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At least 307 records · Page 17Linked to original sources
PubMed1993

The abnormal phosphorylation of tau protein at Ser-202 in Alzheimer disease recapitulates phosphorylation during development.

Tau is a neuronal phosphoprotein whose expression is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult brain, with the fetal isoform corresponding to the shortest of the adult isoforms. Phosphorylation of tau is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer disease, the six adult tau isoforms become abnormally phosphorylated and form the paired helical filament, the major fibrous component of the characteristic neurofibrillary lesions. We show here that Ser-202 (in the numbering of the longest human brain tau isoform) is a phosphorylation site that distinguishes fetal from adult tau and we identify it as one of the abnormal phosphorylation sites in Alzheimer disease. The abnormal phosphorylation of tau at Ser-202 in Alzheimer disease thus recapitulates normal phosphorylation during development.

Adult↗
PubMed1998

Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: involvement of MEK upstream of Bad phosphorylation.

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras-mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.

Apoptosis↗
PubMed1995

The phosphorylated ribosomal protein S7 in Tetrahymena is homologous with mammalian S4 and the phosphorylated residues are located in the C-terminal region. Structural characterization of proteins separated by two-dimensional polyacrylamide gel electrophoresis.

A single basic ribosomal protein, protein S7, can be multiply phosphorylated in the ciliated protozoan Tetrahymena. Induction of phosphorylation is highly regulated, and the phosphorylation proceeds in a strictly sequential manner. The first site to be phosphorylated is a serine residue and the second a threonine. In this paper we report the complete primary structure of Tetrahymena thermophila ribosomal protein S7 including identification of the phosphorylated serine and threonine residues. Most of the sequence information was obtained from peptides generated by in situ digestion of S7 in two-dimensional gels using an approach that combined traditional protein chemistry with mass spectrometry. T. thermophila ribosomal protein S7 has a molecular mass of 29,459 Da and contains 259 amino acid residues. Phosphorylation takes place on Ser258 and Thr248 in the C-terminal region of the protein. Alignment of T. thermophila ribosomal protein S7 with known ribosomal proteins yielded the surprising result that T. thermophila S7 is homologous, not with mammalian ribosomal protein S6, but with mammalian ribosomal protein S4. These findings clearly distinguish the pattern of phosphorylation of ribosomal proteins in Tetrahymena from all other eukaryotes analyzed to date.

Amino Acid Sequence↗
PubMed1995

Phosphorylation and activation of Ca(2+)-calmodulin-dependent protein kinase IV by Ca(2+)-calmodulin-dependent protein kinase Ia kinase. Phosphorylation of threonine 196 is essential for activation.

Purified pig brain Ca(2+)-calmodulin (CaM)-dependent protein kinase Ia kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164) enhances, by up to 24-fold, the activity of recombinant CaM kinase IV in a reaction also requiring Ca(2+)-CaM and MgATP. The addition of brain extract, although capable of activating CaM kinase IV by itself, provides no further activation beyond that induced by purified CaM kinase Ia kinase, consistent with the lack of a requirement of additional components for activation. Activation is accompanied by the development of significant (38%) Ca(2+)-CaM-independent CaM kinase IV activity. In parallel fashion to its activation, CaM kinase IV is phosphorylated in a CaM kinase Ia kinase-, Ca(2+)-CaM-, and MgATP-dependent manner. Phosphorylation occurs on multiple serine and threonine residues with a Ser-P:Thr-P ratio of approximately 3:1. The identical requirements for phosphorylation and activation and a linear relationship between extent of phosphorylation of CaM kinase IV and its activation state indicate that CaM kinase IV activation is induced by its phosphorylation. Replacement of Thr-196 of CaM kinase IV with a nonphosphorylatable alanine by site-directed mutagenesis abolishes both the phosphorylation and activation of CaM kinase IV, demonstrating that Thr-196 phosphorylation is essential for activation.

Amino Acid Sequence↗
PubMed1995

Tumor necrosis factor alpha-induced phosphorylation of insulin receptor substrate-1 (IRS-1). Possible mechanism for suppression of insulin-stimulated tyrosine phosphorylation of IRS-1.

