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Journal articles on the topic 'Phosphorylation'
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1
Hizli, Asli A., Yong Chi, Jherek Swanger, John H. Carter, Yi Liao, Markus Welcker, Alexey G. Ryazanov, and Bruce E. Clurman. "Phosphorylation of Eukaryotic Elongation Factor 2 (eEF2) by Cyclin A–Cyclin-Dependent Kinase 2 Regulates Its Inhibition by eEF2 Kinase." Molecular and Cellular Biology 33, no. 3 (November 26, 2012): 596–604. http://dx.doi.org/10.1128/mcb.01270-12.
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ABSTRACTProtein synthesis is highly regulated via both initiation and elongation. One mechanism that inhibits elongation is phosphorylation of eukaryotic elongation factor 2 (eEF2) on threonine 56 (T56) by eEF2 kinase (eEF2K). T56 phosphorylation inactivates eEF2 and is the only known normal eEF2 functional modification. In contrast, eEF2K undergoes extensive regulatory phosphorylations that allow diverse pathways to impact elongation. We describe a new mode of eEF2 regulation and show that its phosphorylation by cyclin A–cyclin-dependent kinase 2 (CDK2) on a novel site, serine 595 (S595), directly regulates T56 phosphorylation by eEF2K. S595 phosphorylation varies during the cell cycle and is required for efficient T56 phosphorylationin vivo. Importantly, S595 phosphorylation by cyclin A-CDK2 directly stimulates eEF2 T56 phosphorylation by eEF2Kin vitro, and we suggest that S595 phosphorylation facilitates T56 phosphorylation by recruiting eEF2K to eEF2. S595 phosphorylation is thus the first known eEF2 modification that regulates its inhibition by eEF2K and provides a novel mechanism linking the cell cycle machinery to translational control. Because all known eEF2 regulation is exerted via eEF2K, S595 phosphorylation may globally couple the cell cycle machinery to regulatory pathways that impact eEF2K activity.
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Coulonval, Katia, Hugues Kooken, and Pierre P. Roger. "Coupling of T161 and T14 phosphorylations protects cyclin B–CDK1 from premature activation." Molecular Biology of the Cell 22, no. 21 (November 2011): 3971–85. http://dx.doi.org/10.1091/mbc.e11-02-0136.
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Mitosis is triggered by the abrupt dephosphorylation of inhibitory Y15 and T14 residues of cyclin B1–bound cyclin-dependent kinase (CDK)1 that is also phosphorylated at T161 in its activation loop. The sequence of events leading to the accumulation of fully phosphorylated cyclin B1–CDK1 complexes remains unclear. Two-dimensional gel electrophoresis allowed us to determine whether T14, Y15, and T161 phosphorylations occur on same CDK1 molecules and to characterize the physiological occurrence of their seven phosphorylation combinations. Intriguingly, in cyclin B1–CDK1, the activating T161 phosphorylation never occurred without the T14 phosphorylation. This strict association could not be uncoupled by a substantial reduction of T14 phosphorylation in response to Myt1 knockdown, suggesting some causal relationship. However, T14 phosphorylation was not directly required for T161 phosphorylation, because Myt1 knockdown did uncouple these phosphorylations when leptomycin B prevented cyclin B1–CDK1 complexes from accumulating in cytoplasm. The coupling mechanism therefore depended on unperturbed cyclin B1–CDK1 traffic. The unexpected observation that the activating phosphorylation of cyclin B1–CDK1 was tightly coupled to its T14 phosphorylation, but not Y15 phosphorylation, suggests a mechanism that prevents premature activation by constitutively active CDK-activating kinase. This explained the opposite effects of reduced expression of Myt1 and Wee1, with only the latter inducing catastrophic mitoses.
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ADAMS, Ryan A., Xinran LIU, David S. WILLIAMS, and Alexandra C. NEWTON. "Differential spatial and temporal phosphorylation of the visual receptor, rhodopsin, at two primary phosphorylation sites in mice exposed to light." Biochemical Journal 374, no. 2 (September 1, 2003): 537–43. http://dx.doi.org/10.1042/bj20030408.
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Phosphorylation of rhodopsin critically controls the visual transduction cascade by uncoupling it from the G-protein transducin. The kinase primarily responsible for this phosphorylation is rhodopsin kinase, a substrate-regulated kinase that phosphorylates light-activated rhodopsin. Protein kinase C has been implicated in controlling the phosphorylation of both light-activated and dark-adapted rhodopsin. Two of the major rhodopsin phosphorylation sites in vivo, Ser334 and Ser338, are effective protein kinase C phosphorylation sites in vitro, while the latter is preferentially phosphorylated by rhodopsin kinase in vitro. Using phosphospecific antibodies against each of these two sites, we show that both sites are under differential spatial and temporal regulation. Exposure of mice to light results in rapid phosphorylation of Ser338 that is evenly distributed along the rod outer segment. Phosphorylation of Ser334 is considerably slower, begins at the base of the rod outer segment, and spreads to the top of the photoreceptor over time. In addition, we show that phosphorylation of both sites is abolished in rhodopsin kinase−/− mice, revealing an absolute requirement for rhodopsin kinase to phosphorylate rhodopsin. This requirement may reflect the need for priming phosphorylations at rhodopsin kinase sites allowing for subsequent phosphorylation by protein kinase C at Ser334. In this regard, treatment of mouse retinas with phorbol esters results in a 4-fold increase in phosphorylation on Ser334, with no significant effect on the phosphorylation of Ser338. Our results are consistent with light triggering rapid priming phosphorylations of rhodopsin by rhodopsin kinase, followed by a slower phosphorylation on Ser334, which is regulated by protein kinase C.
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Vanoosthuyse, Vincent, and Kevin G. Hardwick. "The Complexity of Bub1 Regulation: Phosphorylation, Phosphorylation, Phosphorylation." Cell Cycle 2, no. 2 (March 7, 2003): 118–19. http://dx.doi.org/10.4161/cc.2.2.343.
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Pant, Harish C., and Veeranna. "Neurofilament phosphorylation." Biochemistry and Cell Biology 73, no. 9-10 (September 1, 1995): 575–92. http://dx.doi.org/10.1139/o95-063.
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Neurofilament proteins (NFPs) are highly phosphorylated molecules in the axonal compartment of the adult nervous system. The phosphorylation of NFP is considered an important determinant of filament caliber, plasticity, and stability. This process reflects the function of NFs during the lifetime of a neuron from differentiation in the embryo through long-term activity in the adult until aging and environmental insult leads to pathology and ultimately death. NF function is modulated by phosphorylation–dephosphorylation in each of these diverse neuronal states. In this review, we have summarized some of these properties of NFP in adult nervous tissue, mostly from work in our own laboratory. Identification of sites phosphorylated in vivo in high molecular weight NFP (NF-H) and properties of NF-associated and neural-specific kinases phosphorylating specific sites in NFP are described. A model to explain the role of NF phosphorylation in determining filament caliber, plasticity, and stability is proposed.Key words: neurofilament proteins, phosphorylation, kinases, phosphatases, regulators, inhibitors, multimesic complex, domains.
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Bhattacharyya, Sumit, Alip Borthakur, Arivarasu N. Anbazhagan, Shivani Katyal, Pradeep K. Dudeja, and Joanne K. Tobacman. "Specific effects of BCL10 Serine mutations on phosphorylations in canonical and noncanonical pathways of NF-κB activation following carrageenan." American Journal of Physiology-Gastrointestinal and Liver Physiology 301, no. 3 (September 2011): G475—G486. http://dx.doi.org/10.1152/ajpgi.00071.2011.
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To determine the impact of B cell leukemia/lymphoma (BCL) 10 on the phosphorylation of crucial mediators in NF-κB-mediated inflammatory pathways, human colonic epithelial cells were exposed to carrageenan (CGN), a sulfated polysaccharide commonly used as a food additive and known to induce NF-κB nuclear translocation by both canonical and noncanonical pathways. Phosphorylations of intermediates in inflammatory cascades, including NF-κB-inducing kinase (NIK) at Thr559, transforming growth factor-β-activating kinase (TAK) 1 at Thr184, Thr187, and Ser192, and inhibitory factor κBα (IκBα) at Ser32, were examined following mutation of BCL10 at Ser138 and at Ser218. Specific phosphoantibodies were used for detection by enzyme-linked immunosorbent assay, immunoblot, and confocal microscopy of differences in phosphorylation following transfection by mutated BCL10. Both mutations demonstrated dominant-negative effects, with inhibition of phospho(Ser32)-IκBα to less than control levels. Both of the BCL10 mutations reduced the CGN-induced increases in nuclear RelA and p50, but only the Ser138 mutation inhibited the CGN-induced increases in nuclear RelB and p52 and in NIK Thr559 phosphorylation. Hence, the phosphorylation of BCL10 Ser138, but not Ser218, emerged as a critical event in activation of the noncanonical pathway of NF-κB activation. Either BCL10 Ser138 or Ser218 mutation inhibited the phosphorylation of TAK1 at Thr184 and at Thr187, but not at Ser192. These findings indicate that BCL10 phosphorylations act upstream of phosphorylations of NIK, TAK1, and IκBα and differentially affect the canonical and noncanonical pathways of NF-κB activation.
