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Phosphorylation of the compartmentalized PKA substrate TAF15 regulates RNA-Protein interactions

ABSTRACT: Spatiotemporal-controlled second messengers alter molecular interactions of central signaling nodes for ensuring physiological signal transmission. One prototypical second messenger molecule which modulates kinase signal transmission is the cyclic-adenosine monophosphate (cAMP). The main proteinogenic cellular effectors of cAMP are compartmentalized protein kinase A (PKA) complexes. Their cell-type specific compositions precisely coordinate substrate phosphorylation and proper signal propagation which is indispensable for numerous cell-type specific functions. Here we present evidence that TAF15, which is implicated in the etiology of amyotrophic lateral sclerosis, represents a novel nuclear PKA substrate. In cross-linking and immunoprecipitation experiments (iCLIP) we showed that TAF15 phosphorylation alters the binding to target transcripts related to mRNA maturation, splicing and protein-binding related functions. TAF15 appears to be one of multiple PKA substrates that undergo RNA-binding dynamics upon phosphorylation. We observed that the activation of the cAMP-PKA signaling axis caused a change in the composition of a collection of RNA species that interact with TAF15. This observation appears to be a broader principle in the regulation of molecular interactions, as we identified a significant enrichment of RNA-binding proteins within endogenous PKA complexes. We assume that phosphorylation of RNA-binding domains adds another layer of regulation to binary protein-RNAs interactions with consequences to RNA features including binding specificities, localization, abundance and composition.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Malignant Cell, Colon

DISEASE(S): Colon Cancer

SUBMITTER: Omar Torres

LAB HEAD: Eduard Stefan

PROVIDER: PXD048869 | Pride | 2024-04-04

REPOSITORIES: Pride

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Project description:Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3’5’cyclic AMP (cAMP)-dependent Protein Kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in Acrodysostosis. How mutations on PDEs and RIα interfere with regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit. PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα:PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: Translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis Step 2) Processivity: Binding of free cAMP from the cytosol at both CNBs of RIα and Step 3) Product (5’AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that trigger subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, (PDE8 T690P) and RIα (T207A) that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and Fluorescence Polarization (FP) reveal that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5’AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5’AMP release from the PDE, T207A greatly slowed release of substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα:PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.

Project description:The second messenger cAMP acts via protein kinase A (PKA) to induce apoptosis by mechanisms that are poorly understood. Here, we assessed a role for mitochondria and analyzed gene expression in cAMP/PKA-promoted apoptosis by comparing wild-type (WT) S49 lymphoma cells and the S49 variant, D- (cAMP-deathless), which lacks cAMP-promoted apoptosis but has wild-type levels of PKA activity and cAMP-promoted G1 growth arrest. Treatment of WT, but not D-, S49 cells with 8-CPT-cAMP for 24 h induced loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and Smac and increase in caspase-3 activity. Gene expression analysis (using Affymetrix 430 2.0 Arrays) revealed that WT and D- cells incubated with 8-CPT-cAMP have similar, but non-identical, extents of cAMP-regulated gene expression at 2h (~800 transcripts) and 6h (~1000 transcripts) (|Fold|>2, P<0.06); by contrast, at 24h ~2500 and ~1100 transcripts were changed in WT and D- cells, respectively. Using an approach that combined regression analysis, clustering and functional annotation to identify transcripts that showed differential expression between WT and D- cells, we found differences in cAMP-mediated regulation of mRNAs involved in transcriptional repression, apoptosis, the cell cycle, RNA splicing, Golgi and lysosomes. The 2 cell lines differed in CREB phosphorylation and expression of the transcriptional inhibitor Icer and in cAMP-regulated expression of genes in the Inhibitor of apoptosis (IAP) and Bcl families. The findings indicate that cAMP/PKA-promoted apoptosis of lymphoid cells occurs via mitochondrial-mediated events and imply that such apoptosis involves gene networks in multiple biochemical pathways. Experiment Overall Design: S49 D- cells were treated with 8-CPT-cAMP over the course of 24 hours. Cells were prepared for hybridization to microarrays at times 0-untreated, 2h, 6h, and 24h after treatment with 8-CPT-cAMP. Experimental replicates were as follows n=4 for time 0 and n=3 for 2, 6, and 24h post treatment.

Project description:Musante2017 - Switching behaviour of PP2A inhibition by ARPP-16 - mutual inhibitions and PKA inhibits MAST3 This model is described in the article: Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition. Musante V, Li L, Kanyo J, Lam TT, Colangelo CM, Cheng SK, Brody AH, Greengard P, Le Novère N, Nairn AC. Elife 2017 Jun; 6: Abstract: ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition. This model is hosted on BioModels Database and identified by: BIOMD0000000644. To cite BioModels Database, please use: Chelliah V et al. BioModels: ten-year anniversary. Nucl. Acids Res. 2015, 43(Database issue):D542-8. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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Phosphorylation of the compartmentalized PKA substrate TAF15 regulates RNA-Protein interactions

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