Tumor necrosis factor-alpha (TNF) has been suggested to be the mediator of insulin resistance in infection, tumor cachexia, and obesity. We have previously shown that TNF diminishes insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1). The current work examines potential mechanisms that mediate this event. TNF effect on IRS-1 in Fao hepatoma cells was not associated with a significant reduction in insulin receptor tyrosine kinase activity as measured in vitro but impaired the association of IRS-1 with phosphatidylinositol 3-kinase, localizing TNF impact to IRS-1. TNF did not increase protein-tyrosine phosphatase activity and protein-tyrosine phosphatase inhibition by vanadate did not change TNF effect on IRS-1 tyrosine phosphorylation, suggesting that protein-tyrosine phosphatases are not involved in this TNF effect. In contrast, TNF increased IRS-1 phosphorylation on serine residues, leading to a decrease in its electrophoretic mobility. TNF effect on IRS-1 tyrosine phosphorylation was not abolished by inhibiting protein kinase C using staurosporine, while inactivation of Ser/Thr phosphatases by calyculin A and okadaic acid mimicked it. Our data suggest that TNF induces serine phosphorylation of IRS-1 through inhibition of serine phosphatases or activation of serine kinases other than protein kinase C. This increased serine phosphorylation interferes with insulin-induced tyrosine phosphorylation of IRS-1 and impairs insulin action.

Alkaloids↗
PubMed1996

Phosphorylation of the inositol 1,4,5-trisphosphate receptor. Cyclic GMP-dependent protein kinase mediates cAMP and cGMP dependent phosphorylation in the intact rat aorta.

The effects of cyclic GMP (cGMP) and activation of cGMP-dependent protein kinase (PKG) on the phosphorylation of the inositol 1,4, 5-trisphosphate (IP3) receptor were examined in intact rat aorta using the technique of back phosphorylation. Aorta treated with the nitric oxide donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, or the selective PKG activator, 8-(4-para-chlorophenylthio)-cGMP (8-CPT-cGMP), demonstrated increased IP3 receptor phosphorylation in situ, which was both time- and concentration-dependent with a stoichiometry of 0.5 mol of phosphate/mol of receptor above control. Treatment of aorta with the adenyl cyclase activator, forskolin, also demonstrated increased phosphorylation of the IP3 receptor on the PKG site, although the selective cAMP-dependent protein kinase activator, 8-(4-para-chlorophenylthio)-cAMP (8-CPT-cAMP), did not increase the phosphorylation of the IP3 receptor. Moreover, the PKG selective inhibitor, KT 5823, inhibited both sodium nitroprusside and forskolin-induced IP3 receptor phosphorylation more potently than the selective cAMP-dependent protein kinase inhibitor, KT 5720, suggesting that PKG mediates the increase in IP3 receptor phosphorylation by both cyclic nucleotides in intact aorta. These results provide further support for the notion that PKG is activated by both cAMP and cGMP in intact vascular smooth muscle and that PKG performs a critical role in cyclic nucleotide-dependent relaxation of blood vessels.

Alkaloids↗
PubMed1996

p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2.

The Ca2+-sensitive 85-kDa cytosolic phospholipase A2 (cPLA2) is responsible for thrombin-stimulated mobilization of arachidonic acid for the synthesis of thromboxane A2 in human platelets. We have previously shown that thrombin activates p38 kinase, a recently discovered new member of the mitogen-activated protein kinase family (Kramer, R. M., Roberts, E. F., Strifler, B. A., and Johnstone, E. M. (1995) J. Biol. Chem. 270, 27395-27398) and also induces phosphorylation of cPLA2, thereby increasing its intrinsic catalytic activity. In the present study we have examined the role of p38 kinase in the phosphorylation and activation of cPLA2 in stimulated platelets. We have observed that activation of p38 kinase accompanies receptor-mediated events in platelets and coincides with cPLA2 phosphorylation. Furthermore, in the presence of inhibitors of p38 kinase, the proline-directed phosphorylation of cPLA2 was completely blocked in platelets stimulated with the thrombin receptor agonist peptide SFLLRN and was suppressed during the early (up to 2 min) phase of platelet stimulation caused by thrombin. Unexpectedly, we found that prevention of proline-directed phosphorylation of cPLA2 in stimulated platelets did not attenuate its ability to release arachidonic acid from platelet phospholipids. We conclude that: 1) cPLA2 is a physiological target of p38 kinase; 2) p38 kinase is involved in the early phosphorylation of cPLA2 in stimulated platelets; and 3) proline-directed phosphorylation of cPLA2 is not required for its receptor-mediated activation.

Arachidonic Acid↗
PubMed1996

Energetic implications for protein phosphorylation. Conformational stability of HPr variants that mimic phosphorylated forms.

