An extranuclear locus of cAMP-dependent protein kinase action is necessary and sufficient for promotion of spiral ganglion neuronal survival by cAMP.
We showed previously that cAMP is a survival-promoting stimulus for cultured postnatal rat spiral ganglion neurons (SGNs) and that depolarization promotes SGN survival in part via recruitment of cAMP signaling. We here investigate the subcellular locus of cAMP prosurvival signaling. Transfection of GPKI, a green fluorescent protein (GFP)-tagged cAMP-dependent protein kinase (PKA) inhibitor, inhibits the ability of the permeant cAMP analog cpt-cAMP [8-(4-chlorophenylthio)-cAMP] to promote survival, indicating that PKA activity is necessary. Transfection of GFP-tagged PKA (GPKA) is sufficient to promote SGN survival, but restriction of GPKA to the nucleus by addition of a nuclear localization signal (GPKAnls) almost completely abrogates its prosurvival effect. In contrast, GPKA targeted to the extranuclear cytoplasm by addition of a nuclear export signal (GPKAnes) promotes SGN survival as effectively as does GPKA. Moreover, GPKI targeted to the nucleus lacks inhibitory effect on SGN survival attributable to cpt-cAMP or depolarization. These data indicate an extranuclear target of PKA for promotion of neuronal survival. Consistent with this, we find that dominant-inhibitory CREB mutants inhibit the prosurvival effect of depolarization but not that of cpt-cAMP. SGN survival is compromised by overexpression of the proapoptotic regulator Bad, previously shown to be phosphorylated in the cytoplasm by PKA. This Bad-induced apoptosis is prevented by cpt-cAMP or by cotransfection of GPKA or of GPKAnes but not of GPKAnls. Thus, cAMP prevents SGN death through a cytoplasmic as opposed to nuclear action, and inactivation of Bad proapoptotic function is a mechanism by which PKA can prevent neuronal death.
Bok
Jinwoong
J
Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242, USA.
Zha
Xiang-Ming
XM
Cho
Yang-Sun
YS
Green
Steven H
SH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D020134:Catalytic Domain / Q000235:genetics; D002467:Cell Nucleus / Q000201:enzymology*; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D019913:Nuclear Localization Signals / Q000235:genetics / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D013136:Spiral Ganglion / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D013873:Thionucleotides / Q000037:antagonists & inhibitors / Q000494:pharmacology; D014162:Transfection; D051025:bcl-Associated Death Protein
C494821:Bad protein, rat; D002352:Carrier Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; D051025:bcl-Associated Death Protein; D049452:Green Fluorescent Proteins; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases