Dataset Information

Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition.

ABSTRACT: Choline kinase beta (CK?) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CK? is important for normal mitochondrial function and muscle development as the lack of the ck? gene in human and mice results in the development of muscular dystrophy. In contrast, CK? is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CK? was found to regulate the enzyme's activity and its subcellular location. This study provides evidence for CK? phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CK? by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CK? phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CK? was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CK? residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CK? phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CK? to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CK?, in concert with CK?, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.

SUBMITTER: Chang CC

PROVIDER: S-EPMC4858151 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition.

Chang Ching Ching CC Few Ling Ling LL Konrad Manfred M See Too Wei Cun WC

PloS one 20160505 5

Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme's activity and its subcellular location. This study provi ...[more]

PMID: 27149373

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Project description:Detection of the abnormal phosphatidylcholine (PtdCho) metabolism in EOC by analysis of magnetic resonance profile (MRS), showed a significant increase of PCho content in EOC cells as compared with non tumoral counterparts associated with activation of choline kinase (ChoK), the enzyme responsible for PCho production following choline phosphorylation in the PtdCho biosynthetic pathway (Kennedy’s pathway). The α-isoform of ChoK has an essential role in growth control and signal transduction and it has been implicated in the carcinogenic process. Aim of the study is the evaluation of the biological relevance of ChoK expression and activity to define its possible role as a non-invasively detectable EOC biomarker and druggable target. We specifically silenced ChoKα expression by transient RNA interference in two different EOC cell lines and evaluated the biological effects related to metabolic profiles, cell proliferation, cell cycle regulation and alteration of global gene expression to possibly identify new pathways implicated in altered choline metabolism. Following ChoKα silencing we observed a 70% reduction of mRNA and protein expression with a similar reduction in PCho accumulation as assessed by HMRS analysis. The ChoKα silencing was accompanied by 20% inhibition of cell growth and similar percentage of cells blocked in the G1-phase of cell cycle. No alteration in cell morphology and modulation of the main survival signaling pathways (PI3K and MAPK-related signaling) were observed. By gene expression analysis we found 476 differentially expressed genes (p< 0.01; FDR 25%) in silenced cells. By transcriptome analysis of CHKA silenced cells as compared to controls, among the most relevant co-repressed genes we found CyclinA, related to regulation of cell cycle progression, and cytokines genes (IL-6 and IL-8) related to inflammation and EOC aggressiveness. Acyl-CoA synthetase medium-chain family member 3 (ACSM3), phosphatidic acid phosphatase type 2 (PPAP2A) and Osteoprotegerin were among the most up-regulated genes. All co-modulated genes have been validated by RTqPCR also in independent biological replicates. Ingenuity System Pathways Analysis (IPA) identified a network of molecules most significantly affected by CHKA silencing whose main functions is related to cell morphology cellular assembly and organization, cellular function and maintenance. Also, the cellular functions predicted to be mostly affected by silencing were cellular movement and cell death that are expected to be respectively decreased and increased in silenced cells. a total of 12 samples were analyzed: Two EOC cell line SKOV3 and INTOV11 were transiently silenced with unrelated or CHKA-specific siRNA pool. Three independent experiment were run for each cell line.

Project description:Mitochondrial protein phosphorylation is a well-recognized metabolic control mechanism, with the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases and phosphatases of bacterial origin. However, despite the growing number of reported mitochondrial phosphoproteins, the identity of the protein kinases mediating these phosphorylation events remains largely unknown. The detection of mitochondrial protein kinases is complicated by the low concentration of kinase relative to that of the target protein, the lack of specific antibodies, and contamination from associated, but nonmatrix, proteins. In this study, we use blue native gel electrophoresis (BN-PAGE) to isolate rat and porcine heart mitochondrial complexes for screening of protein kinase activity. To detect kinase activity, one-dimensional BN-PAGE gels were exposed to [?-(32)P]ATP and then followed by sodium dodecyl sulfate gel electrophoresis. Dozens of mitochondrial proteins were labeled with (32)P in this setting, including all five complexes of oxidative phosphorylation and several citric acid cycle enzymes. The nearly ubiquitous (32)P protein labeling demonstrates protein kinase activity within each mitochondrial protein complex. The validity of this two-dimensional BN-PAGE method was demonstrated by detecting the known PDH kinases and phosphatases within the PDH complex band using Western blots and mass spectrometry. Surprisingly, these same approaches detected only a few additional conventional protein kinases, suggesting a major role for autophosphorylation in mitochondrial proteins. Studies on purified Complex V and creatine kinase confirmed that these proteins undergo autophosphorylation and, to a lesser degree, tenacious (32)P-metabolite association. In-gel Complex IV activity was shown to be inhibited by ATP, and partially reversed by phosphatase activity, consistent with an inhibitory role for protein phosphorylation in this complex. Collectively, this study proposes that many of the mitochondrial complexes contain an autophosphorylation mechanism, which may play a functional role in the regulation of these multiprotein units.

Dataset Information

Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition.

Publications

Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition.

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