The HPr protein from Bacillus subtilis is a key protein in the phosphoenolpyruvate-sugar transport system. HPr has two biological phosphorylation sites. The active site histidine is transiently phosphorylated in the phosphotransferase reaction while phosphorylation of serine 46 diminishes the activity of HPr. Here, we use protein engineering and equilibrium protein folding experiments to determine if the two phosphorylation events are energetically coupled. Our approach is to use structural mimics of the two phosphorylated forms of HPr, where histidine 15 is replaced by a negatively charged glutamate and serine 46 is changed to an aspartate, both alone and in combination. The thermodynamic analysis of the differences in conformational stability between the single and double mutants shows that the two phosphorylation sites are not energetically coupled in the HPr protein. We also show that single mutants of the active site histidine residue can have dramatic effects on the conformational stability of HPr. Combined with structural information, the method employed here will be of general use in unraveling the biological effects of phosphorylation on protein activity.

Bacillus subtilis↗
PubMed1997

G protein-coupled receptors mediate two functionally distinct pathways of tyrosine phosphorylation in rat 1a fibroblasts. Shc phosphorylation and receptor endocytosis correlate with activation of Erk kinases.

The Ras-dependent activation of Erk kinases by G protein-coupled receptors (GPCRs) is thought to involve tyrosine phosphorylation of docking proteins that serve as scaffolds for the plasma membrane recruitment of Ras guanine nucleotide exchange factors, such as the Grb2-mSos complex. We have investigated the role of two GPCR-regulated tyrosine phosphoproteins, p125(FAK) (FAK) and Shc, in the Ras-dependent activation of Erk kinases by endogenously expressed GPCRs in Rat 1a fibroblasts. Several lines of evidence suggest that tyrosine phosphorylation of FAK and Shc are independently regulated. The GPCRs for lysophosphatidic acid (LPA), thrombin, and bombesin mediate equivalent increases in FAK tyrosine phosphorylation and FAK-Grb2 association. In contrast, only LPA and thrombin receptors significantly stimulate Shc tyrosine phosphorylation and Shc-Grb2 complex formation. Tyrosine phosphorylation of FAK is pertussis toxin-insensitive, can be mimicked by calcium ionophore, and is inhibited by treatment with cytochalasin D, which depolymerizes the actin cytoskeleton. In contrast, tyrosine phosphorylation of Shc is inhibited by pertussis toxin treatment, is not induced by calcium ionophore, and is insensitive to cytochalasin D. In each case, the rapid stimulation of Erk 1/2 correlates with tyrosine phosphorylation of Shc but not of FAK. The dissociation of FAK-Grb2 complex formation from receptor-mediated activation of Erk 1/2 indicates that recruitment of Grb2-mSos to the plasma membrane is not sufficient to mediate rapid Erk activation. Using four mechanistically distinct inhibitors of clathrin-mediated endocytosis, concanavalin A, hypertonic medium, depletion of intracellular potassium, and monodansylcadaverine, we find that GPCR-mediated Erk 1/2 activation is also endocytosis-dependent. Thus, we propose that an additional step involving vesicle-mediated endocytosis is required for the rapid, Ras-dependent activation of Erk kinases in fibroblasts.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Identification of in vitro phosphorylation sites in the growth cone protein SCG10. Effect Of phosphorylation site mutants on microtubule-destabilizing activity.

SCG10 is a neuron-specific, membrane-associated protein that is highly concentrated in growth cones of developing neurons. Previous studies have suggested that it is a regulator of microtubule dynamics and that it may influence microtubule polymerization in growth cones. Here, we demonstrate that in vivo, SCG10 exists in both phosphorylated and unphosphorylated forms. By two-dimensional gel electrophoresis, two phosphoisoforms were detected in neonatal rat brain. Using in vitro phosphorylated recombinant protein, four phosphorylation sites were identified in the SCG10 sequence. Ser-50 and Ser-97 were the target sites for protein kinase A, Ser-62 and Ser-73 for mitogen-activated protein kinase and Ser-73 for cyclin-dependent kinase. We also show that overexpression of SCG10 induces a disruption of the microtubule network in COS-7 cells. By expressing different phosphorylation site mutants, we have dissected the roles of the individual phosphorylation sites in regulating its microtubule-destabilizing activity. We show that nonphosphorylatable mutants have increased activity, whereas mutants in which phosphorylation is mimicked by serine-to-aspartate substitutions have decreased activity. These data suggest that the microtubule-destabilizing activity of SCG10 is regulated by phosphorylation, and that SCG10 may link signal transduction of growth or guidance cues involving serine/threonine protein kinases to alterations of microtubule dynamics in the growth cone.

