Correction: Phosphorylation of CK1δ: identification of Ser370 as the major phosphorylation site targeted by PKA in vitro and in vivo

ABSTRACT Human RNPS1 was originally characterized as a pre-mRNA splicing activator in vitro and was shown to regulate alternative splicing in vivo. RNPS1 was also identified as a protein component of the splicing-dependent mRNP complex, or exon-exon junction complex (EJC), and a role for RNPS1 in postsplicing processes has been proposed. Here we demonstrate that RNPS1 incorporates into active spliceosomes, enhances the formation of the ATP-dependent A complex, and promotes the generation of both intermediate and final spliced products. RNPS1 is phosphorylated in vivo and interacts with the CK2 (casein kinase II) protein kinase. Serine 53 (Ser-53) of RNPS1 was identified as the major phosphorylation site for CK2 in vitro, and the same site is also phosphorylated in vivo. The phosphorylation status of Ser-53 significantly affects splicing activation in vitro, but it does not perturb the nuclear localization of RNPS1. In vivo experiments indicated that the phosphorylation of RNPS1 at Ser-53 influences the efficiencies of both splicing and translation. We propose that RNPS1 is a splicing regulator whose activator function is controlled in part by CK2 phosphorylation.

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The major phosphorylation site of nucleophosmin (B23) is phosphorylated by a nuclear kinase II

Biochemical Journal ◽

10.1042/bj2700549 ◽

1990 ◽

Vol 270 (2) ◽

pp. 549-552 ◽

Cited By ~ 32

Author(s):

P K Chan ◽

Q R Liu ◽

E Durban

Keyword(s):

Hela Cells ◽

Peptide Mapping ◽

Phosphorylation Site ◽

Type Ii ◽

Mapping Analysis ◽

Major Phosphorylation Site

Nucleophosmin (B23) was phosphorylated in vitro with [gamma-32P]ATP and a nuclear kinase (type II) purified from HeLa cells. The phosphorylation was inhibited by heparin and by 2,3-diphosphoglycerate. Peptide mapping analysis indicated that the phosphorylation site in vitro was identical to that in vivo. Purified nucleoli have a similar kinase that phosphorylated nucleophosmin at the same site. These results indicated that nucleophosmin is phosphorylated in vivo by a nucleolar kinase (type II).

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Phosphorylation of Serine 303 Is a Prerequisite for the Stress-Inducible SUMO Modification of Heat Shock Factor 1

Molecular and Cellular Biology ◽

10.1128/mcb.23.8.2953-2968.2003 ◽

2003 ◽

Vol 23 (8) ◽

pp. 2953-2968 ◽

Cited By ~ 205

Author(s):

Ville Hietakangas ◽

Johanna K. Ahlskog ◽

Annika M. Jakobsson ◽

Maria Hellesuo ◽

Niko M. Sahlberg ◽

...

Keyword(s):

Heat Shock ◽

Phosphorylation Site ◽

Stress Granules ◽

Heat Shock Factor ◽

Transcriptional Level ◽

Heat Shock Factor 1 ◽

Protection Mechanism ◽

Sumo Modification

ABSTRACT The heat shock response, which is accompanied by a rapid and robust upregulation of heat shock proteins (Hsps), is a highly conserved protection mechanism against protein-damaging stress. Hsp induction is mainly regulated at transcriptional level by stress-inducible heat shock factor 1 (HSF1). Upon activation, HSF1 trimerizes, binds to DNA, concentrates in the nuclear stress granules, and undergoes a marked multisite phosphorylation, which correlates with its transcriptional activity. In this study, we show that HSF1 is modified by SUMO-1 and SUMO-2 in a stress-inducible manner. Sumoylation is rapidly and transiently enhanced on lysine 298, located in the regulatory domain of HSF1, adjacent to several critical phosphorylation sites. Sumoylation analyses of HSF1 phosphorylation site mutants reveal that specifically the phosphorylation-deficient S303 mutant remains devoid of SUMO modification in vivo and the mutant mimicking phosphorylation of S303 promotes HSF1 sumoylation in vitro, indicating that S303 phosphorylation is required for K298 sumoylation. This finding is further supported by phosphopeptide mapping and analysis with S303/7 phosphospecific antibodies, which demonstrate that serine 303 is a target for strong heat-inducible phosphorylation, corresponding to the inducible HSF1 sumoylation. A transient phosphorylation-dependent colocalization of HSF1 and SUMO-1 in nuclear stress granules provides evidence for a strictly regulated subnuclear interplay between HSF1 and SUMO.

