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Journal of Neurochemistry Jul 2010Brain aromatic L-amino acid decarboxylase (AAAD) is subject to regulation, and phosphorylation might be involved in the short-term activation of the enzyme. Sites for...
Brain aromatic L-amino acid decarboxylase (AAAD) is subject to regulation, and phosphorylation might be involved in the short-term activation of the enzyme. Sites for serine/threonine phosphorylation are present in the deduced amino acid sequence of AAAD, and cAMP-dependent protein kinase phosphorylates and activates neuronal AAAD in vitro. We now report that cGMP-dependent protein kinase (PKG) is able to phosphorylate and activate neuronal AAAD. In an in vitro kinase assay, immunoprecipitated native and recombinant mouse brain AAAD was rapidly phosphorylated by exogenous PKGIalpha. When added to striatal homogenates, PKGIalpha increased AAAD activity in a temporal fashion similar to phosphorylation. Recombinant AAAD was also activated by the kinase demonstrating a direct effect. Native enzyme activation was moderate and characterized by increased V(max) and K(m) for L-DOPA. A PKG peptide inhibitor prevented AAAD phosphorylation and activation providing specificity, and causally linking the two events. Together, the findings provide evidence for PKGIalpha-dependent phosphorylation and activation of neuronal AAAD in vitro, and introduce AAAD as a putative PKGIalpha substrate. Neuronal AAAD is best known for its role in the biosynthesis of catecholamines, indoleamines and trace amines in the nervous system, and the biological importance of PKGIalpha phosphorylation in these processes remains to be determined.
Topics: Animals; Aromatic-L-Amino-Acid Decarboxylases; Brain; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; Enzyme Activation; In Vitro Techniques; Male; Mice; Neurons; Phosphorylation
PubMed: 20456015
DOI: 10.1111/j.1471-4159.2010.06784.x -
The Journal of Biological Chemistry Jun 2002Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it...
Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it phosphorylates beta-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of beta-catenin by GSK-3 does not require priming in vitro, i.e. it is not dependent on the presence of a phosphoserine, four residues C-terminal to the GSK-3 phosphorylation site. Recently, a means of dissecting GSK-3 activity toward primed and non-primed substrates has been made possible by identification of the R96A mutant of GSK-3beta. This mutant is unable to phosphorylate primed but can still phosphorylate unprimed substrates (Frame, S., Cohen, P., and Biondi R. M. (2001) Mol. Cell 7, 1321-1327). Here we have investigated whether phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin requires priming through prior phosphorylation at Ser(45) in intact cells. We have shown that the Arg(96) mutant does not induce beta-catenin degradation but instead stabilizes beta-catenin, indicating that it is unable to phosphorylate beta-catenin in intact cells. Furthermore, if Ser(45) in beta-catenin is mutated to Ala, beta-catenin is markedly stabilized, and phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin by wild type GSK-3beta is prevented in intact cells. In addition, we have shown that the L128A mutant, which is deficient in phosphorylating Axin in vitro, is still able to phosphorylate beta-catenin in intact cells although it has reduced activity. Mutation of Tyr(216) to Phe markedly reduces the ability of GSK-3beta to phosphorylate and down-regulate beta-catenin. In conclusion, we have found that the Arg(96) mutant has a dominant-negative effect on GSK-3beta-dependent phosphorylation of beta-catenin and that targeting of beta-catenin for degradation requires prior priming through phosphorylation of Ser(45).
Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cytoskeletal Proteins; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Humans; Mutation; Phosphorylation; Structure-Activity Relationship; Trans-Activators; beta Catenin
PubMed: 11967263
DOI: 10.1074/jbc.M201364200 -
Molecular Pharmacology Jun 2017Phosphorylation of G protein-coupled receptors (GPCRs) is a key event for cell signaling and regulation of receptor function. Previously, using tandem mass spectrometry,...