Amino Acid Sequence↗
PubMed1998

Evidence of a functional interaction between serine 3 and serine 25 Mos phosphorylation sites. A dominant inhibitory role of serine 25 phosphorylation on Mos protein kinase.

Recently, we identified the major in vivo phosphorylation site on v-Mos as Ser-56, which is phosphorylated by cyclic AMP dependent protein kinase (PKA). Others have shown that c-Mos phosphorylation at Ser-3 (equivalent to Ser-34 in v-Mos) is important for the interaction of c-Mos with its substrate MEK and for its stability and cytostatic factor activity in eggs. To investigate the role of Ser-56 phosphorylation, we generated site-directed mutants of v-Mos that would mimic phosphorylation in terms of charge at positions 56 and 34. After mutating serine (S) residues with alanine (A) or glutamic acid (E) in different combinations, various v-Mos mutants were expressed in a rabbit reticulocyte lysate in vitro translation system and in COS-1 or NIH/3T3 cells. The effect of mutations on Mos function was evaluated by in vitro protein kinase assays and by the ability of Mos to cause neoplastic transformation of NIH/3T3 cells. The S56E but not the S56A mutation inhibited v-Mos kinase activity suggesting that Ser-56 phosphorylation has an inhibitory role. As predicted from Xenopus c-Mos studies, S34A but not S34E mutation inhibited v-Mos activity. Studies with the double mutants showed that the S56E mutation but not S56A mutation inhibited v-Mos kinase activity of both S34A and S34E mutants. Interestingly, the S56A mutation blocked the inhibitory effect of the S34A mutation on v-Mos kinase suggesting that in c-Mos the corresponding serine (Ser-25) can influence the regulation of c-Mos by Ser-3. Results showing inhibition of v-Mos kinase activity of the S34E mutant by the S56E mutation is significant as it suggests that doubly phosphorylated Mos at these residues would be inactive. Because residues corresponding to both v-Mos Ser-34 and Ser-56 are evolutionarily conserved in c-Mos, the kinase activity of c-Mos during meiosis may also be regulated in the same manner as v-Mos kinase activity.

3T3 Cells↗
PubMed1998

Activation of p47(PHOX), a cytosolic subunit of the leukocyte NADPH oxidase. Phosphorylation of ser-359 or ser-370 precedes phosphorylation at other sites and is required for activity.

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O-2) at the expense of NADPH in phagocytes and B lymphocytes. The enzyme is dormant in resting cells but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(PHOX) becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that p47(PHOX)-deficient B lymphoblasts expressing the p47(PHOX) S359A/S370A or p47(PHOX) S359K/S370K double mutation show dramatically reduced levels of enzyme activity and phosphorylation of p47(PHOX) as compared with the same cells expressing wild type p47(PHOX). In addition, these mutant p47(PHOX) proteins fails to translocate to the plasma membrane when the cells are stimulated. In contrast, normal phosphorylation and translocation are seen in mutants containing aspartate or glutamate at positions 359 and 370, but oxidase activity is still greatly reduced. These results imply that a negative charge at position 359 and/or 370 is sufficient to allow the phosphorylation and translocation of p47(PHOX) to take place but that features unique to a phosphorylated hydroxyamino acid are required to support O-2 production. These findings, plus those from an earlier study (Inanami, O., Johnson, J. L., McAdara, J. K., El Benna, J., Faust, L. P., Newburger, P. E., and Babior, B. M. (1998) J. Biol. Chem. 273, 9539-9543), suggest that oxidase activation requires 1) the sequential phosphorylation of at least two serines on p47(PHOX): Ser-359 or Ser-370, followed by Ser-303 or Ser-304; and 2) the translocation of p47(PHOX) to the membrane at some point after the first phosphorylation takes place.

Arachidonic Acid↗
PubMed1998

The C-terminal conserved domain of MARCKS is phosphorylated in vivo by proline-directed protein kinase. Application of ion trap mass spectrometry to the determination of protein phosphorylation sites.

