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Kenneth B. Storey - One of the best experts on this subject based on the ideXlab platform.
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a hydrogen peroxide safety valve the Reversible Phosphorylation of catalase from the freeze tolerant north american wood frog rana sylvatica
Biochimica et Biophysica Acta, 2016Co-Authors: Neal J. Dawson, Kenneth B. StoreyAbstract:Abstract Background The North American wood frog, Rana sylvatica , endures whole body freezing while wintering on land and has developed multiple biochemical adaptations to elude cell/tissue damage and optimize its freeze tolerance. Blood flow is halted in the frozen state, imparting both ischemic and oxidative stress on cells. A potential build-up of H 2 O 2 may occur due to increased superoxide dismutase activity previously discovered. The effect of freezing on catalase (CAT), which catalyzes the breakdown of H 2 O 2 into molecular oxygen and water, was investigated as a result. Methods The present study investigated the purification and kinetic profile of CAT in relation to the Phosphorylation state of CAT from the skeletal muscle of control and frozen R. sylvatica . Results Catalase from skeletal muscle of frozen wood frogs showed a significantly higher V max (1.48 fold) and significantly lower K m for H 2 O 2 (0.64 fold) in comparison to CAT from control frogs (5 °C acclimated). CAT from frozen frogs also showed higher overall Phosphorylation (1.73 fold) and significantly higher levels of phosphoserine (1.60 fold) and phosphotyrosine (1.27 fold) compared to control animals. Phosphorylation via protein kinase A or the AMP-activated protein kinase significantly decreased the K m for H 2 O 2 of CAT, whereas protein phosphatase 2B or 2C action significantly increased the K m . Conclusion The physiological consequence of freeze-induced CAT Phosphorylation appears to improve CAT function to alleviate H 2 O 2 build-up in freezing frogs. General significance Augmented CAT activity via Reversible Phosphorylation may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia.
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regulation of liver lactate dehydrogenase by Reversible Phosphorylation in response to anoxia in a freshwater turtle
Comparative Biochemistry and Physiology B, 2012Co-Authors: Zi Jian Xiong, Kenneth B. StoreyAbstract:Lactate dehydrogenase (LDH) is the terminal enzyme of anaerobic glycolysis and key to hypoxia/anoxia survival by most animals. In this study, the effects of anoxic submergence (20 h at 7°C in nitrogen-bubbled water) were assessed on LDH from liver of an anoxia-tolerant freshwater turtle, the red-eared slider (Trachemys scripta elegans). Liver LDH from aerobic and anoxic turtles was purified to homogeneity in two steps. The kinetic properties and thermal stability of purified LDH were analyzed, revealing significant differences between the two enzyme forms in V(max), K(m) pyruvate, and I(50) pyruvate as well as melting temperature determined by differential scanning fluorimetry. The Phosphorylation state of aerobic and anoxic forms of LDH was visualized by ProQ Diamond phosphoprotein staining, the results indicating that the anoxic form had a higher Phosphorylation state. Incubation studies that promoted protein kinase versus protein phosphatase actions showed that changes in the Phosphorylation state of aerobic and anoxic forms mimicked the anoxia-responsive changes in K(m) pyruvate and I(50) pyruvate. The high phosphate form of liver LDH that occurs in anoxic turtles appears to be a less active form. Turtle liver LDH was also subject to another form of posttranslational modification, protein acetylation, with a 70% higher content of acetylated lysine residues on anoxic versus aerobic LDH. This is the first study to show that LDH function in an anoxia-tolerant animal can be differentially modified between aerobic and anoxic states via the mechanism of posttranslational modification.
