Browsing by Subject "Cyclic AMP-Dependent Protein Kinases"
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Item Crosstalk Signaling Between cAMP and mTORC1(August 2021) Melick, Chase Hunter; DeBerardinis, Ralph J.; Cobb, Melanie H.; Pan, Duojia; Jewell, Jenna L.The mammalian target of rapamycin complex 1 (mTORC1) senses multiple stimuli to regulate anabolic and catabolic processes. G-protein-coupled receptors (GPCRs) paired to Gs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA), which phosphorylates Raptor at Ser 791 resulting in potent mTORC1 inhibition. We identified a novel mTORC1-interacting protein called A-kinase anchoring protein 8L (AKAP8L). Using biochemical assays, we found that the N-terminal region of AKAP8L binds to mTORC1 in the cytoplasm. Importantly, loss of AKAP8L decreased mTORC1-mediated processes such as translation, cell growth and cell proliferation. AKAPs anchor protein kinase A (PKA) through PKA regulatory subunits, and we show that AKAP8L can anchor PKA through regulatory subunit I (RI). Full-length AKAP8L restored mTORC1-regulated biology, whereas AKAP8L missing the N-terminal region that confers interaction with mTORC1 did not. Additionally, we have shown that H89 (N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide), a well-characterized ATP-mimetic kinase inhibitor, renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors across multiple cell lines. Moreover, H89 prevented the dephosphorylation of AKT and S6K1 under nutrient depleted conditions. PKA and other known H89-targeted kinases do not alter the phosphorylation status of S6K1 and AKT. Pharmacological inhibition of some phosphatases also enhanced S6K1 and AKT phosphorylation. These findings suggest a new unknown target for H89 by which it sustains the phosphorylation status of S6K1 and AKT, resulting in mTOR signaling. Lastly, we identified A-kinase anchoring protein 13 (AKAP13) as a crucial scaffold involved in GPCR-Gs signaling to mTORC1. AKAP13 potently enhances Raptor Ser 791 phosphorylation and inhibits mTORC1 activity. Consistently, in cells where Raptor Ser 791 is mutated to Ala, AKAP13 is unable to supress mTORC1 activity. AKAP13 mediates mTORC1-induced cell proliferation, cell size and colony formation. Interestingly, AKAP13 expression inversely correlates with mTORC1 activation and positively correlates with overall lung adenocarcinoma patient survival. Our results place the GPCR-Gas signaling pathway to mTORC1 as a potential target that may be beneficial for human diseases with hyperactivated mTORC1.Item Integrated Regulation of PKA by Fast and Slow Neurotransmission(2016-12-19) Thomas, Rachel Elizabeth; Powell, Craig M.; Bibb, James A.; Huber, Kimberly M.; Sternweis, Paul C.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.