MARCKS, the major protein kinase C substrate in various cells and tissues, binds to calmodulin, acidic membrane phospholipids, and actin filaments, and these interactions are regulated by protein phosphorylation. We have previously shown that MARCKS purified from bovine brain is phosphorylated not only by protein kinase C but also by so-called proline-directed protein kinases in the well conserved N-terminal half of the molecule (Taniguchi, H., Manenti, S., Suzuki, M., and Titani, K. (1994) J. Biol. Chem. 269, 18299-18302). Although the presence of other phosphorylation sites in the C-terminal peptide was also noticed, the ambiguity in the C-terminal domain of the bovine protein hampered a more detailed analysis. In the present study, we analyzed MARCKS purified from rat brain by electrospray ionization/ion trap mass spectrometry. The results obtained revealed two additional novel phosphorylation sites in the C-terminal region. Both phosphorylation sites (Ser291 and Ser299) are immediately followed by proline, suggesting that these sites are also phosphorylated by the proline-directed protein kinase(s). Since Ser299 is within the C-terminal domain, which is well conserved among various species, the function of the domain, whatever it is, seems to be controlled by phosphorylation.

Amino Acid Sequence↗
PubMed1999

Differential effects of CC chemokines on CC chemokine receptor 5 (CCR5) phosphorylation and identification of phosphorylation sites on the CCR5 carboxyl terminus.

The binding of CC chemokines to CC chemokine receptor 5 (CCR5) triggers cellular responses that, generally, are only transient in nature. To explore the potential role of G protein-coupled receptor kinases (GRKs) in the regulation of CCR5, we performed phosphorylation experiments in a rat basophilic leukemia cell line stably expressing CCR5. The ability of various CCR5 ligands to stimulate calcium mobilization in these cells correlated with their ability to induce receptor phosphorylation, desensitization, internalization, and GRK association with the receptor. Aminooxypentane-RANTES, a potent inhibitor of human immunodeficiency virus infection, has been proposed to act through enhanced CCR5 internalization and inhibition of receptor recycling. Aminooxypentane-RANTES profoundly induced CCR5 phosphorylation, but had no effect on CCR1. In permeabilized rat basophilic leukemia CCR5 cells, monoclonal antibodies with specificity for GRK2/3 inhibited RANTES-induced receptor phosphorylation. Consistent with a role for these kinases in CCR5 regulation, 1-2 x 10(5) copies of GRK2 or GRK3 were found to be expressed in peripheral blood leukocytes. Phosphoamino acid analysis revealed that RANTES-induced CCR5 phosphorylation selectively occurs on serine residues. Our findings with receptor mutants indicate that serine residues at positions 336, 337, 342, and 349 represent GRK phosphorylation sites on CCR5. This study demonstrates that chemokines differ in their ability to induce CCR5 phosphorylation and desensitization and provides a molecular mechanism for the agonist-induced attenuation of CCR5 signaling.

Animals↗
PubMed1999

Activated G protein-coupled receptor induces tyrosine phosphorylation of STAT3 and agonist-selective serine phosphorylation via sustained stimulation of mitogen-activated protein kinase. Resultant effects on cell proliferation.

The peptide hormone somatostatin exhibits antiproliferative activity by interacting with the G protein-coupled sst2 or sst5 receptor types. We show here that somatostatin at the human recombinant sst4 receptor induced a concentration-dependent increase in proliferation (EC50 20 nM) with a maximal response 5-fold greater than that produced by its synthetic analog, L-362,855. Analysis of the phosphorylation status of extracellular signal-regulated kinase (ERK)1 and ERK2 showed temporal differences in the changes evoked by the agonists. Phosphorylation induced by somatostatin (100 nM) peaked 10 min after the application and produced a response that continued for at least 4 h. In contrast, L-362,855 (1 microM) showed transient phosphorylation that had declined to basal levels by 1 h. However, both agonists induced rapid and sustained tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3) which was pertussis toxin-insensitive. Serine phosphorylation of STAT3 was only apparent after somatostatin treatment and was abolished by pertussis toxin or PD 98059, together with the associated increases in proliferation. Mitogen-activated protein/ERK kinase-1 inhibition also decreased the time interval over which somatostatin-induced ERK phosphorylation was observed (<2 h). We conclude that the difference in the magnitude of the proliferative response evoked by the two agonists at the sst4 receptor can be accounted for by their differential ability to phosphorylate STAT3 on serine residues and supports the concept that selective signaling can be achieved through pharmacological diversity.

Animals↗
PubMed2001

Multimerization of phosphorylated and non-phosphorylated ArcA is necessary for the response regulator function of the Arc two-component signal transduction system.