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Regulation of tail muscle arginine kinase by Reversible Phosphorylation in an anoxia-tolerant crayfish
Journal of Comparative Physiology B, 2011Co-Authors: Neal J. Dawson, Kenneth B. StoreyAbstract:Freshwater crayfish, Orconectes virilis , can experience periodic exposures to hypoxia or anoxia due to low water flow (in summer) or ice cover (in winter) in their natural habitat. Hypoxia/anoxia disrupts energy metabolism and triggers mechanisms that to support ATP levels while often also suppressing ATP use. Arginine kinase (AK) (E.C. 2.7.3.3) is a crucial enzyme involved in energy metabolism in muscle, gating the use of phosphagen stores to buffer ATP levels. The present study investigated AK from tail muscle of O. virilis identifying changes to kinetic properties, Phosphorylation state and structural stability between the enzyme from aerobic control and 20 h anoxic crayfish. Muscle AK from anoxia-exposed crayfish showed a significantly higher (by 59%) K _m for l -arginine and a lower I_50 value for urea than the aerobic form. Several lines of evidence indicated that AK was converted to a high phosphate form under anoxia: (a) aerobic and anoxic forms of AK showed well-separated elution peaks on DEAE ion exchange chromatography, (b) ProQ Diamond phosphoprotein staining showed a 64% higher bound phosphate content on anoxic AK compared with the aerobic form, and (c) treatment of anoxic AK with alkaline phosphatase reduced K _m l -arginine to aerobic levels whereas incubation of aerobic AK with protein kinase A catalytic subunit raised the K _m to anoxic levels. The physiological consequence of anoxia-induced AK Phosphorylation may be to suppress AK activity in the phosphagen-synthesizing direction and, together with reduced cellular pH and ATP levels, promote the phosphagen-catabolizing direction under anoxic conditions. This is first time that AK has been shown to be regulated by Reversible Phosphorylation.
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regulation of hexokinase by Reversible Phosphorylation in skeletal muscle of a freeze tolerant frog
Comparative Biochemistry and Physiology B, 2011Co-Authors: Christopher A Dieni, Kenneth B. StoreyAbstract:Abstract Hexokinase (HK) was isolated from hind leg skeletal muscle of the wood frog, Rana sylvatica, a freeze tolerant species that uses glucose as a cryoprotectant. Analysis of kinetic parameters (Km and Vmax) of HK showed significant increases in Km glucose (from 144 ± 4.4 to 248 ± 12.0 μM) and Km ATP (from 248 ± 8.5 to 330 ± 20.9 μM), as well as a decrease in Vmax (from 86.1 ± 0.40 to 52 ± 0.49 mU mg− 1 of protein) in frogs following freezing exposure, indicating lower affinity for HK substrates and lower enzyme activity in this state. Subsequent analyses indicated that differential Phosphorylation of HK between the two states was responsible for the altered kinetic properties. HK was analyzed by SDS-PAGE; phosphoprotein staining revealed a 33% decrease in phosphate content of HK from frozen frogs but immunoblotting showed no change in total HK protein content. Muscle extracts from control and frozen frogs were incubated with ions and second messengers to stimulate the actions of protein kinases and protein phosphatases, with results indicating that HK can be phosphorylated by protein kinases A and C, and AMP-activated protein kinase, and can be dephosphorylated by protein phosphatases 1, 2A and 2C. The data indicate that in control frogs, HK is in a higher phosphate form and displays a high substrate affinity and high activity, whereas in frozen frogs HK is less phosphorylated, with lower substrate affinity and lower activity. Studies also showed that HK affinity for ATP decreases further in response to low temperature, but that high cryoprotective glucose concentrations can prevent these changes in affinity. Finally, the activity and structure of HK from frozen frogs is more sensitive to non-compatible osmolytes than the enzyme in control frogs.