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Carty, DJ, DL Freas, and AR Gear. "ADP causes subsecond changes in protein phosphorylation of platelets." Blood 70, no. 2 (August 1, 1987): 511–15. http://dx.doi.org/10.1182/blood.v70.2.511.511.
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Abstract We developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 47-kiloDalton (kD) proteins was analyzed during the first 5 seconds of platelet response to ADP from 0.5 to 10.0 mumol/L and compared with the progress of aggregation. The onset time for aggregation and phosphorylation of both proteins was less than 1 second; 20-K phosphorylation was increased greater than 200% and 47-K phosphorylation was increased 50%. The ADP sensitivity of 20-K phosphorylation was greater than that of 47-K phosphorylation (P less than .025), and of that of aggregation (P less than .01), with Ka values of 0.7, 1.0, and 1.2 mumol/L of ADP, respectively. The cyclooxygenase inhibitor indomethacin had no effect on aggregation, but inhibited both phosphorylations. Its inhibition of 20-K phosphorylation was greater than that of 47-K phosphorylation. Platelet activation by ADP thus induced biochemical changes well before 1 second. The quenched- flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet activation.
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Carty, DJ, DL Freas, and AR Gear. "ADP causes subsecond changes in protein phosphorylation of platelets." Blood 70, no. 2 (August 1, 1987): 511–15. http://dx.doi.org/10.1182/blood.v70.2.511.bloodjournal702511.
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We developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 47-kiloDalton (kD) proteins was analyzed during the first 5 seconds of platelet response to ADP from 0.5 to 10.0 mumol/L and compared with the progress of aggregation. The onset time for aggregation and phosphorylation of both proteins was less than 1 second; 20-K phosphorylation was increased greater than 200% and 47-K phosphorylation was increased 50%. The ADP sensitivity of 20-K phosphorylation was greater than that of 47-K phosphorylation (P less than .025), and of that of aggregation (P less than .01), with Ka values of 0.7, 1.0, and 1.2 mumol/L of ADP, respectively. The cyclooxygenase inhibitor indomethacin had no effect on aggregation, but inhibited both phosphorylations. Its inhibition of 20-K phosphorylation was greater than that of 47-K phosphorylation. Platelet activation by ADP thus induced biochemical changes well before 1 second. The quenched- flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet activation.
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Kabachnik, M. I., L. S. Zakharov, E. I. Goryunov, and I. Yu Kudryavtsev. "Catalytic phosphorylation of polyfluoroalkanols. 11. ?-Polyfluoroalkylbenzyldichlorophosphates as phosphorylating agents in the catalytic phosphorylation of primary polyfluoroalkanols." Bulletin of the Academy of Sciences of the USSR Division of Chemical Science 38, no. 7 (July 1989): 1522–26. http://dx.doi.org/10.1007/bf00978451.
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Langlais, Paul, Zhengping Yi, and Lawrence J. Mandarino. "The Identification of Raptor as a Substrate for p44/42 MAPK." Endocrinology 152, no. 4 (February 15, 2011): 1264–73. http://dx.doi.org/10.1210/en.2010-1271.
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Abstract The adaptor protein raptor is the functional identifier for mammalian target of rapamycin (mTOR) complex 1 (mTORC1), acting to target mTOR to specific substrates for phosphorylation and regulation. Using HPLC-electrospray ionization tandem mass spectrometry, we confirmed the phosphorylation of raptor at Ser696, Thr706, Ser721, Ser722, Ser855, Ser859, Ser863, Thr865, Ser877, Ser881, Ser883, and Ser884 and identified Tyr692, Ser699, Thr700, Ser704, Ser854, Ser857, Ser882, Ser886, Ser887, and Thr889 as new, previously unidentified raptor phosphorylation sites. Treatment of cells with insulin increased the phosphorylation of raptor at Ser696, Ser855, Ser863, and Thr865 and suppressed the phosphorylation of Ser722. Ser696 phosphorylation was insensitive to mTOR inhibition with rapamycin, whereas treatment of cells with the MAPK inhibitor PD98059 inhibited the insulin-stimulated phosphorylation of raptor at Ser696. In vitro incubation of raptor with p42 MAPK significantly increased raptor phosphorylation (P < 0.01), whereas phosphorylation of a Ser696Ala mutant was decreased (P < 0.05), suggesting MAPK is capable of directly phosphorylating raptor at Ser696. Mutation of Ser696 to alanine interfered with insulin-stimulated phosphorylation of the mTOR downstream substrate p70S6 kinase. Incubation of cells with the MAPK inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor wortmannin decreased the insulin stimulated phosphorylation of raptor, suggesting that the MAPK and phosphatidylinositol 3-kinase pathways may merge at mTORC1.
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Violin, Jonathan D., Jin Zhang, Roger Y. Tsien, and Alexandra C. Newton. "A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C." Journal of Cell Biology 161, no. 5 (June 2, 2003): 899–909. http://dx.doi.org/10.1083/jcb.200302125.
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Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of ∼10 s. Novel FRET-based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+ and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.
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Vary, Thomas C., and Christopher J. Lynch. "Meal feeding enhances formation of eIF4F in skeletal muscle: role of increased eIF4E availability and eIF4G phosphorylation." American Journal of Physiology-Endocrinology and Metabolism 290, no. 4 (April 2006): E631—E642. http://dx.doi.org/10.1152/ajpendo.00460.2005.
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Feeding promotes protein accretion in skeletal muscle through a stimulation of the mRNA translation initiation phase of protein synthesis either secondarily to nutrient-induced rises in insulin or owing to direct effects of nutrients themselves. The present set of experiments establishes the effects of meal feeding on potential signal transduction pathways that may be important in accelerating mRNA translation initiation. Gastrocnemius muscle from male Sprague-Dawley rats trained to consume a meal consisting of rat chow was sampled before, during, and after the meal. Meal feeding enhanced the assembly of the active eIF4G·eIF4E complex, which returned to basal levels within 3 h of removal of food. The increased assembly of the active eIF4G·eIF4E complex was associated with a marked 10-fold rise in phosphorylation of eIF4G(Ser1108) and a decreased assembly of inactive 4E-BP1·eIF4E complex. The reduced assembly of 4E-BP1·eIF4E complex was associated with a 75-fold increase in phosphorylation of 4E-BP1 in the γ-form during feeding. Phosphorylation of S6K1 on Ser789 was increased by meal feeding, although the extent of phosphorylation was greater at 0.5 h after feeding than after 1 h. Phosphorylation of mammalian target of rapamycin (mTOR) on Ser2448 or Ser2481, an upstream kinase responsible for phosphorylating both S6K1 and 4E-BP1, was increased at all times during meal feeding, although the extent of phosphorylation was greater at 0.5 h after feeding than after 1 h. Phosphorylation of PKB, an upstream kinase responsible for phosphorylating mTOR, was elevated only after 0.5 h of meal feeding for Thr308, whereas phosphorylation Ser473 was significantly elevated at only 0.5 and 1 h after initiation of feeding. We conclude from these studies that meal feeding stimulates two signal pathways in skeletal muscle that lead to elevated eIF4G·eIF4E complex assembly through increased phosphorylation of eIF4G and decreased association of 4E-BP1 with eIF4E.
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Norling, L. L., and M. Landt. "Comparison of Ca2+-dependent phosphorylation in viable dispersed brain cells with calmodulin-dependent protein kinase activity in cell-free preparations of rat brain." Biochemical Journal 232, no. 3 (December 15, 1985): 629–35. http://dx.doi.org/10.1042/bj2320629.