To adapt to anaerobic conditions, Escherichia coli operates the Arc two-component signal transduction system, consisting of a sensor kinase, ArcB, and a response regulator, ArcA. ArcA is converted to the active form, phosphorylated ArcA (ArcA-P), by ArcB-mediated phosphorylation. The active ArcA-P binds to the promoter regions of target genes, thereby regulating their transcriptional activities. The phosphoryl group of ArcA-P is unstable with a half-life of 30 min. However, we were able to inhibit the dephosphorylation for more than 12 h by the addition of EDTA; this allowed us to characterize ArcA-P. Gel-filtration and glycerol sedimentation experiments demonstrated that ArcA exists as a homo-dimer. ArcA phosphorylated by either ArcB or carbamyl phosphate multimerizes to form a tetramer of dimers; this multimer binds to the ArcA DNA binding site. Isoelectric focusing gel electrophoresis and nitrocellulose-filter binding analyses indicated that the ArcA multimer is composed of both ArcA-P and ArcA in a ratio, 1:1. The ArcA(D54E) mutant protein was unable to be phosphorylated by ArcB. This defect resulted in the inability of ArcA(D54E) to form a multimer or to bind to the ArcA DNA binding site. These results indicate that phosphorylation of ArcA induces multimerization prior to DNA binding, and the multimerization is a prerequisite for binding. Our results suggest a novel model that phosphorylation of ArcA by ArcB regulates multimerization of ArcA, which in turn functions as a response regulator.

Bacterial Outer Membrane Proteins↗
PubMed2001

Osmotic shock induces G1 arrest through p53 phosphorylation at Ser33 by activated p38MAPK without phosphorylation at Ser15 and Ser20.

Osmotic shock induced transient stabilization of p53, possibly due to increased degradation of Mdm2. Stabilized p53 was activated by p38(MAPK), resulting in G(1) arrest through induction of p21(WAF1). Among the postulated phosphorylation sites involved in p53 stabilization or activation (Ser(15), Ser(20), Ser(33), and Ser(46)), only Ser(33) was phosphorylated. Furthermore, interaction of p53 with the transcriptional coactivator p300 was induced, and Lys(382) of p53 was acetylated. Although inhibition of p38(MAPK) did not prevent nuclear accumulation of p53, phosphorylation of Ser(33) was markedly suppressed by SB203580, a specific inhibitor of p38(MAPK). Under these conditions, acetylation of Lys(382) and induction of p21(WAF1) were also inhibited, and cells with elevated levels of p53 showed normal cell cycle progression. Activated p38(MAPK) phosphorylated endogenous p53 at Ser(33) in living cells. In stable transformants expressing dominant negative MKK6, an upstream protein kinase of p38(MAPK), p53 stabilization was induced normally following osmotic shock, but phosphorylation of Ser(33), acetylation of Lys(382), and induction of p21(WAF1) were almost completely inhibited. These results suggest that phosphorylation at Ser(33) by p38(MAPK) is critical for activation of p53 following osmotic shock. Phosphorylation of neither Ser(15) nor Ser(20) was needed in this activation.

Acetylation↗
PubMed2001

Phosphorylation of pyrimidine deoxynucleoside analog diphosphates: selective phosphorylation of L-nucleoside analog diphosphates by 3-phosphoglycerate kinase.

D-Nucleoside analogs, which are in the natural configuration, as well as the L-nucleoside analogs, are clinically relevant antiviral and anticancer agents. Metabolism of L-nucleoside analog diphosphates to the triphosphates, however, remains unexplored. Studies with recombinant nm23-H1 and -H2 isoforms indicated that L-nucleoside analog diphosphates were not phosphorylated by their nucleoside diphosphate kinase (NDPK) activity. Therefore, roles of creatine kinase, 3-phosphoglycerate kinase, and pyruvate kinase were evaluated using preparations from commercial sources and human HepG2 cells. Phosphorylation of L-OddC, L-SddC, L-Fd4C, L-FMAU, and L-ddC were compared with D-deoxynucleoside analogs, AraC, dFdC, and D-FMAU, and D-dideoxynucleoside analogs, ddC and d4T. Results based on preparations from HepG2 cells showed that L-nucleoside analog diphosphates were selectively phosphorylated by 3-phosphoglycerate kinase, whereas, D-deoxynucleoside analog diphosphates were phosphorylated by NDPK. Interestingly, ddCDP and d4TDP were substrates for creatine kinase, but were not phosphorylated by NDPK. In conclusion, it is proposed that specificity of the phosphorylating enzymes toward the nucleoside analog diphosphates is dependent on the configuration of the analog (L or D) and the presence or absence of 3'-hydroxyl group in the sugar moiety. The enzymatic process of phosphorylation of L- and D-nucleoside analog diphosphates is different in cells.

Adenosine Diphosphate↗