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Reversible Phosphorylation regulation of nadph linked polyol dehydrogenase in the freeze avoiding gall moth epiblema scudderiana role in glycerol metabolism
Archives of Insect Biochemistry and Physiology, 2011Co-Authors: Helen A Holden, Kenneth B. StoreyAbstract:Larvae of the goldenrod gall moth, Epiblema scudderiana, use a freeze avoidance strategy of cold hardiness to survive the winter. A key metabolic adaption that supports subzero survival is the accumulation of large amounts of glycerol as a colligative antifreeze. Production of glycerol relies on polyol dehydrogenase (PDH) which catalyzes the NADPH-dependent conversion of glyceraldehyde into glycerol. Kinetic analysis of PDH from E. scudderiana revealed significant changes in properties as a result of subzero temperature acclimation; the Km for glyceraldehyde in 5°C-acclimated larvae was 7.0 mM and doubled in − 15°C-exposed larvae. This change suggested that PDH is regulated by a state-dependent covalent modification. Indeed, high and low Km forms could be interconverted by incubating larval extracts in vitro under conditions that stimulated either endogenous protein kinases or protein phosphatases. Protein kinase incubations doubled the Km glyceraldehyde of the 5°C enzyme, whereas protein phosphatase incubations decreased the Km of the − 15°C enzyme by about 50%. PDH was purified by ion exchange and affinity chromatography steps and then subjected to electrophoresis. Staining with ProQ Diamond phosphoprotein stain showed a much higher phosphate content of PDH from − 15°C-acclimated larvae, a result that was further confirmed by immunoblotting that showed a much greater phosphoserine content on the − 15°C enzyme. These experiments established that PDH is regulated by state-dependent Reversible Phosphorylation in E. scudderiana and suggest that this regulatory mechanism makes a significant contribution to controlling the synthesis, maintenance, and degradation of glycerol pools over the winter months. © 2011 Wiley Periodicals, Inc.
Josef Krieglstein - One of the best experts on this subject based on the ideXlab platform.
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Reversible Phosphorylation of histidine residues in proteins from vertebrates
Science Signaling, 2009Co-Authors: Susanne Klumpp, Josef KrieglsteinAbstract:Signaling by kinases and phosphatases that act on serine, threonine, and tyrosine residues of proteins is among the most extensively studied regulatory mechanisms in mammalian cells, and research focused in this area is ongoing. We are just beginning to appreciate that such signaling mechanisms are extended and enriched by the Reversible Phosphorylation of histidine residues. The most exciting developments in this field to date come from studies on the beta subunit of heterotrimeric guanosine triphosphate-binding proteins (G proteins), the enzyme adenosine 5'-triphosphate-citrate lyase, and now the Ca(2+)-activated K(+) channel KCa3.1, all of which are targeted by nucleoside diphosphate kinase (which phosphorylates histidines) and protein histidine phosphatase (which dephosphorylates phosphorylated histidines).
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Reversible Phosphorylation of histidine residues in vertebrate proteins
Biochimica et Biophysica Acta, 2005Co-Authors: Susanne Klumpp, Josef KrieglsteinAbstract:Knowledge on kinases and phosphatases acting on serine, threonine and tyrosine residues of vertebrate proteins is huge. These enzymes are still under intensive investigation at present. This is in sharp contrast to what is known about kinases and phosphatases acting on histidine, arginine, lysine and aspartate residues in vertebrate proteins. It also is in contrast to extensive studies of histidine/aspartate Phosphorylation in prokaryotes. This minireview briefly summarizes what we have learned about the Reversible Phosphorylation of histidine residues in mammals. It is described how the field developed during 40 years of science. The article especially highlights the discovery of the first protein histidine phosphatase from vertebrates. Having identified and characterized a protein histidine phosphatase provides at least one desperately required tool to handle and study Phosphorylation and dePhosphorylation of histidine residues in vertebrates in more detail. Recent evidence even suggests an involvement of histidine Phosphorylation in signal transduction.
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Protein kinase CK2 phosphorylates BAD at threonine-117.
Neurochemistry International, 2004Co-Authors: Susanne Klumpp, Anette Mäurer, Dagmar Aichele, Lorenzo A Pinna, Josef KrieglsteinAbstract:Reversible Phosphorylation of the 22 kDa BAD protein is crucial for cell survival. Five Phosphorylation sites, all serines, had been identified. Here we report on number six. It is threonine-117 phosphorylated by the constitutively active kinase, CK2. Phosphoamino acid analysis and phospho-specific antibodies confirmed Thr117 as additional Phosphorylation site. Immunoprecipitation furthermore revealed that BAD is phosphorylated at Thr117 in cultured cortical neurons. PP1, PP2A and PP2C dephosphorylated BAD at Thr117, but PP2B did not. The discovery of the constitutively active CK2 phosphorylating BAD is shedding an unexpected light in the otherwise strictly signal-regulated Phosphorylation events on BAD.
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protein phosphatase type 2c dephosphorylates bad
Neurochemistry International, 2003Co-Authors: Susanne Klumpp, Dagmar Selke, Josef KrieglsteinAbstract:Reversible Phosphorylation modulates a cells' susceptibility to apoptosis. The Phosphorylation status of BAD, a member of the Bcl-2 protein family, is an important checkpoint governing life-or-death decisions: Phosphorylation of serine residues 112, 136 and 155 on BAD prevents apoptosis. Here we report that BAD is a substrate for PP2C. Ser(155) is involved in heterodimerization with Bcl-X(L). We could demonstrate that PP1, PP2A and PP2C act on this site in vitro. However, only PP2C gives priority to P-Ser(155) compared to P-Ser(112) and P-Ser(136) on BAD. The results indicate that PP2C is an additional factor triggering the pro-apoptotic function of BAD.
Susanne Klumpp - One of the best experts on this subject based on the ideXlab platform.
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Reversible Phosphorylation of histidine residues in proteins from vertebrates
Science Signaling, 2009Co-Authors: Susanne Klumpp, Josef KrieglsteinAbstract:Signaling by kinases and phosphatases that act on serine, threonine, and tyrosine residues of proteins is among the most extensively studied regulatory mechanisms in mammalian cells, and research focused in this area is ongoing. We are just beginning to appreciate that such signaling mechanisms are extended and enriched by the Reversible Phosphorylation of histidine residues. The most exciting developments in this field to date come from studies on the beta subunit of heterotrimeric guanosine triphosphate-binding proteins (G proteins), the enzyme adenosine 5'-triphosphate-citrate lyase, and now the Ca(2+)-activated K(+) channel KCa3.1, all of which are targeted by nucleoside diphosphate kinase (which phosphorylates histidines) and protein histidine phosphatase (which dephosphorylates phosphorylated histidines).
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Reversible Phosphorylation of histidine residues in vertebrate proteins
Biochimica et Biophysica Acta, 2005Co-Authors: Susanne Klumpp, Josef KrieglsteinAbstract:Knowledge on kinases and phosphatases acting on serine, threonine and tyrosine residues of vertebrate proteins is huge. These enzymes are still under intensive investigation at present. This is in sharp contrast to what is known about kinases and phosphatases acting on histidine, arginine, lysine and aspartate residues in vertebrate proteins. It also is in contrast to extensive studies of histidine/aspartate Phosphorylation in prokaryotes. This minireview briefly summarizes what we have learned about the Reversible Phosphorylation of histidine residues in mammals. It is described how the field developed during 40 years of science. The article especially highlights the discovery of the first protein histidine phosphatase from vertebrates. Having identified and characterized a protein histidine phosphatase provides at least one desperately required tool to handle and study Phosphorylation and dePhosphorylation of histidine residues in vertebrates in more detail. Recent evidence even suggests an involvement of histidine Phosphorylation in signal transduction.
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Protein kinase CK2 phosphorylates BAD at threonine-117.
Neurochemistry International, 2004Co-Authors: Susanne Klumpp, Anette Mäurer, Dagmar Aichele, Lorenzo A Pinna, Josef KrieglsteinAbstract:Reversible Phosphorylation of the 22 kDa BAD protein is crucial for cell survival. Five Phosphorylation sites, all serines, had been identified. Here we report on number six. It is threonine-117 phosphorylated by the constitutively active kinase, CK2. Phosphoamino acid analysis and phospho-specific antibodies confirmed Thr117 as additional Phosphorylation site. Immunoprecipitation furthermore revealed that BAD is phosphorylated at Thr117 in cultured cortical neurons. PP1, PP2A and PP2C dephosphorylated BAD at Thr117, but PP2B did not. The discovery of the constitutively active CK2 phosphorylating BAD is shedding an unexpected light in the otherwise strictly signal-regulated Phosphorylation events on BAD.
