Integrated Regulation of PKA by Fast and Slow Neurotransmission



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The neuronal cyclin-dependent kinase 5 (Ckd5) has been shown to be a major regulator of key signaling events downstream of dopamine receptors in the brain. One critical example of such a pathway is the cAMP-dependent protein kinase, PKA, which plays a well-established role in synaptic plasticity. In addition, recent studies have implicated PKA in the regulation of mood via the brain's mesocorticolimbic pathway. Using in vitro kinase assays and mass spectrometry (MS), we identified a novel Cdk5 phosphorylation site at amino acid residue thr69 (T69) that is unique to the Type II β isoform of the regulatory subunit of PKA, RIIβ. Upon examination of rat brain lysate, RIIβ is shown to be the predominantly expressed isoform within the central nervous system (CNS); the prefrontal cortex, striatum (both ventral and dorsal), and hippocampus exhibit notably high levels of the protein. Immunohistochemistry (IHC) demonstrates expression of RIIβ in all hippocampus subregions - though interestingly only dendritic projections of the granule cell layer of the dentate gyrus (DG) show staining with a phospho-state specific antibody to the T69 site - and ubiquitously throughout the striatal medium spiny neurons (MSNs). These regions are also rich in Cdk5, and the two proteins display co-localization in the dendrites of cultured striatal MSNs. In acute striatal slices, inhibition of Cdk5 resulted in a decrease in T69 phosphorylation, demonstrating in vivo regulation of the site by Cdk5. Via site-directed mutagenesis we generated a full-length T69D RIIβ phosphomimic protein. Compared with unphosphorylated RIIβ, the T69D phosphomimic was a poorer substrate for the PKA catalytic subunit at an activating "autophosphorylation" site, S114. These results were further supported by pharmacological treatments of acute striatal slices, which demonstrated T69/S114 phosphorylation antagonism. As expected from previous studies suggesting that S114 phosphorylation altered PKAcat-RIIβ interactions, a S114D RIIβ phosphomimic was indeed less efficient at inhibiting PKA catalytic subunit activity. Therefore, we believe that Cdk5-dependent phosphorylation of RIIβ acts to inhibit PKA holoenzyme activity via intramolecular changes. It may also affect spatial regulation of PKA. In an RIIβ overlay assay, the T69D phosphomimic demonstrated reduced binding to an important scaffold protein, A kinase anchoring protein 150 (AKAP150). Therefore, we believe through phosphorylation at the T69 site, Cdk5 influences PKA signaling via inter- as well as intra-molecular mechanisms. In order to functionally assess the role of Cdk5-dependent RIIβ phosphorylation, we developed a small interfering peptide (siP) approach that allows us to selectively target Cdk5-RIIβ interactions. We found that the T69 site was negatively modulated by glutamatergic and dopaminergic transmission in the nucleus accumbens (NAc), and that the RIIβ−siP was able to substitute for the glutamatergic input required for dopamine-dependent sustained striatal PKA activation. Moreover, infusion of the peptide into the NAc of rats boosted in vivo PKA activation. The limbic system is critical for establishing emotional salience of, and guiding behavioral output in response to, stressful stimuli. Following acute or chronic stress, levels of phospho-T69 RIIβ in the NAc were altered. Infusion of the RIIβ-siP into the NAc improved the animals' behavioral stress responses in a PKA-dependent fashion. Together, these data support the role of phospho-T69 RIIβ in modulation of PKA activity, thereby impacting striatal neuronal functioning and stress-related behaviors.

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