Phosphorylation of G protein-coupled receptors (GPCRs) is a key event for cell signaling and regulation of receptor function. Previously, using tandem mass spectrometry, we identified two phosphorylation sites at the distal C-terminal tail of the chemokine receptor CXCR4, but were unable to determine which specific residues were phosphorylated. Here, we demonstrate that serines (Ser) 346 and/or 347 (Ser-346/7) of CXCR4 are phosphorylated upon stimulation with the agonist CXCL12 as well as a CXCR4 pepducin, ATI-2341. ATI-2341, a G heterotrimer-biased CXCR4 agonist, induced more robust phosphorylation of Ser-346/7 compared with CXCL12. Knockdown of G protein-coupled receptor kinase (GRK) 2, GRK3, or GRK6 reduced CXCL12-induced phosphorylation of Ser-346/7 with GRK3 knockdown having the strongest effect, while inhibition of the conventional protein kinase C (PKC) isoforms, particularly PKC, reduced phosphorylation of Ser-346/7 induced by either CXCL12 or ATI-2341. The loss of GRK3- or PKC-mediated phosphorylation of Ser-346/7 impaired the recruitment of -arrestin to CXCR4. We also found that a pseudo-substrate peptide inhibitor for PKC effectively inhibited CXCR4 phosphorylation and signaling, most likely by functioning as a nonspecific CXCR4 antagonist. Together, these studies demonstrate the role Ser-346/7 plays in arrestin recruitment and initiation of receptor desensitization and provide insight into the dysregulation of CXCR4 observed in patients with various forms of WHIM syndrome.
Topics: G-Protein-Coupled Receptor Kinase 3; HEK293 Cells; Humans; Isoenzymes; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; Receptors, CXCR4; Serine; beta-Arrestins
PubMed: 28331048
DOI: 10.1124/mol.116.106468 -
Molecular & Cellular Proteomics : MCP Feb 2018Reversible protein phosphorylation is one of the major mechanisms in the regulation of protein expression and protein activity, controlling physiological functions of...
Reversible protein phosphorylation is one of the major mechanisms in the regulation of protein expression and protein activity, controlling physiological functions of the important human pathogen Phosphorylations at serine, threonine and tyrosine are known to influence for example protein activity in central metabolic pathways and the more energy-rich phosphorylations at histidine, aspartate or cysteine can be found as part of two component system sensor domains or mediating bacterial virulence. In addition to these well-known phosphorylations, the phosphorylation at arginine residues plays an essential role. Hence, the deletion mutant COL Δ (protein tyrosine phosphatase B) was studied because the protein PtpB is assumed to be an arginine phosphatase. A gel-free approach was applied to analyze the changes in the phosphoproteome of the deletion mutant Δ and the wild type in growing cells, thereby focusing on the occurrence of phosphorylation on arginine residues. In order to enhance the reliability of identified phosphorylation sites at arginine residues, a subset of arginine phosphorylated peptides was chemically synthesized. Combined spectral libraries based on phosphoenriched samples, synthetic arginine phosphorylated peptides and classical proteome samples provide a sophisticated tool for the analysis of arginine phosphorylations. This way, 212 proteins phosphorylated on serine, threonine, tyrosine or arginine residues were identified within the mutant Δ and 102 in wild type samples. Among them, 207 arginine phosphosites were identified exclusively within the mutant Δ, widely distributed along the whole bacterial metabolism. This identification of putative targets of PtpB allows further investigation of the physiological relevance of arginine phosphorylations and provides the basis for reliable quantification of arginine phosphorylations in bacteria.
Topics: Arginine; Bacterial Proteins; Peptide Library; Peptides; Phosphorylation; Staphylococcus aureus
PubMed: 29183913
DOI: 10.1074/mcp.RA117.000378 -
Journal of Visualized Experiments : JoVE May 2018Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number...
Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.
Topics: CDC2 Protein Kinase; Humans; Mass Spectrometry; Phosphorylation
PubMed: 29782014
DOI: 10.3791/57674 -
Genes Aug 2022(1) Background: RNA binding motif 20 (RBM20) regulates mRNA splicing specifically in muscle tissues. Missense mutations in the arginine/serine (RS) domain of RBM20 lead...
