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The Plant Cell Jul 2021Calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is an important positive regulator of abscisic acid (ABA) and abiotic stress signaling in plants and is...
Calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is an important positive regulator of abscisic acid (ABA) and abiotic stress signaling in plants and is believed to act upstream of mitogen-activated protein kinase (MAPK) in ABA signaling. However, it is unclear how CCaMK activates MAPK in ABA signaling. Here, we show that OsDMI3, a rice (Oryza sativa) CCaMK, directly interacts with and phosphorylates OsMKK1, a MAPK kinase (MKK) in rice, in vitro and in vivo. OsDMI3 was found to directly phosphorylate Thr-25 in the N-terminus of OsMKK1, and this Thr-25 phosphorylation is OsDMI3-specific in ABA signaling. The activation of OsMKK1 and its downstream kinase OsMPK1 is dependent on Thr-25 phosphorylation of OsMKK1 in ABA signaling. Moreover, ABA treatment induces phosphorylation in the activation loop of OsMKK1, and the two phosphorylations, in the N-terminus and in the activation loop, are independent. Further analyses revealed that OsDMI3-mediated phosphorylation of OsMKK1 positively regulates ABA responses in seed germination, root growth, and tolerance to both water stress and oxidative stress. Our results indicate that OsMKK1 is a direct target of OsDMI3, and OsDMI3-mediated phosphorylation of OsMKK1 plays an important role in activating the MAPK cascade and ABA signaling.
Topics: Abscisic Acid; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Oryza; Oxidative Stress; Phosphorylation; Phosphothreonine; Plant Proteins; Protein Binding; Signal Transduction; Stress, Physiological; Water
PubMed: 33630095
DOI: 10.1093/plcell/koab071 -
Scientific Reports Oct 2020It is widely accepted that FAM20C functions as a Golgi casein kinase and has large numbers of kinase substrates within the secretory pathway. It has been previously...
It is widely accepted that FAM20C functions as a Golgi casein kinase and has large numbers of kinase substrates within the secretory pathway. It has been previously reported that FAM20C is required for maintenance of healthy periodontal tissues. However, there has been no report that any extracellular matrix molecules expressed in periodontal tissues are indeed substrates of FAM20C. In this study, we sought to identify the binding partner(s) of FAM20C. FAM20C wild-type (WT) and its kinase inactive form D478A proteins were generated. These proteins were electrophoresed and the Coomassie Brilliant Blue (CBB)-positive bands were analyzed to identify FAM20C-binding protein(s) by Mass Spectrometry (MS) analysis. Periostin was found by the analysis and the binding between FAM20C and Periostin was investigated in cell cultures and in vitro. We further determined the binding region(s) within Periostin responsible for FAM20C-binding. Immunolocalization of FAM20C and Periostin was examined using mouse periodontium tissues by immunohistochemical analysis. In vitro kinase assay was performed using Periostin and FAM20C proteins to see whether FAM20C phosphorylates Periostin in vitro. We identified Periostin as one of FAM20C-binding proteins by MS analysis. Periostin interacted with FAM20C in a kinase-activity independent manner and the binding was direct in vitro. We further identified the binding domain of FAM20C in Periostin, which was mapped within Fasciclin (Fas) I domain 1-4 of Periostin. Immunolocalization of FAM20C was observed in periodontal ligament (PDL) extracellular matrix where that of Periostin was also immunostained in murine periodontal tissues. FAM20C WT, but not D478A, phosphorylated Periostin in vitro. Consistent with the overlapped expression pattern of FAM20C and Periostin, our data demonstrate for the first time that Periostin is a direct FAM20C-binding partner and that FAM20C phosphorylates Periostin in vitro.
Topics: Amino Acid Sequence; Animals; Casein Kinase I; Cell Adhesion Molecules; Cell Line; Extracellular Matrix Proteins; HEK293 Cells; Humans; Mice; Mice, Inbred C57BL; Periodontal Ligament; Phosphorylation; Protein Kinases; Secretory Pathway
PubMed: 33051588
DOI: 10.1038/s41598-020-74400-6 -
Eukaryotic Cell May 2015Candida albicans is an important human fungal pathogen in both immunocompetent and immunocompromised individuals. C. albicans regulation has been studied in many...