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Regulation of GABAergic synapse formation and plasticity by GSK3β-dependent phosphorylation of gephyrin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1011824108 ◽

2010 ◽

Vol 108 (1) ◽

pp. 379-384 ◽

Cited By ~ 116

Author(s):

Shiva K. Tyagarajan ◽

Himanish Ghosh ◽

Gonzalo E. Yévenes ◽

Irina Nikonenko ◽

Claire Ebeling ◽

...

Keyword(s):

Synaptic Plasticity ◽

Hippocampal Neurons ◽

Glycogen Synthase ◽

Phosphorylation Site ◽

Scaffolding Protein ◽

Scaffolding Proteins ◽

Gabaergic Synapse ◽

Gabaergic Synapses

Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3β (GSK3β) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3β inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca2+-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.

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Phosphoenolpyruvate carboxylase during grape berry development: protein level, enzyme activity and regulation

Functional Plant Biology ◽

10.1071/pp99141 ◽

2000 ◽

Vol 27 (3) ◽

pp. 221 ◽

Cited By ~ 8

Author(s):

Paraskevi Diakou ◽

Laurence Svanella ◽

Philippe Raymond ◽

Jean-Pierre Gaudillère ◽

Annick Moing

Keyword(s):

Malic Acid ◽

Protein Level ◽

Phosphoenolpyruvate Carboxylase ◽

Phosphorylation Site ◽

Acid Synthesis ◽

Grape Berry ◽

Vitis Vinifera L ◽

Normal Acid

The protein level and regulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, involved in malic acid synthesis) was studied during the fruit development of two grape (Vitis vinifera L.) varieties, ‘Cabernet Sauvignon’ and ‘Gora Chirine’, with berries of normal and low organic acid content, respectively. The protein level and in vitro activity were higher in the low-acid variety than in the normal-acid variety for most stages. In vivo PEPC activity, measured using 14 CO2 labelling, was significantly higher in the low-acid variety than in the normal-acid variety about 1 week before and 1 week after veraison (the day which corresponds to the onset of ripening). However, partitioning into malate was the same for both varieties. Antibodies raised against the N-terminal part of SorghumPEPC recognised the grape berry PEPC, indicating the presence of the consensus phosphorylation site involved in PEPC regulation. PEPC phosphorylation status was estimated by studying sensitivity to pH and malate. Grape berry PEPC appeared more sensitive to low pH and malate during ripening (IC50 malate, 0.2–0.7 mM) compared to during the earlier stages of development (IC50 malate, 1.2–2 mM) for both varieties. Therefore, in the normal-acid variety, PEPC seems to participate in controlling malic acid accumulation but does not seem to control the differences in malic acid concentration observed between the two varieties.

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Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.2000.278.4.c718 ◽

2000 ◽

Vol 278 (4) ◽

pp. C718-C726 ◽

Cited By ~ 78

Author(s):

Jason C. Hedges ◽

Brian C. Oxhorn ◽

Michael Carty ◽

Leonard P. Adam ◽

Ilia A. Yamboliev ◽

...