(1) Background: RNA binding motif 20 (RBM20) regulates mRNA splicing specifically in muscle tissues. Missense mutations in the arginine/serine (RS) domain of RBM20 lead to abnormal gene splicing and have been linked to severe dilated cardiomyopathy (DCM) in human patients and animal models. Interestingly, many of the reported DCM-linked missense mutations in RBM20 are in a highly conserved RSRSP stretch within the RS domain. Recently, it was found that the two Ser residues within this stretch are constitutively phosphorylated, yet the identity of the kinase(s) responsible for phosphorylating these residues, as well as the function of RSRSP phosphorylation, remains unknown. (2) Methods: The ability of three known SR protein kinases (SRPK1, CLK1, and AKT2) to phosphorylate the RBM20 RSRSP stretch and regulate target gene splicing was evaluated by using both in vitro and in vivo approaches. (3) Results: We found that all three kinases phosphorylated S638 and S640 in the RSRSP stretch and regulated RBM20 target gene splicing. While SRPK1 and CLK1 were both capable of directly phosphorylating the RS domain in RBM20, whether AKT2-mediated control of the RS domain phosphorylation is direct or indirect could not be determined. (4) Conclusions: Our results indicate that SR protein kinases regulate the splicing of a cardiomyopathy-relevant gene by modulating phosphorylation of the RSRSP stretch in RBM20. These findings suggest that SR protein kinases may be potential targets for the treatment of RBM20 cardiomyopathy.
Topics: Animals; Arginine; Cardiomyopathy, Dilated; Humans; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; RNA, Messenger; RNA-Binding Proteins; Serine
PubMed: 36140694
DOI: 10.3390/genes13091526 -
Chang Gung Medical Journal 2008Protein phosphorylation has been known to be a pivotal modification regulating many cellular activities and functions. Except for several conventional techniques, mass... (Review)
Review
Protein phosphorylation has been known to be a pivotal modification regulating many cellular activities and functions. Except for several conventional techniques, mass spectrometry-based strategies are increasingly considered as vital tools that can be utilized to characterize phosphorylated peptides or proteins. In this article, we summarized currently available mass spectrometry-based techniques for the analysis of phosphorylation. Due to the low abundance of phosphopeptides, enrichment steps such as specific antibodies, immobilized metal affinity chromatography, and specific tags are crucial for their use in detection. Since the non-specific binding of the enrichment techniques are constantly of major concerns, phosphatase treatment, neutral loss scan, or precursor ion scan enable the recognition of the phosphopeptide signals. In addition, quantitative methods including isotope labeling and mass tags are also discussed. Phosphoproteome analysis seems to provide elucidation of signaling networks and global decipherment of cell activities, which require powerful analytical methods for complete and routine identification of the phosphorylation event. Despite that numerous approaches have been exploited, comprehensive analysis of protein phosphorylation remains a challenging task. With the progressively more improvements of instruments and methodologies, we can foresee the implementation of a comprehensive approach for the analysis of phosphorylation states of proteins.
Topics: Mass Spectrometry; Phosphorylation; Proteins
PubMed: 18782944
DOI: No ID Found -
The Biochemical Journal May 2020Loss of function mutations in the PTEN-induced kinase 1 (PINK1) kinase are causal for autosomal recessive Parkinson's disease (PD) whilst gain of function mutations in...
Loss of function mutations in the PTEN-induced kinase 1 (PINK1) kinase are causal for autosomal recessive Parkinson's disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies, we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor and GTPase activating protein. In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2 in vitro. Strikingly, we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 of recombinant nucleotide-bound Rab8A in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in transfected HEK293 cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and nuclear magnetic resonance structure of Ser111-phosphorylated Rab1B. The structures reveal that the phosphorylated SF3 motif does not induce any major changes, but may interfere with effector-Switch II interactions through intramolecular H-bond formation and/or charge effects with Arg79. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A indicating a potential cross-talk between PINK1-regulated mitochondrial homeostasis and LRRK2 signalling that requires further investigation in vivo.