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Using two depolarizing agents, veratrine and high concentrations of extracellular KCl, we studied depolarization-stimulated phosphorylations in 32P-labelled dispersed brain tissue in order to identify phosphoprotein substrates for Ca2+ - and calmodulin-dependent protein kinase activity at the cellular level, for comparison with findings in cell-free preparations. In intact brain cells, the only prominent depolarization-stimulated phosphorylation was a 77 kDa protein separated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This phosphorylation was dependent on external Ca2+, since chelation of Ca2+ in media with 6 mM-EGTA or the presence of verapamil (a Ca2+ -channel blocker) in the incubation media inhibited depolarization-stimulated phosphorylation of the 77 kDa protein. Phosphorylation of the 77 kDa protein also appeared to be dependent on calmodulin, because depolarization-stimulated phosphorylation was significantly decreased (P less than 0.05) when 100 microM-trifluoperazine was present in the incubation media. Polymyxin B, an inhibitor of Ca2+- and phospholipid-dependent phosphorylation, and 12-O-tetradecanoylphorbol 13-acetate, the phorbol ester enhancing Ca2+- and phospholipid-dependent phosphorylation, had no effect on the phosphorylation of the 77 kDa protein. The 77 kDa phosphoprotein was identified as a protein previously named synapsin I [Ueda, Maeno & Greengard (1973) J. Biol. Chem 248, 8295-8305] on the basis of similar migration of native and proteolytic fragments of the 77 kDa protein with those of authentic synapsin I on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Whereas several studies with cell-free preparations showed that 57 kDa and 54 kDa endogenous phosphoproteins were the most prominent species phosphorylated in a Ca2+ and calmodulin-dependent manner, these results indicate that synapsin is the most prominent Ca2+-and calmodulin-dependent phosphorylation in intact cells. The phosphorylations of 54 kDa and 57 kDa proteins may not be as important in vivo, but instead occur as a result of the disruption of cellular integrity inherent in preparation of cell-free subfractions of brain tissue.
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Dusi, S., M. Donini, and F. Rossi. "Tyrosine phosphorylation and activation of NADPH oxidase in human neutrophils: a possible role for MAP kinases and for a 75 kDa protein." Biochemical Journal 304, no. 1 (November 15, 1994): 243–50. http://dx.doi.org/10.1042/bj3040243.
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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.
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KNEBEL, Axel, Claire E. HAYDON, Nick MORRICE, and Philip COHEN. "Stress-induced regulation of eukaryotic elongation factor 2 kinase by SB 203580-sensitive and −insensitive pathways." Biochemical Journal 367, no. 2 (October 15, 2002): 525–32. http://dx.doi.org/10.1042/bj20020916.
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Eukaryotic elongation factor 2 (eEF2) kinase, the enzyme that inactivates eEF2, is controlled by phosphorylation. Previous work showed that stress-activated protein kinase 4 (SAPK4, also called p38Δ) inhibits eEF2 kinase in vitro by phosphorylating Ser-359, while ribosomal protein S6 kinases inhibit eEF2 kinase by phosphorylating Ser-366 [Knebel, Morrice and Cohen (2001) EMBO J. 20, 4360—4369; Wang, Li, Williams, Terada, Alessi and Proud (2001) EMBO J. 20, 4370—4379]. In the present study we have examined the effects of the protein synthesis inhibitor anisomycin and tumour necrosis factor-α (TNF-α) on the phosphorylation of eEF2 kinase. We demonstrate that Ser-359, Ser-366 and two novel sites (Ser-377 and Ser-396) are all phosphorylated in human epithelial KB cells, but only the phosphorylation of Ser-359 and Ser-377 increases in response to these agonists and correlates with the dephosphorylation (activation) of eEF2. Ser-377 is probably a substrate of MAPKAP-K2/K3 (mitogen-activated protein kinase-activated protein kinase 2/kinase 3) in cells, because eEF2 kinase is phosphorylated efficiently by these protein kinases in vitro and phosphorylation of this site, induced by TNF-α and low (but not high) concentrations of anisomycin, is prevented by SB 203580, which inhibits SAPK2a/p38, their ‘upstream’ activator. The phosphorylation of Ser-359 induced by high concentrations of anisomycin is probably catalysed by SAPK4/p38Δ in cells, because no other stress-activated, proline-directed protein kinase tested phosphorylates this site in vitro and phosphorylation is insensitive to SB 203580. Interestingly, the phosphorylation of Ser-359 induced by TNF-α or low concentrations of anisomycin is suppressed by SB 203580, indicating that phosphorylation is also mediated by a novel pathway. Since the phosphorylation of Ser-377 does not inhibit eEF2 kinase in vitro, our results suggest that anisomycin or TNF-α inhibit eEF2 kinase via the phosphorylation of Ser-359.
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Harper, Mary-Ellen, and Martin D. Brand. "Hyperthyroidism stimulates mitochondrial proton leak and ATP turnover in rat hepatocytes but does not change the overall kinetics of substrate oxidation reactions." Canadian Journal of Physiology and Pharmacology 72, no. 8 (August 1, 1994): 899–908. http://dx.doi.org/10.1139/y94-127.
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Thyroid hormones have well-known effects on oxidative phosphorylation, but there is little quantitative information on their important sites of action. We have used top-down elasticity analysis, an extension of metabolic control analysis, to identify the sites of action of thyroid hormones on oxidative phosphorylation in rat hepatocytes. We divided the oxidative phosphorylation system into three blocks of reactions: the substrate oxidation subsystem, the phosphorylating subsystem, and the mitochondrial proton leak subsystem and have identified those blocks of reactions whose kinetics are significantly changed by hyperthyroidism. Our results show significant effects on the kinetics of the proton leak and the phosphorylating subsystems. Quantitative analyses revealed that 43% of the increase in resting respiration rate in hyperthyroid hepatocytes compared with euthyroid hepatocytes was due to differences in the proton leak and 59% was due to differences in the activity of the phosphorylating subsystem. There were no significant effects on the substrate oxidation subsystem. Changes in nonmitochondrial oxygen consumption accounted for −2% of the change in respiration rate. Top-down control analysis revealed that the distribution of control over the rates of mitochondrial oxygen consumption, ATP synthesis and consumption, and proton leak and over mitochondrial membrane potential (Δψm) was similar in hepatocytes from hyperthyroid and littermate-paired euthyroid controls. The results of this study include the first complete top-down elasticity and control analyses of oxidative phosphorylation in hepatocytes from hyperthyroid rats.Key words: thyroid hormones, oxidative phosphorylation, mitochondria, proton leak, thermogenesis.
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Cohen, M. E., G. W. Sharp, and M. Donowitz. "Suggestion of a role for calmodulin and phosphorylation in regulation of rabbit ileal electrolyte transport: effects of promethazine." American Journal of Physiology-Gastrointestinal and Liver Physiology 251, no. 5 (November 1, 1986): G710—G717. http://dx.doi.org/10.1152/ajpgi.1986.251.5.g710.
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Suggestion of a role for protein phosphorylation in the regulation of intestinal active NaCl transport was found by studying the effects of low concentrations of promethazine on Ca2+-calmodulin (CaM)-dependent protein phosphorylation of ileal microvillus membranes and on active ileal electrolyte transport. Ca2+-CaM increased the phosphorylation of six microvillus peptides (Mr 137,000, 116,000, 77,000, 58,000, 53,000, and 50,000) in a concentration-dependent manner. Promethazine inhibited the Ca2+-CaM-induced increases in each of these phosphorylations. The effect of promethazine was concentration dependent, with concentrations of 5–12 microM (mean 8 microM) causing 50% inhibition. Promethazine also caused a concentration-dependent increase in net Cl absorption and decrease in the ileal short-circuit current, with 9 microM promethazine causing a change in short-circuit current 50% of maximum. The promethazine effect on microvillus membrane phosphorylation was specific, since neither cAMP- and cGMP-induced phosphorylation in the microvillus membrane nor the stimulation by Ca2+-CaM of myosin light chain kinase phosphorylation of myosin light chain were affected by promethazine. The similar, and unusual sensitivity to low concentrations of promethazine on ileal microvillus membrane phosphorylation increased by Ca2+-CaM and on ileal electrolyte transport is consistent with Ca2+-CaM-dependent microvillus membrane phosphorylation being involved in the regulation of active electrolyte transport in ileal absorptive cells.
