfection, cell extracts were prepared, and luciferase assays were done using the Luciferase Assay System (Promega). Luciferase activities were normalized with respect to parallel ␤-gal activities, to correct for differences in transfection efficiency. ␤-gal assays were performed using the ␤-Galactosidase Enzyme Assay System (Promega). The plasmid pRc/CMV-⌬93GK was kindly provided by R. Pilz (University of California, San Diego) (10), the pAP-1-luciferase plasmid by J. Pierce (NCI) (11), the pGAL4DB-c-Jun and pG5-luciferase plasmids by A. Minden (Columbia University), and the pCEP4-HA-MEKK1-WT and pCEP4-HA-MEKK1-D1369A plasmids by M. H. Cobb (University of Texas Southwestern Medical Center) (12). The pCMV-␤-gal plasmid was purchased from Stratagene. JNK1 Kinase Assays-The cells were lysed in a lysis buffer (20 mM Tris-HCl, pH 7.5, 0.5% Nonidet P-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 20 mM ␤-glycerophosphate, 25% glycerol) and then JNK1 was immunoprecipitated for 2 h with an anti-JNK1 antibody (Santa Cruz) and assayed for in vitro kinase activity with GST-c-Jun-(1-79) (New England Biolabs) as the substrate, in a kinase reaction buffer (20 mM HEPES, pH 7.5, 10 mM MgCl 2 , 1 mM DTT, 20 M ATP, 20 mM ␤-glycerophosphate, 1 Ci of [␥-32 P]ATP), for 20 min, as described previously (8). The reaction mixture was then subjected to 10% SDS-PAGE. For transient transfection experiments, subconfluent cultures of cells in 10-cm plates were transfected, using Lipofectin (Life Technologies, Inc.), with 2.5 g of the pCMV5-M2-JNK1 plasmid and either 5 g of pRc/CMV-⌬93GK or pCEP4-HA-MEKK1 plasmid DNA. The pcDNA3 plasmid DNA was used as a control. Twenty-four hours after transfection, the cells were lysed in the lysis buffer described above and then JNK1 was immunoprecipitated with an anti-FLAG antibody (Sigma) for 2 h and assayed for in vitro kinase activity, as described above. The intensities of the bands on gels were determined with a Phos-phorImager (Molecular Dynamics), and the ratio of the treated sample to the control untreated sample was expressed as "relative kinase activity" or "fold activation." The experiments were repeated three times with similar results. PKG Assays-Purified recombinant PKG I␣ (100 units, Calbiochem)