Keyword(s):

Smooth Muscle ◽

Map Kinase ◽

Map Kinases ◽

Isometric Force ◽

Phosphorylation Site ◽

Smooth Muscle Contraction ◽

Tracheal Smooth Muscle ◽

Muscarinic Agonists

Phosphorylation of h-caldesmon has been proposed to regulate airway smooth muscle contraction. Both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases phosphorylate h-caldesmon in vitro. To determine whether both enzymes phosphorylate caldesmon in vivo, phosphorylation-site-selective antibodies were used to assay phosphorylation of MAP kinase consensus sites. Stimulation of cultured tracheal smooth muscle cells with ACh or platelet-derived growth factor increased caldesmon phosphorylation at Ser789 by about twofold. Inhibiting ERK MAP kinase activation with 50 μM PD-98059 blocked agonist-induced caldesmon phosphorylation completely. Inhibiting p38 MAP kinases with 25 μM SB-203580 had no effect on ACh-induced caldesmon phosphorylation. Carbachol stimulation increased caldesmon phosphorylation at Ser789 in intact tracheal smooth muscle, which was blocked by the M2 antagonist AF-DX 116 (1 μM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 ± 1.4%, thus dissociating caldesmon phosphorylation from contraction. Activation of M2 receptors leads to activation of ERK MAP kinases and phosphorylation of caldesmon with little or no functional effect on isometric force. P38 MAP kinases are also activated by muscarinic agonists, but they do not phosphorylate caldesmon in vivo.

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Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation

Blood ◽

10.1182/blood.v87.12.5287.bloodjournal87125287 ◽

1996 ◽

Vol 87 (12) ◽

pp. 5287-5296 ◽

Cited By ~ 45

Author(s):

YL Zu ◽

Y Ai ◽

A Gilchrist ◽

ME Labadia ◽

RI Sha'afi ◽

...

Keyword(s):

Protein Kinase ◽

Enzymatic Activity ◽

Map Kinase ◽

Phorbol Ester ◽

Map Kinases ◽

Phosphorylation Site ◽

Human Neutrophils ◽

Inhibitory Peptide

In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen- activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase- activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP- stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.

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Functional Interactions between Herpesvirus Oncoprotein MEQ and Cell Cycle Regulator CDK2

Journal of Virology ◽

10.1128/jvi.73.5.4208-4219.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 4208-4219 ◽

Cited By ~ 29

Author(s):

Juinn-Lin Liu ◽

Ying Ye ◽

Zheng Qian ◽

Yongyi Qian ◽

Dennis J. Templeton ◽

...

Keyword(s):

Cell Cycle ◽

Phosphorylation Site ◽

Fibroblast Cell ◽

S Phase ◽

Morphological Transformation ◽

Necrosis Factor Alpha ◽

Coiled Bodies ◽

Cdk Phosphorylation

ABSTRACT Marek’s disease virus, an avian alphaherpesvirus, has been used as an excellent model to study herpesvirus oncogenesis. One of its potential oncogenes, MEQ, has been demonstrated to transform a rodent fibroblast cell line, Rat-2, in vitro by inducing morphological transformation and anchorage- and serum-independent growth and by protecting cells from apoptosis induced by tumor necrosis factor alpha, C2-ceramide, UV irradiation, or serum deprivation. In this report, we show that there is a cell cycle-dependent colocalization of MEQ protein and cyclin-dependent kinase 2 (CDK2) in coiled bodies and the nucleolar periphery during the G1/S boundary and early S phase. To our knowledge, this is the first demonstration that CDK2 is found to localize to coiled bodies. Such an in vivo association and possibly subsequent phosphorylation may result in the cytoplasmic translocation of MEQ protein. Indeed, MEQ is expressed in both the nucleus and the cytoplasm during the G1/S boundary and early S phase. In addition, we were able to show in vitro phosphorylation of MEQ by CDKs. We have mapped the CDK phosphorylation site of MEQ to be serine 42, a residue in the proximity of the bZIP domain. An indirect-immunofluorescence study of the MEQ S42D mutant, in which the CDK phosphorylation site was mutated to a charged residue, reveals more prominent cytoplasmic localization. This lends further support to the notion that the translocation of MEQ is regulated by phosphorylation. Furthermore, phosphorylation of MEQ by CDKs drastically reduces the DNA binding activity of MEQ, which may in part account for the lack of retention of MEQ oncoprotein in the nucleus. Interestingly, the localization of CDK2 in coiled bodies and the nucleolar periphery is observed only in MEQ-transformed Rat-2 cells, implicating MEQ in modifying the subcellular localization of CDK2. Taken together, our data suggest that there is a novel reciprocal modulation between the herpesvirus oncoprotein MEQ and CDK2.