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Geraghty, Kathryn M., Shuai Chen, Jean E. Harthill, Adel F. Ibrahim, Rachel Toth, Nick A. Morrice, Franck Vandermoere, Greg B. Moorhead, D. Grahame Hardie, and Carol MacKintosh. "Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR." Biochemical Journal 407, no. 2 (September 25, 2007): 231–41. http://dx.doi.org/10.1042/bj20070649.
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AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA–AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.
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Solomon, M. J., T. Lee, and M. W. Kirschner. "Role of phosphorylation in p34cdc2 activation: identification of an activating kinase." Molecular Biology of the Cell 3, no. 1 (January 1992): 13–27. http://dx.doi.org/10.1091/mbc.3.1.13.
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Phosphorylation of p34cdc2 can both positively and negatively regulate its kinase activity. We have mapped two phosphorylation sites in Xenopus p34cdc2 to Thr-14 and Tyr-15 within the putative ATP-binding region of p34cdc2. Mutation of these sites to Ala-14 and Phe-15 has no effect on the final histone H1 kinase activity of the cyclin/p34cdc2 complex. Phosphopeptide analysis shows that there is at least one more site of phosphorylation on p34cdc2. When Thr-161 is changed to Ala, two phosphopeptide spots disappear and it is no longer possible to activate the H1 kinase activity of p34cdc2. We suggest that Thr-161 is a third site of phosphorylation, which is required for kinase activity. All three phosphorylations are induced by cyclin. None of the phosphorylations appears to be required for binding to cyclin, as indicated by the ability of the triple mutant, Ala-14, Phe-15, Ala-161, to bind cyclin. The activating phosphorylation that requires Thr- or Ser-161 occurs even in a catalytically inactive K33R mutant of p34cdc2 and hence does not appear to be the result of intramolecular autophosphorylation. We have detected an activity in Xenopus extracts required for activation of p34cdc2 and present evidence that this is a p34cdc2 activating kinase which, in a cyclin-dependent manner, probably directly phosphorylates Thr-161.
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Scheid, Michael P., Paola A. Marignani, and James R. Woodgett. "Multiple Phosphoinositide 3-Kinase-Dependent Steps in Activation of Protein Kinase B." Molecular and Cellular Biology 22, no. 17 (September 1, 2002): 6247–60. http://dx.doi.org/10.1128/mcb.22.17.6247-6260.2002.
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ABSTRACT The protein kinase B (PKB)/Akt family of serine kinases is rapidly activated following agonist-induced stimulation of phosphoinositide 3-kinase (PI3K). To probe the molecular events important for the activation process, we employed two distinct models of posttranslational inducible activation and membrane recruitment. PKB induction requires phosphorylation of two critical residues, threonine 308 in the activation loop and serine 473 near the carboxyl terminus. Membrane localization of PKB was found to be a primary determinant of serine 473 phosphorylation. PI3K activity was equally important for promoting phosphorylation of serine 473, but this was separable from membrane localization. PDK1 phosphorylation of threonine 308 was primarily dependent upon prior serine 473 phosphorylation and, to a lesser extent, localization to the plasma membrane. Mutation of serine 473 to alanine or aspartic acid modulated the degree of threonine 308 phosphorylation in both models, while a point mutation in the substrate-binding region of PDK1 (L155E) rendered PDK1 incapable of phosphorylating PKB. Together, these results suggest a mechanism in which 3′ phosphoinositide lipid-dependent translocation of PKB to the plasma membrane promotes serine 473 phosphorylation, which is, in turn, necessary for PDK1-mediated phosphorylation of threonine 308 and, consequentially, full PKB activation.
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Benes, Cyril, and Stephen P. Soltoff. "Modulation of PKCδ tyrosine phosphorylation and activity in salivary and PC-12 cells by Src kinases." American Journal of Physiology-Cell Physiology 280, no. 6 (June 1, 2001): C1498—C1510. http://dx.doi.org/10.1152/ajpcell.2001.280.6.c1498.
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Protein kinase C (PKC) δ becomes tyrosine phosphorylated in rat parotid acinar cells exposed to muscarinic and substance P receptor agonists, which initiate fluid secretion in this salivary cell. Here we examine the signaling components of PKCδ tyrosine phosphorylation and effects of phosphorylation on PKCδ activity. Carbachol- and substance P-promoted increases in PKCδ tyrosine phosphorylation were blocked by inhibiting phospholipase C (PLC) but not by blocking intracellular Ca2+ concentration elevation, suggesting that diacylglycerol, rather than d- myo-inositol 1,4,5-trisphosphate production, positively modulated this phosphorylation. Stimuli-dependent increases in PKCδ activity in parotid and PC-12 cells were blocked in vivo by inhibitors of Src tyrosine kinases. Dephosphorylation of tyrosine residues by PTP1B, a protein tyrosine phosphatase, reduced the enhanced PKCδ activity. Lipid cofactors modified the tyrosine phosphorylation-dependent PKCδ activation. Two PKCδ regulatory sites (Thr-505 and Ser-662) were constitutively phosphorylated in unstimulated parotid cells, and these phosphorylations were not altered by stimuli that increased PKCδ tyrosine phosphorylation. These results demonstrate that PKCδ activity is positively modulated by tyrosine phosphorylation in parotid and PC-12 cells and suggest that PLC-dependent effects of secretagogues on salivary cells involve Src-related kinases.
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Vendelbo, M. H., A. B. Møller, J. T. Treebak, L. C. Gormsen, L. J. Goodyear, J. F. P. Wojtaszewski, J. O. L. Jørgensen, N. Møller, and N. Jessen. "Sustained AS160 and TBC1D1 phosphorylations in human skeletal muscle 30 min after a single bout of exercise." Journal of Applied Physiology 117, no. 3 (August 1, 2014): 289–96. http://dx.doi.org/10.1152/japplphysiol.00044.2014.
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Background: phosphorylation of AS160 and TBC1D1 plays an important role for GLUT4 mobilization to the cell surface. The phosphorylation of AS160 and TBC1D1 in humans in response to acute exercise is not fully characterized. Objective: to study AS160 and TBC1D1 phosphorylation in human skeletal muscle after aerobic exercise followed by a hyperinsulinemic euglycemic clamp. Design: eight healthy men were studied on two occasions: 1) in the resting state and 2) in the hours after a 1-h bout of ergometer cycling. A hyperinsulinemic euglycemic clamp was initiated 240 min after exercise and in a time-matched nonexercised control condition. We obtained muscle biopsies 30 min after exercise and in a time-matched nonexercised control condition ( t = 30) and after 30 min of insulin stimulation ( t = 270) and investigated site-specific phosphorylation of AS160 and TBC1D1. Results: phosphorylation on AS160 and TBC1D1 was increased 30 min after the exercise bout, whereas phosphorylation of the putative upstream kinases, Akt and AMPK, was unchanged compared with resting control condition. Exercise augmented insulin-stimulated phosphorylation on AS160 at Ser341and Ser704270 min after exercise. No additional exercise effects were observed on insulin-stimulated phosphorylation of Thr642and Ser588on AS160 or Ser237and Thr596on TBC1D1. Conclusions: AS160 and TBC1D1 phosphorylations were evident 30 min after exercise without simultaneously increased Akt and AMPK phosphorylation. Unlike TBC1D1, insulin-stimulated site-specific AS160 phosphorylation is modified by prior exercise, but these sites do not include Thr642and Ser588. Together, these data provide new insights into phosphorylation of key regulators of glucose transport in human skeletal muscle.
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Gaplovska-Kysela, Katarina, and Andrea Sevcovicova. "Phosphorylation." Cell Cycle 12, no. 5 (March 2013): 716. http://dx.doi.org/10.4161/cc.23910.
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BABY, Y., M. TSUHAKO, and N. YOZA. "ChemInform Abstract: Phosphorylation of Biomolecules with Inorganic Phosphorylating Agents." ChemInform 25, no. 25 (August 19, 2010): no. http://dx.doi.org/10.1002/chin.199425285.
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Greiwe, Julia F., Thomas C. R. Miller, Julia Locke, Fabrizio Martino, Steven Howell, Anne Schreiber, Andrea Nans, John F. X. Diffley, and Alessandro Costa. "Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase." Nature Structural & Molecular Biology 29, no. 1 (December 28, 2021): 10–20. http://dx.doi.org/10.1038/s41594-021-00698-z.
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AbstractLoading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.
