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JPST000234-1 Identifications of protein kinase A substrates with quantitative phosphoproteomics and primary-sequence based scoring.(H-89) [Reanalysis: JPST000234]

ABSTRACT: Protein kinase A (PKA) is a serine/threonine kinase who plays essential roles in the complex signalling pathways. To better understand the functionalities, it is necessary to elucidate direct interplays between PKA and their substrates in living system. In order to screen them in a high-throughput manner, we firstly quantified the change of phosphoproteome in the cells of which PKA activity was perturbed by drag stimulations. LC-MS/MS analysis identified 2,755 and 3,191 phosphopeptides from either experiments with activator or inhibitor of PKA. To exclude indirect targets of PKA, we built the simple model to characterize the kinase sequential specificity to its substrate recognitions based on the known kinase-substrate relationships. Finally, by applying the metric to the sites of which phosphorylation were changed due to the drug stimulations, we identified 29 reliable candidates of PKA targeting residues in living cells including 8 known substrates. Moreover, 18 sites out of them were confirmed to be site-specifically phosphorylated in vitro. Altogether this study proposed confident list of PKA substrate and expanded the knowledge of PKA signalling network. [Original project description]

ORGANISM(S): Hela Cell

SUBMITTER: Yasushi Ishihama

PROVIDER: RPXD034682 | JPOST Repository | Sat Mar 25 00:00:00 GMT 2023

REPOSITORIES: jPOST

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Project description:The pattern of gene transcription in Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA), triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. To investigate the requirement of PKA for glucose control of gene expression, we have analyzed global transcription in strains devoid of PKA activity. In S. cerevisiae three genes, TPK1, TPK2, TPK3, encode catalytic subunits of PKA and the triple mutant tpk1 tpk2 tpk3 is unviable. We have worked, therefore, with two strains, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4, that bear suppressor mutations,. We have identified different classes of genes that can be induced, or repressed, by glucose in the absence of PKA. Among these genes, some are also controlled by a redundant signalling pathway involving PKA activation, while others do not respond to an increase in cAMP concentration. On the other hand, among genes which do not respond to glucose in the absence of PKA, some show a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways. The goal of the present study was to investigate the occurrence of PKA-independent glucose signalling in S. cerevisiae. To this end, we have used global transcription analysis to study the effects of glucose on yeast strains completely devoid of PKA activity. In S. cerevisiae three genes TPK1, TPK2,and TPK3 encode catalytic subunits of PKA. While strains expressing only one of these genes grow normally, a triple null mutant (tpk1 tpk2 tpk3) is not viable (Toda et al 1987). Identification of different mutations able to suppress the growth defect of the triple mutant (Garrett and Broach 1989, Reinders et al 1998, Smith et al 1998) has allowed to determine what is the crucial function of PKA. As shown in Fig.1, PKA is needed to counteract the negative effect of the protein kinase Yak1 on yeast growth (Hartley et al 1994, Moriya et al 2001). In the presence of PKA the protein kinase Rim15 (Reinders et al 1998) and the transcription factors Msn2 and Msn4 (Görner et al 1998) can be phosphorylated and exported to the cytoplasm, transcription of the YAK1 gene, which is activated by Msn2/Msn4 (Smith et al 1998), is reduced, Yak1 levels remain low and growth is not hindered. In the absence of PKA, Rim15 remains in the nucleus where it can activate Msn2/Msn4 (Cameroni et al 2004) that turn on YAK1 transcription, thus blocking growth. This explains why strains lacking Rim15, Msn2/Msn4 or Yak1 no longer require PKA for growth. In this work we have used two isogenic strains lacking PKA and carrying the suppressor mutations msn2 msn4 or yak1. Two different suppressor mutants were used with the aim to enable a dissection of effects of the lack of PKA and effects of the suppressor mutations themselves.

Project description:The oocyteâs capacity to complete maturation, to succeed fertilization and to reach the blastocyst stage is what defines the oocyteâs competence. The oocyte acquires this competence working closely with somatic cells of the follicle. Cumulus and granulosa cells provided support for the oocyteâs development and conversely the oocyte influence follicular cell growth and differentiation. Existing studies support the idea that follicular-stimulated hormone and luteinizing hormone play an essential role in oocyte competence acquisition through protein kinase A (PKA) and protein kinase C (PKC) signalling in granulosa cells. Therefore, human-like granulosa cells (KGN) were treated with forskolin 10 Î¼M and phorbol 12-myristate 13-acetate 0.1 Î¼M for 24 hours in order to process a transcriptomic analysis of differentially express genes between treatment. Over 2000 genes were founded to be differentially express at cut-off fold change of 1.5 and a p-value of 0.05. Five major upstreams, EGF, TGFB1, VEGF, FGF2 and HGF were founded to play an important role in competence acquisition thought PKA and PKC signalling. Differentially expressed targeted genes of both signalling pathways were classified in seven major ovarian functions such as PTGS2, IL8 and IL6 in inflammation, STAR, CYP11A1, CYP19A1 in steroidogenesis, VEGFC, VEGFA, CXCR4 in angiogenesis, AREG, EGFR, SPRY2 in differentiation, BAX, BCL2L12, CASP1 in apoptosis, CCND1, CCNB1, CCNB2 in division and MMP1, MMP9, TIMP1 in ovulation. Taken together, the results of this study suggest that PKA and PKC signalling potentiate their effects in some functions such as inflammation and apoptosis while some others are more specific to one or the other protein kinase like differentiation, ovulation and angiogenesis that are thought to be more PKC-dependent in human granulosa cells. 8 samples were analysed, 4 controls compared to 4 Phorbol 12-myristate 13-acetate treatments (total of 4 replicates).

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	161206TN_2-1.maxq.txt	Txt

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JPST000234-1 Identifications of protein kinase A substrates with quantitative phosphoproteomics and primary-sequence based scoring.(H-89) [Reanalysis: JPST000234]

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