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Phosphorylation of the phosphatase PTPROt at Tyr399is a molecular switch that controls osteoclast activity and bone mass in vivo

Science Signaling ◽

10.1126/scisignal.aau0240 ◽

2019 ◽

Vol 12 (563) ◽

pp. eaau0240 ◽

Cited By ~ 2

Author(s):

Lee Roth ◽

Jean Wakim ◽

Elad Wasserman ◽

Moran Shalev ◽

Esther Arman ◽

...

Keyword(s):

Bone Mass ◽

Cultured Cells ◽

Phosphorylation Site ◽

Molecular Switch ◽

Receptor Type ◽

Mouse Strains ◽

Bone Homeostasis ◽

Osteoclast Activity

Bone resorption by osteoclasts is essential for bone homeostasis. The kinase Src promotes osteoclast activity and is activated in osteoclasts by the receptor-type tyrosine phosphatase PTPROt. In other contexts, however, PTPROt can inhibit Src activity. Through in vivo and in vitro experiments, we show that PTPROt is bifunctional and can dephosphorylate Src both at its inhibitory residue Tyr527and its activating residue Tyr416. Whereas wild-type and PTPROt knockout mice exhibited similar bone masses, mice in which a putative C-terminal phosphorylation site, Tyr399, in endogenous PTPROt was replaced with phenylalanine had increased bone mass and reduced osteoclast activity. Osteoclasts from the knock-in mice also showed reduced Src activity. Experiments in cultured cells and in osteoclasts derived from both mouse strains demonstrated that the absence of phosphorylation at Tyr399caused PTPROt to dephosphorylate Src at the activating site pTyr416. In contrast, phosphorylation of PTPROt at Tyr399enabled PTPROt to recruit Src through Grb2 and to dephosphorylate Src at the inhibitory site Tyr527, thus stimulating Src activity. We conclude that reversible phosphorylation of PTPROt at Tyr399is a molecular switch that selects between its opposing activities toward Src and maintains a coherent signaling output, and that blocking this phosphorylation event can induce physiological effects in vivo. Because most receptor-type tyrosine phosphatases contain potential phosphorylation sites at their C termini, we propose that preventing phosphorylation at these sites or its consequences may offer an alternative to inhibiting their catalytic activity to achieve therapeutic benefit.

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The major phosphorylation site of the NADPH oxidase component p67phox is Thr233

Biochemical Journal ◽

10.1042/bj3380099 ◽

1999 ◽

Vol 338 (1) ◽

pp. 99-105 ◽

Cited By ~ 20

Author(s):

Louisa V. FORBES ◽

Oanh TRUONG ◽

Frans B. WIENTJES ◽

Stephen J. MOSS ◽

Anthony W. SEGAL

Keyword(s):

Protein Kinase ◽

Cyanogen Bromide ◽

Tryptic Peptide ◽

Phosphorylation Site ◽

Mitogen Activated Protein Kinase ◽

Rich Domain ◽

Mitogen Activated Protein ◽

Phosphopeptide Mapping ◽

Major Phosphorylation Site

Phosphorylation of p67phox was shown to increase two- to three-fold upon stimulation by PMA, N-formylmethionyl-leucylphenylalanine or serum-opsonized zymosan. Phosphopeptide mapping showed one major tryptic peptide for p67phox immunoprecipitated from resting or stimulated cells. In vitro phosphorylation of p67phox by isolated cytosol or mitogen-activated protein kinase also generated the same phosphopeptide. Results of cyanogen bromide digestion and HPLC–MS suggested that Thr233 was the phosphorylated residue. Mutagenesis of Thr233 to alanine resulted in loss of phosphorylation in vitro. In the present work, Thr233 has been identified as the major phosphorylation site of p67phox, which is situated in a proline-rich domain.

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