Protein kinase G type-Ialpha phosphorylates the apoptosis-regulating protein Bad at serine 155 and protects against apoptosis in N1E-115 cells.
Previous studies from our laboratory have shown that the cGMP/protein kinase G (PKG) signaling pathway plays an essential role in preventing spontaneous apoptosis in neural cells; however, the mechanism is not understood. A potential downstream target of PKG is the apoptosis-regulating protein Bad, which contains a sequence around its serine 155 (ser155 in mouse Bad, equivalent to ser118 in human Bad) predicted to be a consensus motif for PKG-catalyzed phosphorylation. Using both in vitro and cell-based experiments, we determined if PKG phosphorylates Bad at ser155 and if blocking/stimulating PKG-catalyzed Bad phosphorylation causes pro-apoptotic/anti-apoptotic responses. Recombinant PKG type-Ialpha (PKG-Ialpha) was found to directly phosphorylate recombinant Bad at ser155 in vitro. In N1E-115 mouse neural cells, which naturally express PKG-Ialpha as the predominant PKG isoform, addition of 8-Br-cGMP (0.1-1.0 mM), a cell-permeable direct PKG-Ialpha activator, increased ser155 phosphorylation of Bad. ODQ (50 microM), a soluble guanylyl cyclase inhibitor that lowers cGMP/PKG activity, decreased serum-induced ser155 phosphorylation of Bad and induced apoptosis in N1E-115 cells. Treatment with DT-2 and DT-3, selective PKG-Ialpha inhibitors, both decreased Bad ser155 phosphorylation and induced apoptosis. The data indicate that PKG-Ialpha directly phosphorylates Bad at ser155, which may participate in cGMP/PKG-induced anti-apoptotic/cytoprotective effects in neural cells.
Johlfs
Mary G
MG
Cancer Molecular Biology Section, Nevada Cancer Institute, Las Vegas, NV 89135, USA.
Fiscus
Ronald R
RR
D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D002460:Cell Line; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000494:pharmacology; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D053938:DNA Fragmentation; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000378:metabolism; D010069:Oxadiazoles / Q000494:pharmacology; D010766:Phosphorylation; D011810:Quinoxalines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D051025:bcl-Associated Death Protein / Q000378:metabolism*
C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D010069:Oxadiazoles; D011810:Quinoxalines; D011994:Recombinant Proteins; D051025:bcl-Associated Death Protein; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; D012694:Serine; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569718:PRKG1 protein, human; C569720:Prkg1 protein, mouse; D006152:Cyclic GMP