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Almagor, Lior, Ivan S. Ufimtsev, Aruna Ayer, Jingzhi Li, and William I. Weis. "Structural insights into the aPKC regulatory switch mechanism of the human cell polarity protein lethal giant larvae 2." Proceedings of the National Academy of Sciences 116, no. 22 (May 14, 2019): 10804–12. http://dx.doi.org/10.1073/pnas.1821514116.
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Metazoan cell polarity is controlled by a set of highly conserved proteins. Lethal giant larvae (Lgl) functions in apical-basal polarity through phosphorylation-dependent interactions with several other proteins as well as the plasma membrane. Phosphorylation of Lgl by atypical protein kinase C (aPKC), a component of the partitioning-defective (Par) complex in epithelial cells, excludes Lgl from the apical membrane, a crucial step in the establishment of epithelial cell polarity. We present the crystal structures of human Lgl2 in both its unphosphorylated and aPKC-phosphorylated states. Lgl2 adopts a double β-propeller structure that is unchanged by aPKC phosphorylation of an unstructured loop in its second β-propeller, ruling out models of phosphorylation-dependent conformational change. We demonstrate that phosphorylation controls the direct binding of purified Lgl2 to negative phospholipids in vitro. We also show that a coil–helix transition of this region that is promoted by phosphatidylinositol 4,5-bisphosphate (PIP2) is also phosphorylation-dependent, implying a highly effective phosphorylative switch for membrane association.
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Her, J. H., S. Lakhani, K. Zu, J. Vila, P. Dent, T. W. Sturgill, and M. J. Weber. "Dual phosphorylation and autophosphorylation in mitogen-activated protein (MAP) kinase activation." Biochemical Journal 296, no. 1 (November 15, 1993): 25–31. http://dx.doi.org/10.1042/bj2960025.
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p42mapk [mitogen activated protein (MAP) kinase; extracellular signal-regulated protein kinase (ERK)] is a serine/threonine-specific protein kinase that is activated by dual tyrosine and threonine phosphorylation in response to diverse agonists. Both the tyrosine and threonine phosphorylations are necessary for full enzymic activity. A MAP kinase activator recently purified and cloned has been shown to be a protein kinase (MAP kinase kinase) that is able to induce the dual phosphorylation of MAP kinase on both the regulatory tyrosine and threonine sites in vitro. In the present paper we have utilized MAP kinase mutants altered in the sites of regulatory phosphorylation to show, both in vivo and in vitro, that phosphorylation of the tyrosine and the threonine can occur independently of one another, with no required order of phosphorylation. We also utilized kinase-defective variants of MAP kinase with mutations in either the ATP-binding loop or the catalytic loop, and obtained data suggesting that the activity or structure of the catalytic loop of MAP kinase plays an important role in its own dual phosphorylation.
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Hamáková, Kateřina, David Potěšil, Ondřej Bernatik, Igor Červenka, Matěj Rádsetoulal, Vitězslav Bryja, and Zbyněk Zdráhal. "Semiquantitative Assessment of Dishevelled-3 Phosphorylation Status by Mass Spectrometry." Hungarian Journal of Industry and Chemistry 46, no. 1 (July 1, 2018): 3–6. http://dx.doi.org/10.1515/hjic-2018-0002.
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Abstract The focus of this paper is the human Dishevelled 3 protein (hDvl3), an essential component of the Wnt signalling pathway that contributes to their regulation. Mass spectrometry-based analysis of hDvl3 phosphorylations induced by eight associated kinases was performed revealing several dozens of phosphorylation sites. The main outcome of this study was the description of Dvl phosphorylation “patterns” induced by individual kinases
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Maik-Rachline, Galia, Shmuel Shaltiel, and Rony Seger. "Extracellular phosphorylation converts pigment epithelium–derived factor from a neurotrophic to an antiangiogenic factor." Blood 105, no. 2 (January 15, 2005): 670–78. http://dx.doi.org/10.1182/blood-2004-04-1569.
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Abstract The pigment epithelium–derived factor (PEDF) belongs to the superfamily of serine protease inhibitors (serpin). There have been 2 distinct functions attributed to this factor, which can act either as a neurotrophic or as an antiangiogenic factor. Besides its localization in the eye, PEDF was recently reported to be present also in human plasma. We found that PEDF purified from plasma is a phosphoprotein, which is extracellularly phosphorylated by protein kinase CK2 (CK2) and to a lesser degree, intracellularly, by protein kinase A (PKA). CK2 phosphorylates PEDF on 2 main residues, Ser24 and Ser114, and PKA phosphorylates PEDF on one residue only, Ser227. The physiologic relevance of these phosphorylations was determined using phosphorylation site mutants. We found that both CK2 and PKA phosphorylations of PEDF markedly affect its physiologic function. The fully CK2 phosphorylation site mutant S24, 114E abolished PEDF neurotrophic activity but enhanced its antiangiogenic activity, while the PKA phosphorylation site mutant S227E reduced PEDF antiangiogenic activity. This is a novel role of extracellular phosphorylation that is shown here to completely change the nature of PEDF from a neutrophic to an antiangiogenic factor.
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Amano, Mutsuki, Yoko Kanazawa, Kei Kozawa, and Kozo Kaibuchi. "Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase." Biomolecules 12, no. 2 (January 18, 2022): 159. http://dx.doi.org/10.3390/biom12020159.
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Protein kinases exert physiological functions through phosphorylating their specific substrates; however, the mode of kinase–substrate recognition is not fully understood. Rho-kinase is a Ser/Thr protein kinase that regulates cytoskeletal reorganization through phosphorylating myosin light chain (MLC) and the myosin phosphatase targeting subunit 1 (MYPT1) of MLC phosphatase (MLCP) and is involved in various diseases, due to its aberrant cellular contraction, morphology, and movement. Despite the importance of the prediction and identification of substrates and phosphorylation sites, understanding of the precise regularity in phosphorylation preference of Rho-kinase remains far from satisfactory. Here we analyzed the Rho-kinase–MYPT1 interaction, to understand the mode of Rho-kinase substrate recognition and found that the three short regions of MYPT1 close to phosphorylation sites (referred to as docking motifs (DMs); DM1 (DLQEAEKTIGRS), DM2 (KSQPKSIRERRRPR), and DM3 (RKARSRQAR)) are important for interactions with Rho-kinase. The phosphorylation levels of MYPT1 without DMs were reduced, and the effects were limited to the neighboring phosphorylation sites. We further demonstrated that the combination of pseudosubstrate (PS) and DM of MYPT1 (PS1 + DM3 and PS2 + DM2) serves as a potent inhibitor of Rho-kinase. The present information will be useful in identifying new substrates and developing selective Rho-kinase inhibitors.
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Zheng, Yupeng, Sam John, James J. Pesavento, Jennifer R. Schultz-Norton, R. Louis Schiltz, Sonjoon Baek, Ann M. Nardulli, Gordon L. Hager, Neil L. Kelleher, and Craig A. Mizzen. "Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II." Journal of Cell Biology 189, no. 3 (May 3, 2010): 407–15. http://dx.doi.org/10.1083/jcb.201001148.
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Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. In this study, we show that specific sites in H1.2 and H1.4 of human cells are phosphorylated only during mitosis or during both mitosis and interphase. Antisera generated to individual H1.2/H1.4 interphase phosphorylations reveal that they are distributed throughout nuclei and enriched in nucleoli. Moreover, interphase phosphorylated H1.4 is enriched at active 45S preribosomal RNA gene promoters and is rapidly induced at steroid hormone response elements by hormone treatment. Our results imply that site-specific interphase H1 phosphorylation facilitates transcription by RNA polymerases I and II and has an unanticipated function in ribosome biogenesis and control of cell growth. Differences in the numbers, structure, and locations of interphase phosphorylation sites may contribute to the functional diversity of H1 variants.
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Thornton, Tina, and Mercedes Rincon. "The role of p38 MAPK/GSK3β signaling in T and B lymphocytes undergoing programmed DNA recombination (111.47)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 111.47. http://dx.doi.org/10.4049/jimmunol.188.supp.111.47.