Candida albicans is an important human fungal pathogen in both immunocompetent and immunocompromised individuals. C. albicans regulation has been studied in many contexts, including morphological transitions, mating competence, biofilm formation, stress resistance, and cell wall synthesis. Analysis of kinase- and phosphatase-deficient mutants has made it clear that protein phosphorylation plays an important role in the regulation of these pathways. In this study, to further our understanding of phosphorylation in C. albicans regulation, we performed a deep analysis of the phosphoproteome in C. albicans. We identified 19,590 unique peptides that corresponded to 15,906 unique phosphosites on 2,896 proteins. The ratios of serine, threonine, and tyrosine phosphosites were 80.01%, 18.11%, and 1.81%, respectively. The majority of proteins (2,111) contained at least two detected phosphorylation sites. Consistent with findings in other fungi, cytoskeletal proteins were among the most highly phosphorylated proteins, and there were differences in Gene Ontology (GO) terms for proteins with serine and threonine versus tyrosine phosphorylation sites. This large-scale analysis identified phosphosites in protein components of Mediator, an important transcriptional coregulatory protein complex. A targeted analysis of the phosphosites in Mediator complex proteins confirmed the large-scale studies, and further in vitro assays identified a subset of these phosphorylations that were catalyzed by Cdk8 (Ssn3), a kinase within the Mediator complex. These data represent the deepest single analysis of a fungal phosphoproteome and lay the groundwork for future analyses of the C. albicans phosphoproteome and specific phosphoproteins.
Topics: Candida albicans; Fungal Proteins; Phosphoproteins; Phosphorylation; Proteome; Proteomics; Serine; Threonine
PubMed: 25750214
DOI: 10.1128/EC.00011-15 -
The Biochemical Journal Nov 1994Challenge of neutrophils with concanavalin A (ConA), formyl-methionyl-leucyl-phenylalanine (FMLP), and phorbol 12-myristate 13-acetate (PMA) induced the tyrosine...
Challenge of neutrophils with concanavalin A (ConA), formyl-methionyl-leucyl-phenylalanine (FMLP), and phorbol 12-myristate 13-acetate (PMA) induced the tyrosine phosphorylation of several proteins. Among these proteins we have identified two mitogen-activated protein kinase (MAPK) isoforms of 43 kDa (p43 MAPK) and 45 kDa (p45 MAPK) molecular mass. Moreover here we show that: (1) FMLP induced the tyrosine phosphorylation of the p43 MAPK, and ConA that of p45 MAPK, while PMA induced the tyrosine phosphorylation of both p43 and p45 MAPK; all these agonists induced the tyrosine phosphorylation of a 75 kDa protein (p75). (2) With FMLP or ConA as agonists, tyrosine phosphorylations of MAPK and p75 can be involved in the process of NADPH oxidase activation. On the contrary, PMA can activate the respiratory burst independently of these phosphorylations. (3) In Ca(2+)-depleted neutrophils, where phospholipid hydrolysis did not take place, ConA or FMLP did not activate the respiratory burst, but while ConA induced the tyrosine phosphorylation of p45 MAPK and p75, FMLP was not able to phosphorylate p43 MAPK and p75. (4) As previously observed in our laboratory, a double stimulation of Ca(2+)-depleted neutrophils with ConA plus FMLP induced a respiratory burst in the absence of activation of second messengers derived from phospholipase C, D and A2 activity. This respiratory burst was accompanied by tyrosine phosphorylation of both p43 and p45 MAPKs. These results indicate that when FMLP is the agonist, both the tyrosine phosphorylation of p43 MAPK and p75, and the activation of NADPH oxidase, are coupled to Ca(2+)-dependent mechanisms. On the contrary, ConA can induce the tyrosine phosphorylation of p45 MAPK and p75 independently of calcium, but an unknown Ca(2+)-dependent mechanism is necessary for the activation of NADPH oxidase by this agonist. This mechanism could be substituted by the induction of tyrosine phosphorylation of both p43 MAPK and p45 MAPK when Ca(2+)-depleted neutrophils are stimulated with ConA plus FMLP.
Topics: Calcium-Calmodulin-Dependent Protein Kinases; Concanavalin A; Enzyme Activation; Genistein; Humans; Isoflavones; N-Formylmethionine Leucyl-Phenylalanine; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophils; Oxygen Consumption; Phosphorylation; Tyrosine
PubMed: 7998940
DOI: 10.1042/bj3040243 -
European Journal of Biochemistry Oct 1993Mitogen-activated protein-kinase (MAP) kinase-activated protein kinases 1 and 2 (MAPKAP kinase-1, MAPKAP kinase-2), were found to phosphorylate bacterially expressed...
Mitogen-activated protein-kinase (MAP) kinase-activated protein kinases 1 and 2 (MAPKAP kinase-1, MAPKAP kinase-2), were found to phosphorylate bacterially expressed human tyrosine hydroxylase in vitro at comparable rates to other proteins thought to be physiological substrates of these protein kinases. The phosphorylation of all four alternatively spliced forms of human tyrosine hydroxylase by MAPKAP kinases-1 and -2 reached plateau values at 1 mol/mol subunit and 2 mol/mol subunit, respectively; the sites of phosphorylation were identified as Ser40 (MAPKAP kinase-1) and Ser19 and Ser40 (MAPKAP kinase-2). In contrast to calmodulin-dependent protein kinase-II, which phosphorylates Ser19 faster than Ser40, MAPKAP kinase-2 phosphorylated Ser40 about twice as fast as Ser19. The maximal activation of tyrosine hydroxylase by MAPKAP kinase-1 or-2 was about 3-fold, and activation by MAPKAP kinases-1 and -2 or calmodulin-dependent protein kinase-II correlated with the extent of phosphorylation of Ser40. The four alternatively spliced forms of human tyrosine hydroxylase were phosphorylated at Ser31 by MAP kinase, but at markedly different rates (3 = 4 > 1 >> 2). Forms 3 and 4 were phosphorylated rapidly and stoichiometrically by MAP kinase doubling the activity, while phosphorylation of form 1 by MAP kinase to 0.4 mol/mol subunit increased activity by 40%. The effect on activity of phosphorylating both Ser31 and Ser40 was not additive. The possible roles of MAPKAP kinase-1, MAPKAP kinase-2 and MAP kinase in the regulation of tyrosine hydroxylase in vivo are discussed.
Topics: Alternative Splicing; Amino Acid Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinase 1; Molecular Sequence Data; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Ribosomal Protein S6 Kinases, 90-kDa; Serine; Tyrosine 3-Monooxygenase
PubMed: 7901013
DOI: 10.1111/j.1432-1033.1993.tb18297.x -
The Journal of Biological Chemistry Feb 1992We have partially purified a protein kinase that phosphorylates muscarinic receptors (mAChR) in the presence of agonists and have shown that the phosphorylation is...
We have partially purified a protein kinase that phosphorylates muscarinic receptors (mAChR) in the presence of agonists and have shown that the phosphorylation is stimulated by the beta gamma subunits of the GTP binding protein Go (Haga, K., and Haga, T. (1990) FEBS Lett. 268, 43-47). We report here that rhodopsin is also phosphorylated in a light-dependent manner by the same kinase preparation and that beta gamma subunits derived from Gs, Gi, and Go stimulate the phosphorylation of both rhodopsin and mAChRs. The rhodopsin- and mAChR-phosphorylating activities were eluted in the same fractions using a purification procedure that is essentially the same as that used for the purification of beta-adrenergic receptor kinase (Benovic, J.L., Strasser, R.H., Caron, M.G., and Lefkowitz, R.J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2797-2801) and were inhibited by low concentrations of heparin, an inhibitor of beta-adrenergic receptor kinase, (IC50 = 15 nM), suggesting that both mAChR and rhodopsin are phosphorylated by the same or very similar kinase(s) belonging to the beta-adrenergic receptor kinase family. G protein beta gamma subunits increased the Vmax of the phosphorylation of rhodopsin 12-fold. Kinetic data were consistent with the assumptions that the protein kinase (mAChR kinase) binds rhodopsin and beta gamma subunits in a random order and that the reaction rate is proportional to concentration of the ternary complex. By contrast, the light-dependent phosphorylation of rhodopsin by the rhodopsin kinase was not stimulated by the beta gamma subunits. These results indicate that beta gamma subunits may interact with and activate the mAChR kinase but not rhodopsin kinase and suggest that the beta gamma subunit of G proteins may take part in the desensitization of G protein-linked receptors.
Topics: Animals; Brain; Carbachol; Eye Proteins; G-Protein-Coupled Receptor Kinase 1; GTP-Binding Proteins; Light; Phosphorylation; Protein Kinases; Receptors, Muscarinic; Retina; Rhodopsin; Rod Cell Outer Segment; Swine
PubMed: 1733928
DOI: No ID Found -
Plant Signaling & Behavior Aug 2011Phytochrome (phy) family of photoreceptors is a broad sensor of environmental light signals that promote photomorphogenic development of plants. Phytochrome Interacting... (Review)
Review
Phytochrome (phy) family of photoreceptors is a broad sensor of environmental light signals that promote photomorphogenic development of plants. Phytochrome Interacting Factors (PIFs), bHLH family of transcription factors, repress photomorphogenesis in the dark in an overlapping manner. Phytochromes interact with PIFs in response to light and induce rapid phosphorylation, poly-ubiquitylation and degradation of PIFs through the ubiquitin/26S proteasome pathway to promote photomorphogenesis. Structure-function analyses with PIF family members revealed that multiple domains are necessary for the light-induced phosphorylation and degradation of PIFs. CK2, a ubiquitious Ser/Thr kinase, phosphorylates PIF1 independent of light. In addition, PIF1 mutants deficient in CK2 phosphorylation sites are still robustly phosphorylated but not efficiently degraded in response to light. These data suggest that multiple kinases phosphorylate PIF1 to promote light-induced degradation and photomorphogenesis.
Topics: Basic Helix-Loop-Helix Transcription Factors; Casein Kinase II; Gene Expression Regulation, Plant; Light; Phosphorylation; Phytochrome; Plant Proteins; Protein Serine-Threonine Kinases; Proteolysis
PubMed: 21758014
DOI: 10.4161/psb.6.8.16049 -
The Journal of Biological Chemistry Aug 2000The beta-amyloid precursor protein (betaAPP) is one of the rare proteins known to be phosphorylated within its ectodomain. We have shown previously that betaAPP can be...
The beta-amyloid precursor protein (betaAPP) is one of the rare proteins known to be phosphorylated within its ectodomain. We have shown previously that betaAPP can be phosphorylated within secretory vesicles and at the cell surface (Walter, J., Capell, A., Hung, A. Y. , Langen, H., Schnölzer, M., Thinakaran, G., Sisodia, S. S., Selkoe, D. J., and Haass, C. (1997) J. Biol. Chem. 272, 1896-1903). We have now specifically characterized the phosphorylation of cell surface-located betaAPP and identified two ectoprotein kinases that phosphorylate betaAPP at the outer face of the plasma membrane. By using selective protein kinase inhibitors and by investigating the usage of ATP and GTP as cosubstrates, we demonstrate that membrane-bound betaAPP as well as secreted forms of betaAPP can be phosphorylated by casein kinase (CK) 1- and CK2-like ectoprotein kinases. The ectodomain of betaAPP was also phosphorylated by purified CK1 and CK2 in vitro, but not by protein kinases A and C. Phosphorylation of betaAPP by ectoprotein kinases and by purified CK1 and CK2 occurred within an acidic domain in the N-terminal half of the protein. Heparin strongly inhibited the phosphorylation of cell-surface betaAPP by ecto-CK1 and ecto-CK2, indicating a regulatory role of this extracellular matrix component in betaAPP phosphorylation.
Topics: Amyloid beta-Protein Precursor; Casein Kinase II; Casein Kinases; Cell Membrane; Heparin; Humans; Models, Biological; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Protein Structure, Tertiary
PubMed: 10806211
DOI: 10.1074/jbc.M002850200 -
The Journal of Biological Chemistry Oct 2018The Nem1-Spo7 protein phosphatase plays a role in lipid synthesis by controlling the membrane localization of Pah1, the diacylglycerol-producing phosphatidate (PA)...
The Nem1-Spo7 protein phosphatase plays a role in lipid synthesis by controlling the membrane localization of Pah1, the diacylglycerol-producing phosphatidate (PA) phosphatase that is crucial for the synthesis of triacylglycerol in the yeast By dephosphorylating Pah1, Nem1-Spo7 facilitates its translocation to the nuclear/endoplasmic reticulum membrane for catalytic activity. Like its substrate Pah1, Nem1-Spo7 is phosphorylated in the cell, but the specific protein kinases involved remain to be identified. In this study, we demonstrate that the Nem1-Spo7 complex is phosphorylated by protein kinase A (PKA), which is associated with active cell growth, metabolic activity, and membrane phospholipid synthesis. phosphorylation of purified Nem1-Spo7 and of their synthetic peptides revealed that both subunits of the phosphatase complex are PKA substrates. Using phosphoamino acid and phosphopeptide-mapping analyses coupled with site-directed mutagenesis, we identified Ser-140 and Ser-210 of Nem1 and Ser-28 of Spo7 as PKA-targeted phosphorylation sites. Immunodetection of the phosphatase complex from the cell with anti-PKA substrate antibody confirmed the phosphorylations of Nem1 and Spo7 on the serine residues. Lipid-labeling analysis of cells bearing phosphorylation-deficient alleles of and indicated that the PKA phosphorylation of the phosphatase complex stimulates phospholipid synthesis and attenuates the synthesis of triacylglycerol. This work advances the understanding of how PKA-mediated posttranslational modifications of Nem1 and Spo7 regulate lipid synthesis in yeast.
Topics: Alkaline Phosphatase; Cyclic AMP-Dependent Protein Kinases; Membrane Proteins; Mutagenesis, Site-Directed; Nuclear Proteins; Phosphatidate Phosphatase; Phosphorylation; Protein Processing, Post-Translational; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Serine
PubMed: 30201607
DOI: 10.1074/jbc.RA118.005348 -
European Journal of Biochemistry Aug 1997DNA-dependent protein kinase (DNA-PK) has been known to catalyze phosphorylation of a number of regulatory factors involved in DNA replication and transcription such as...
Phosphorylation of human general transcription factors TATA-binding protein and transcription factor IIB by DNA-dependent protein kinase--synergistic stimulation of RNA polymerase II basal transcription in vitro.
DNA-dependent protein kinase (DNA-PK) has been known to catalyze phosphorylation of a number of regulatory factors involved in DNA replication and transcription such as simian virus 40 T antigen, p53, c-Myc, Sp1, and RNA polymerase II (Pol II). We examined the possibility that DNA-PK phosphorylates the general transcription factors TATA-binding protein (TBP) and transcription factor (TF) IIB, which play key roles in the formation of transcription initiation complex with Pol II. By using a highly purified preparation of DNA-PK from Raji cells, both TBP and TFIIB were shown to be phosphorylated in vitro by DNA-PK. We then investigated the effect of the phosphorylation of these factors on Pol II basal transcription. Stepwise analysis of preinitiation complex formation by electrophoretic mobility shift assay revealed that the phosphorylation of TBP and TFIIB by DNA-PK did not affect the formation of promoter (P)-TBP and P-TBP-TFIIB complexes but synergistically stimulated the formation of P-TBP-TFIIB-TFIIF-Pol II complex. Similarly, combination of the phosphorylated TBP and TFIIB synergistically stimulated Pol II basal transcription from adenovirus major late promoter. These observations suggest that DNA-PK could positively regulate the Pol II basal transcription by phosphorylating TBP and TFIIB.
Topics: DNA-Activated Protein Kinase; DNA-Binding Proteins; Humans; Nuclear Proteins; Phosphorylation; Protein Serine-Threonine Kinases; RNA Polymerase II; TATA Box; TATA-Box Binding Protein; Transcription Factor TFIIB; Transcription Factors; Transcription, Genetic
PubMed: 9288944
DOI: 10.1111/j.1432-1033.1997.01166.x