Topics: HEK293 Cells; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mitochondria; Parkinsonian Disorders; Phosphorylation; Protein Kinases; Serine; Threonine; rab GTP-Binding Proteins
PubMed: 32227113
DOI: 10.1042/BCJ20190664 -
European Journal of Biochemistry Jul 2002The p25(rum1) is an inhibitor of Cdc2 kinase expressed in fission yeast and plays an important role in cell-cycle control. As its amino-acid sequence suggests that...
Rum1, an inhibitor of cyclin-dependent kinase in fission yeast, is negatively regulated by mitogen-activated protein kinase-mediated phosphorylation at Ser and Thr residues.
The p25(rum1) is an inhibitor of Cdc2 kinase expressed in fission yeast and plays an important role in cell-cycle control. As its amino-acid sequence suggests that p25(rum1) has putative phosphorylation sites for mitogen-activated protein kinase (MAPK), we investigated the ability of MAPK to phosphorylate p25(rum1). Direct in vitro kinase assay using GST-fusion proteins of wild-type as well as various mutants of p25(rum1) demonstrated that MAPK phosphorylates the N-terminal portion of p25(rum1) and residues Thr13 and Ser19 are major phosphorylation sites for MAPK. In addition, phosphorylation of p25(rum1) by MAPK revealed markedly reduced Cdc2 kinase inhibitor ability of the protein. Together with the fact that replacement of both Thr13 and Ser19 with Glu, which mimics the phosphorylated state of these residues, also significantly reduces the activity of p25(rum1) as a Cdc2 inhibitor, it was suggested that the phosphorylation of Thr13 and Ser19 negatively regulates the function of p25(rum1). Further evidence indicates that phosphorylation of Thr13 and Ser19 may retain a negative effect on the function of p25(rum1) even in vivo. Therefore, MAPK may regulate the function of p25(rum1) via phosphorylation of its Thr and Ser residues and thus participate in cell cycle control in fission yeast.
Topics: Amino Acid Substitution; Animals; CDC2 Protein Kinase; Cell Cycle; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Processing, Post-Translational; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Starfish
PubMed: 12135491
DOI: 10.1046/j.1432-1033.2002.03033.x -
PloS One 2012O-linked N-acetylglucosamine glycosylations (O-GlcNAc) and O-linked phosphorylations (O-phosphate), as two important types of post-translational modifications, often...
O-linked N-acetylglucosamine glycosylations (O-GlcNAc) and O-linked phosphorylations (O-phosphate), as two important types of post-translational modifications, often occur on the same protein and bear a reciprocal relationship. In addition to the well documented phosphorylations that control Akt activity, Akt also undergoes O-GlcNAcylation, but the interplay between these two modifications and the biological significance remain unclear, largely due to the technique challenges. Here, we applied a two-step analytic approach composed of the O-GlcNAc immunoenrichment and subsequent O-phosphate immunodetection. Such an easy method enabled us to visualize endogenous glycosylated and phosphorylated Akt subpopulations in parallel and observed the inhibitory effect of Akt O-GlcNAcylations on its phosphorylation. Further studies utilizing mass spectrometry and mutagenesis approaches showed that O-GlcNAcylations at Thr 305 and Thr 312 inhibited Akt phosphorylation at Thr 308 via disrupting the interaction between Akt and PDK1. The impaired Akt activation in turn resulted in the compromised biological functions of Akt, as evidenced by suppressed cell proliferation and migration capabilities. Together, this study revealed an extensive crosstalk between O-GlcNAcylations and phosphorylations of Akt and demonstrated O-GlcNAcylation as a new regulatory modification for Akt signaling.
Topics: Cell Line; Cells, Cultured; Glycosylation; Humans; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Signal Transduction
PubMed: 22629392
DOI: 10.1371/journal.pone.0037427