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Abstract In addition to environmentally induced DNA double strand breaks (DSBs), developmentally programmed DSBs are also generated in T and B cells during V(D)J recombination. We have shown that in response to DSBs, p38 MAPK is activated and translocated to the nucleus. Although typically associated with cell death, we found that p38 MAPK promoted survival of cells following DNA damage by phosphorylating GSK3β at a novel residue (S389). GSK3β is a constitutively active kinase that can promote death by phosphorylating and targeting survival factors for destruction. GSK3β has been shown to be inhibited by the phosphorylation of S9 by Akt. Our biochemical analysis showed that phosphorylation of GSK3β at S389 resulted in inhibition of GSK3β similar to the S9 phosphorylation of GSK3β. Here we show that in response to DSBs in the development of T and B cells, activation of p38 MAPK leads to S389 phosphorylation and inactivation of GSK3β. We also show that unlike S9 phosphorylation that occurs in the cytosol, S389 phosphorylation occurs within the nucleus. To investigate the role of this pathway in vivo, we generated knockin mice (KI) where the S389 has been mutated to alanine. Lymphocytes for our KI mice appear to be more sensitive to death in response to DSBs. Our data indicate that GSK3β inactivation by p38 MAPK is selectively induced by DSBs as a mechanism to inhibit GSK3β activity within the nucleus allowing for the accumulation of survival factors.
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Soltys, Carrie-Lynn M., Suzanne Kovacic, and Jason R. B. Dyck. "Activation of cardiac AMP-activated protein kinase by LKB1 expression or chemical hypoxia is blunted by increased Akt activity." American Journal of Physiology-Heart and Circulatory Physiology 290, no. 6 (June 2006): H2472—H2479. http://dx.doi.org/10.1152/ajpheart.01206.2005.
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AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac energy substrate utilization and can be negatively regulated by Akt activation in the heart. It has recently been shown that Akt directly phosphorylates AMPKα1/α2 on Ser485/491 in vitro and prevents the AMPK kinase (AMPKK) LKB1 from phosphorylating AMPKα at its primary activation site, Thr172 (S Horman, D Vertommen, R Heath, D Neumann, V Mouton, A Woods, U Schlattner, T Wallimann, D Carling, L Hue, and MH Rider. J Biol Chem 281: 5335–5340, 2006). To determine whether this is also the case in the cardiac myocyte, neonatal rat cardiac myocytes (NRCM) were infected with a recombinant adenovirus expressing a constitutively active mutant of Akt1 (myrAkt1) and then with or without adenoviruses expressing the active LKB1 complex. Expression of myrAkt1 blunted LKB1-induced phosphorylation of AMPKα at Thr172, which resulted in a dramatic decrease in phosphorylation of AMPK's target, acetyl CoA-carboxylase. This decrease in AMPK activity was associated with prior Akt1-dependent phosphorylation of AMPKα1/α2 at Ser485/491. To investigate whether Akt1 activation was also able to prevent other AMPKKs from phosphorylating AMPKα, we subjected NRCM to chemical hypoxia and noted a marked increase in phosphorylation of AMPKα at Thr172, despite no change in LKB1 activity. NRCM expressing myrAkt1 demonstrated increased phosphorylation of AMPKα1/α2 at Ser485/491 and a complete inhibition of chemical hypoxia-induced phosphorylation of AMPKα at Thr172. Taken together, our data show that activation of Akt1 is able to prevent activation of cardiac AMPK by LKB1 and at least one other AMPKK, likely by prior phosphorylation of AMPKα1/α2 at Ser485/491.
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Lakkireddy, Dr Suresh. "MOLECULAR ADVANCEMENTS IN PROTEIN PHOSPHORYLATION METHODOLOGIES: A RAPID REVIEW." Era's Journal of Medical Research 10, no. 2 (December 2023): 35–38. http://dx.doi.org/10.24041/ejmr2023.33.
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Phosphorylation, the reversible addition of phosphate groups to proteins, plays a pivotal role in regulating cellular processes, including signal transduction, cell cycle progression, and metabolism. Understanding the dynamics of phosphorylation events is crucial for unraveling complex signaling networks and identifying potential therapeutic targets in various diseases. Phosphorylation immunoassays have emerged as powerful tools for the detection and quantification of phosphorylated proteins, enabling researchers to gain insights into intricate cellular signaling pathways. This review provides an in-depth exploration of the recent advances, methodologies, and applications of phosphorylation immunoassays, highlighting their significance in advancing our understanding of cellular signaling.
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Akiyama, T., T. Saito, H. Ogawara, K. Toyoshima, and T. Yamamoto. "Tumor promoter and epidermal growth factor stimulate phosphorylation of the c-erbB-2 gene product in MKN-7 human adenocarcinoma cells." Molecular and Cellular Biology 8, no. 3 (March 1988): 1019–26. http://dx.doi.org/10.1128/mcb.8.3.1019-1026.1988.
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Treatment of human adenocarcinoma MKN-7 cells with epidermal growth factor (EGF) or phorbol tetradecanoate acetate (TPA) stimulated phosphorylation of the c-erbB-2 gene product. EGF induced a rapid increase in phosphotyrosine followed by relatively gradual increases in phosphoserine and phosphothreonine. On the other hand, the TPA-induced increase in phosphorylations occurred exclusively on serine and threonine residues. Tryptic phosphopeptide mapping analysis suggested that treatments with EGF and TPA induced phosphorylation of many common sites in the c-erbB-2 gene product. However, in contrast to TPA, EGF increased the phosphorylation of the c-erbB-2 protein in cells whose protein kinase C had been down-regulated by long-term pretreatment with TPA, suggesting that EGF and TPA induce phosphorylation by different mechanisms. Since the c-erbB-2 gene product did not show detectable EGF-binding activity, phosphorylation of tyrosine of the c-erbB-2 gene product might be catalyzed directly by the EGF receptor kinase that was activated by EGF.
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Akiyama, T., T. Saito, H. Ogawara, K. Toyoshima, and T. Yamamoto. "Tumor promoter and epidermal growth factor stimulate phosphorylation of the c-erbB-2 gene product in MKN-7 human adenocarcinoma cells." Molecular and Cellular Biology 8, no. 3 (March 1988): 1019–26. http://dx.doi.org/10.1128/mcb.8.3.1019.
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Treatment of human adenocarcinoma MKN-7 cells with epidermal growth factor (EGF) or phorbol tetradecanoate acetate (TPA) stimulated phosphorylation of the c-erbB-2 gene product. EGF induced a rapid increase in phosphotyrosine followed by relatively gradual increases in phosphoserine and phosphothreonine. On the other hand, the TPA-induced increase in phosphorylations occurred exclusively on serine and threonine residues. Tryptic phosphopeptide mapping analysis suggested that treatments with EGF and TPA induced phosphorylation of many common sites in the c-erbB-2 gene product. However, in contrast to TPA, EGF increased the phosphorylation of the c-erbB-2 protein in cells whose protein kinase C had been down-regulated by long-term pretreatment with TPA, suggesting that EGF and TPA induce phosphorylation by different mechanisms. Since the c-erbB-2 gene product did not show detectable EGF-binding activity, phosphorylation of tyrosine of the c-erbB-2 gene product might be catalyzed directly by the EGF receptor kinase that was activated by EGF.
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Ahn, Jae Suk, Andrea Musacchio, Marina Mapelli, Jake Ni, Leonard Scinto, Ross Stein, Kenneth S. Kosik, and Li-An Yeh. "Development of an Assay to Screen for Inhibitors of Tau Phosphorylation by Cdk5." Journal of Biomolecular Screening 9, no. 2 (March 2004): 122–31. http://dx.doi.org/10.1177/1087057103260594.
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A high-throughput assay for tau phosphorylation by cdk5/p25 is described. Full-length recombinant tau was used as a substrate in the presence of saturating adenosine triphosphate (ATP). Using PHF-1, an antibody directed specifically against 2 tau phosphorylation epitopes (serine 396 and serine 404), an enzyme-linked immunosorbent assay (ELISA)- based colorimetric assay was formatted in 384-well plates. The assay was validated by measuring kinetic parameters for cdk5/p25 catalysis and known inhibitors. Rate constants for the site-specific phosphorylations at the PHF-1 epitopes were determined and suggested preferential phosphorylation at these sites. The performance of this assay in a high-throughput format was demonstrated and used to identify inhibitors of tau phosphorylation at specific epitopes phosphorylated by cdk5/p25. ( Journal of Biomolecular Screening 2004:122-131)
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Vilimek, Dino, and Vincent Duronio. "Cytokine-stimulated phosphorylation of GSK-3 is primarily dependent upon PKCs, not PKB." Biochemistry and Cell Biology 84, no. 1 (February 1, 2006): 20–29. http://dx.doi.org/10.1139/o05-154.
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The regulation of glycogen synthase kinase-3 (GSK-3) by phosphorylation at inhibitory sites has been well documented. In many, but not all, cases, the phosphatidylinositol 3-kinase pathway, and particularly the downstream kinase protein kinase B (PKB) / akt, have been shown to be responsible for GSK-3 phosphorylation. Given that no studies have ever reported cytokine-mediated phosphorylation of GSK-3, we investigated the phosphorylation of this kinase in several hemopoietic cell types in response to either interleukin (IL)-3, IL-4 or granulocyte-macrophage colony stimulating factor (GM-CSF). Each of the cytokines was able to stimulate phosphorylation of the isoforms GSK-3α and GSK-3β. However, only in the case of IL-4 stimulation was there any dependence on PKB for this phosphorylation. We were clearly able to show that PKB was capable of phosphorylating GSK-3 in these cells, but studies using inhibitors of the protein kinase C (PKC) family of kinases have shown that these enzymes are more likely to play a key role in GSK-3 phosphorylation. Cytokine-mediated generation of diacylglycerol was demonstrated, supporting the possible activation of PKC family members. Thus, cytokine-dependent GSK-3 phosphorylation in hemopoietic cells proceeds primarily through PKB independent pathways.Key words: kinase, regulation, signal transduction, cytokine, apoptosis.
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Ogura, Masato, Junko Yamaki, Miwako K. Homma, and Yoshimi Homma. "Mitochondrial c-Src regulates cell survival through phosphorylation of respiratory chain components." Biochemical Journal 447, no. 2 (September 26, 2012): 281–89. http://dx.doi.org/10.1042/bj20120509.
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Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr193 of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr215 of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.
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Shimasaki, Kentaro, Keigo Kumagai, Shota Sakai, Toshiyuki Yamaji, and Kentaro Hanada. "Hyperosmotic Stress Induces Phosphorylation of CERT and Enhances Its Tethering throughout the Endoplasmic Reticulum." International Journal of Molecular Sciences 23, no. 7 (April 5, 2022): 4025. http://dx.doi.org/10.3390/ijms23074025.
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The ceramide transport protein (CERT) delivers ceramide from the endoplasmic reticulum (ER) to the Golgi apparatus, where ceramide is converted to sphingomyelin (SM). The function of CERT is regulated in two distinct phosphorylation-dependent events: multiple phosphorylations in a serine-repeat motif (SRM) and phosphorylation of serine 315 residue (S315). Pharmacological inhibition of SM biosynthesis results in an increase in SRM-dephosphorylated CERT, which serves as an activated form, and an enhanced phosphorylation of S315, which augments the binding of CERT to ER-resident VAMP-associated protein (VAP), inducing the full activation of CERT to operate at the ER–Golgi membrane contact sites (MCSs). However, it remains unclear whether the two phosphorylation-dependent regulatory events always occur coordinately. Here, we describe that hyperosmotic stress induces S315 phosphorylation without affecting the SRM-phosphorylation state. Under hyperosmotic conditions, the binding of CERT with VAP-A is enhanced in an S315 phosphorylation-dependent manner, and this increased binding occurs throughout the ER rather than restrictedly at the ER–Golgi MCSs. Moreover, we found that de novo synthesis of SM with very-long acyl chains preferentially increases via a CERT-independent mechanism under hyperosmotic-stressed cells, providing an insight into a CERT-independent ceramide transport pathway for de novo synthesis of SM.
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Kurihara, Kinji, Nobuo Nakanishi, Marilyn L. Moore-Hoon, and R. James Turner. "Phosphorylation of the salivary Na+-K+-2Cl− cotransporter." American Journal of Physiology-Cell Physiology 282, no. 4 (April 1, 2002): C817—C823. http://dx.doi.org/10.1152/ajpcell.00352.2001.
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We studied the phosphorylation of the secretory Na+-K+-2Cl− cotransporter (NKCC1) in rat parotid acinar cells. We have previously shown that NKCC1 activity in these cells is dramatically upregulated in response to β-adrenergic stimulation and that this upregulation correlates with NKCC1 phosphorylation, possibly due to protein kinase A (PKA). We show here that when ATP is added to purified acinar basolateral membranes (BLM), NKCC1 is phosphorylated as a result of membrane-associated protein kinase activity. Additional NKCC1 phosphorylation is seen when PKA is added to BLMs, but our data indicate that this is due to an effect of PKA on endogenous membrane kinase or phosphatase activities, rather than its direct phosphorylation of NKCC1. Also, phosphopeptide mapping demonstrates that these phosphorylations do not take place at the site associated with the upregulation of NKCC1 by β-adrenergic stimulation. However, this upregulatory phosphorylation can be mimicked by the addition of cAMP to permeabilized acini, and this effect can be blocked by a specific PKA inhibitor. These latter results provide good evidence that PKA is indeed involved in the upregulatory phosphorylation of NKCC1 and suggest that an additional factor present in the acinar cell but absent from isolated membranes is required to bring about the phosphorylation.
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Matusiak, Magdalena, Nina Van Opdenbosch, Lieselotte Vande Walle, Jean-Claude Sirard, Thirumala-Devi Kanneganti, and Mohamed Lamkanfi. "Flagellin-induced NLRC4 phosphorylation primes the inflammasome for activation by NAIP5." Proceedings of the National Academy of Sciences 112, no. 5 (January 20, 2015): 1541–46. http://dx.doi.org/10.1073/pnas.1417945112.
Full textAbstract:
The Nlrc4 inflammasome contributes to immunity against intracellular pathogens that express flagellin and type III secretion systems, and activating mutations in NLRC4 cause autoinflammation in patients. Both Naip5 and phosphorylation of Nlrc4 at Ser533 are required for flagellin-induced inflammasome activation, but how these events converge upon inflammasome activation is not known. Here, we showed that Nlrc4 phosphorylation occurs independently of Naip5 detection of flagellin because Naip5 deletion in macrophages abolished caspase-1 activation, interleukin (IL)-1β secretion, and pyroptosis, but not Nlrc4 phosphorylation by cytosolic flagellin of Salmonella Typhimurium and Yersinia enterocolitica. ASC speck formation and caspase-1 expression also were dispensable for Nlrc4 phosphorylation. Interestingly, Helicobacter pylori flagellin triggered robust Nlrc4 phosphorylation, but failed to elicit caspase-1 maturation, IL-1β secretion, and pyroptosis, suggesting that it retained Nlrc4 Ser533 phosphorylating-activity despite escaping Naip5 detection. In agreement, the flagellin D0 domain was required and sufficient for Nlrc4 phosphorylation, whereas deletion of the S. Typhimurium flagellin carboxy-terminus prevented caspase-1 maturation only. Collectively, this work suggests a biphasic activation mechanism for the Nlrc4 inflammasome in which Ser533 phosphorylation prepares Nlrc4 for subsequent activation by the flagellin sensor Naip5.
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Song, Weimeng, Li Hu, Zhihui Ma, Lei Yang, and Jianming Li. "Importance of Tyrosine Phosphorylation in Hormone-Regulated Plant Growth and Development." International Journal of Molecular Sciences 23, no. 12 (June 13, 2022): 6603. http://dx.doi.org/10.3390/ijms23126603.
Full textAbstract:
Protein phosphorylation is the most frequent post-translational modification (PTM) that plays important regulatory roles in a wide range of biological processes. Phosphorylation mainly occurs on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues, with the phosphorylated Tyr sites accounting for ~1–2% of all phosphorylated residues. Tyr phosphorylation was initially believed to be less common in plants compared to animals; however, recent investigation indicates otherwise. Although they lack typical protein Tyr kinases, plants possess many dual-specificity protein kinases that were implicated in diverse cellular processes by phosphorylating Ser, Thr, and Tyr residues. Analyses of sequenced plant genomes also identified protein Tyr phosphatases and dual-specificity protein phosphatases. Recent studies have revealed important regulatory roles of Tyr phosphorylation in many different aspects of plant growth and development and plant interactions with the environment. This short review summarizes studies that implicated the Tyr phosphorylation in biosynthesis and signaling of plant hormones.
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Tinsley, John H., Elena E. Ustinova, Wenjuan Xu, and Sarah Y. Yuan. "Src-dependent, neutrophil-mediated vascular hyperpermeability and β-catenin modification." American Journal of Physiology-Cell Physiology 283, no. 6 (December 1, 2002): C1745—C1751. http://dx.doi.org/10.1152/ajpcell.00230.2002.
Full textAbstract:
The hyperpermeability response of microvessels in inflammation involves complex signaling reactions and structural modifications in the endothelium. Our goal was to determine the role of Src-family kinases (Src) in neutrophil-mediated venular hyperpermeability and possible interactions between Src and endothelial barrier components. We found that inhibition of Src abolished the increases in albumin permeability caused by C5a-activated neutrophils in intact, perfused coronary venules, as well as in cultured endothelial monolayers. Activated neutrophils increased Src phosphorylation at Tyr416, which is located in the catalytic domain, and decreased phosphorylation at Tyr527 near the carboxyl terminus, events consistent with reports that phosphorylating and transforming activities of Src are upregulated by Tyr416 phosphorylation and negatively regulated by Tyr527 phosphorylation. Furthermore, neutrophil stimulation resulted in association of Src with the endothelial junction protein β-catenin and β-catenin tyrosine phosphorylation. These phenomena were abolished by blockage of Src activity. Taken together, our studies link for the first time neutrophil-induced hyperpermeability to a pathway involving Src kinase activation, Src/β-catenin association, and β-catenin tyrosine phosphorylation in the microvascular endothelium.
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Holt, K. H., B. G. Kasson, and J. E. Pessin. "Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase." Molecular and Cellular Biology 16, no. 2 (February 1996): 577–83. http://dx.doi.org/10.1128/mcb.16.2.577.
Full textAbstract:
The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.
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Bishop, R., R. Martinez, M. J. Weber, P. J. Blackshear, S. Beatty, R. Lim, and H. R. Herschman. "Protein phosphorylation in a tetradecanoyl phorbol acetate-nonproliferative variant of 3T3 cells." Molecular and Cellular Biology 5, no. 9 (September 1985): 2231–37. http://dx.doi.org/10.1128/mcb.5.9.2231-2237.1985.
Full textAbstract:
The 3T3-TNR9 cell line is a variant of Swiss 3T3 cells which does not respond mitogenically to tumor promoters, but does respond mitogenically to epidermal growth factor, fibroblast growth factor, and serum. To elucidate differences between tumor promoters and polypeptide mitogens in the pathway(s) of mitogenesis which might be responsible for the nonresponsiveness of the 3T3-TNR9 cells, we have examined in these cells the early protein phosphorylation events known to be associated with mitogenesis in the parental 3T3 cells. We find that the 3T3-TNR9 cells display levels of tetradecanoyl phorbol acetate binding and of a calcium- and phospholipid-dependent protein kinase activity which are at least the equal of those seen in the parental 3T3 cells, implicating some postreceptor event in the nonmitogenic phenotype. In addition, we find that phosphorylation of the epidermal growth factor receptor and of 80-kDa and 22-kDa proteins, as well as the tyrosine phosphorylation of a 42-kDa protein, all proceed normally in the nonmitogenic variant, even though these phosphorylations must depend on the activation of different kinases. Thus, all these early phosphorylation reactions are intact in the 3T3-TNR9 cells. Although these phosphorylations may be necessary, they clearly are insufficient to trigger mitogenesis.
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Bishop, R., R. Martinez, M. J. Weber, P. J. Blackshear, S. Beatty, R. Lim, and H. R. Herschman. "Protein phosphorylation in a tetradecanoyl phorbol acetate-nonproliferative variant of 3T3 cells." Molecular and Cellular Biology 5, no. 9 (September 1985): 2231–37. http://dx.doi.org/10.1128/mcb.5.9.2231.
Full textAbstract:
The 3T3-TNR9 cell line is a variant of Swiss 3T3 cells which does not respond mitogenically to tumor promoters, but does respond mitogenically to epidermal growth factor, fibroblast growth factor, and serum. To elucidate differences between tumor promoters and polypeptide mitogens in the pathway(s) of mitogenesis which might be responsible for the nonresponsiveness of the 3T3-TNR9 cells, we have examined in these cells the early protein phosphorylation events known to be associated with mitogenesis in the parental 3T3 cells. We find that the 3T3-TNR9 cells display levels of tetradecanoyl phorbol acetate binding and of a calcium- and phospholipid-dependent protein kinase activity which are at least the equal of those seen in the parental 3T3 cells, implicating some postreceptor event in the nonmitogenic phenotype. In addition, we find that phosphorylation of the epidermal growth factor receptor and of 80-kDa and 22-kDa proteins, as well as the tyrosine phosphorylation of a 42-kDa protein, all proceed normally in the nonmitogenic variant, even though these phosphorylations must depend on the activation of different kinases. Thus, all these early phosphorylation reactions are intact in the 3T3-TNR9 cells. Although these phosphorylations may be necessary, they clearly are insufficient to trigger mitogenesis.
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Drepper, Friedel, Jacek Biernat, Senthilvelrajan Kaniyappan, Helmut E. Meyer, Eva Maria Mandelkow, Bettina Warscheid, and Eckhard Mandelkow. "A combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications." Journal of Biological Chemistry 295, no. 52 (October 26, 2020): 18213–25. http://dx.doi.org/10.1074/jbc.ra120.015882.
Full textAbstract:
Abnormal changes of neuronal Tau protein, such as phosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer's disease. Abnormal phosphorylation is thought to precede aggregation and therefore to promote aggregation, but the nature and extent of phosphorylation remain ill-defined. Tau contains ∼85 potential phosphorylation sites, which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, methodological limitations (e.g. in MS of phosphopeptides, or antibodies against phosphoepitopes) led to conflicting results regarding the extent of Tau phosphorylation in cells. Here we present results from a new approach based on native MS of intact Tau expressed in eukaryotic cells (Sf9). The extent of phosphorylation is heterogeneous, up to ∼20 phosphates per molecule distributed over 51 sites. The medium phosphorylated fraction Pm showed overall occupancies of ∼8 Pi (± 5) with a bell-shaped distribution; the highly phosphorylated fraction Ph had 14 Pi (± 6). The distribution of sites was highly asymmetric (with 71% of all P-sites in the C-terminal half of Tau). All sites were on Ser or Thr residues, but none were on Tyr. Other known posttranslational modifications were near or below our detection limit (e.g. acetylation, ubiquitination). These findings suggest that normal cellular Tau shows a remarkably high extent of phosphorylation, whereas other modifications are nearly absent. This implies that abnormal phosphorylations at certain sites may not affect the extent of phosphorylation significantly and do not represent hyperphosphorylation. By implication, the pathological aggregation of Tau is not likely a consequence of high phosphorylation.
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L'Allemain, G., J. H. Her, J. Wu, T. W. Sturgill, and M. J. Weber. "Growth factor-induced activation of a kinase activity which causes regulatory phosphorylation of p42/microtubule-associated protein kinase." Molecular and Cellular Biology 12, no. 5 (May 1992): 2222–29. http://dx.doi.org/10.1128/mcb.12.5.2222-2229.1992.
Full textAbstract:
p42/microtubule-associated protein kinase (p42mapk) is activated by tyrosine and threonine phosphorylation, and its regulatory phosphorylation is likely to be important in signalling pathways involved in growth control, secretion, and differentiation. Here we show that treatment of quiescent 3T3 cells with diverse agonists results in the appearance of an activity capable of causing the in vitro phosphorylation of p42mapk on the regulatory tyrosine and to a lesser extent on the regulatory threonine, resulting in enzymatic activation of the p42mapk. This p42mapk-activating activity is capable of phosphorylating a kinase-defective p42mapk mutant, thus confirming its activity as a kinase.
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L'Allemain, G., J. H. Her, J. Wu, T. W. Sturgill, and M. J. Weber. "Growth factor-induced activation of a kinase activity which causes regulatory phosphorylation of p42/microtubule-associated protein kinase." Molecular and Cellular Biology 12, no. 5 (May 1992): 2222–29. http://dx.doi.org/10.1128/mcb.12.5.2222.
Full textAbstract:
p42/microtubule-associated protein kinase (p42mapk) is activated by tyrosine and threonine phosphorylation, and its regulatory phosphorylation is likely to be important in signalling pathways involved in growth control, secretion, and differentiation. Here we show that treatment of quiescent 3T3 cells with diverse agonists results in the appearance of an activity capable of causing the in vitro phosphorylation of p42mapk on the regulatory tyrosine and to a lesser extent on the regulatory threonine, resulting in enzymatic activation of the p42mapk. This p42mapk-activating activity is capable of phosphorylating a kinase-defective p42mapk mutant, thus confirming its activity as a kinase.