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protein phosphatase type 2c dephosphorylates bad
Neurochemistry International, 2003Co-Authors: Susanne Klumpp, Dagmar Selke, Josef KrieglsteinAbstract:Reversible Phosphorylation modulates a cells' susceptibility to apoptosis. The Phosphorylation status of BAD, a member of the Bcl-2 protein family, is an important checkpoint governing life-or-death decisions: Phosphorylation of serine residues 112, 136 and 155 on BAD prevents apoptosis. Here we report that BAD is a substrate for PP2C. Ser(155) is involved in heterodimerization with Bcl-X(L). We could demonstrate that PP1, PP2A and PP2C act on this site in vitro. However, only PP2C gives priority to P-Ser(155) compared to P-Ser(112) and P-Ser(136) on BAD. The results indicate that PP2C is an additional factor triggering the pro-apoptotic function of BAD.
Baichen Wang - One of the best experts on this subject based on the ideXlab platform.
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structural basis of Reversible Phosphorylation by maize pyruvate orthophosphate dikinase regulatory protein
Plant Physiology, 2016Co-Authors: Lun Jiang, Yibo Chen, Jiangge Zheng, Zhenhang Chen, Yujie Liu, Ye Tao, Zhongzhou Chen, Baichen WangAbstract:Pyruvate orthophosphate dikinase (PPDK) is one of the most important enzymes in C4 photosynthesis. PPDK regulatory protein (PDRP) regulates the inorganic phosphate-dependent activation and ADP-dependent inactivation of PPDK by Reversible Phosphorylation. PDRP shares no significant sequence similarity with other protein kinases or phosphatases. To investigate the molecular mechanism by which PDRP carries out its dual and competing activities, we determined the crystal structure of PDRP from maize (Zea mays). PDRP forms a compact homo-dimer in which each protomer contains two separate N-terminal (NTD) and C-terminal (CTD) domains. The CTD includes several key elements for performing both Phosphorylation and dePhosphorylation activities: the phosphate binding loop (P-loop) for binding the ADP and inorganic phosphate substrates, residues Lys-274 and Lys-299 for neutralizing the negative charge, and residue Asp-277 for protonating and deprotonating the target Thr residue of PPDK to promote nucleophilic attack. Surprisingly, the NTD shares the same protein fold as the CTD and also includes a putative P-loop with AMP bound but lacking enzymatic activities. Structural analysis indicated that this loop may participate in the interaction with and regulation of PPDK. The NTD has conserved intramolecular and intermolecular disulfide bonds for PDRP dimerization. Moreover, PDRP is the first structure of the domain of unknown function 299 enzyme family reported. This study provides a structural basis for understanding the catalytic mechanism of PDRP and offers a foundation for the development of selective activators or inhibitors that may regulate photosynthesis.
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posttranslational modification of maize chloroplast pyruvate orthophosphate dikinase reveals the precise regulatory mechanism of its enzymatic activity
Plant Physiology, 2014Co-Authors: Yibo Chen, Hongxia Wang, Jie Shen, Qing Chao, Zhifang Gao, Xinguang Zhu, Yuefeng Wang, Baichen WangAbstract:In C4 plants, pyruvate orthophosphate dikinase (PPDK) activity is tightly dark/light regulated by Reversible Phosphorylation of an active-site threonine (Thr) residue; this process is catalyzed by PPDK regulatory protein (PDRP). Phosphorylation and dePhosphorylation of PPDK lead to its inactivation and activation, respectively. Here, we show that light intensity rather than the light/dark transition regulates PPDK activity by modulating the Reversible Phosphorylation at Thr-527 (previously termed Thr-456) of PPDK in maize (Zea mays). The amount of PPDK (unphosphorylated) involved in C4 photosynthesis is indeed strictly controlled by light intensity, despite the high levels of PPDK protein that accumulate in mesophyll chloroplasts. In addition, we identified a transit peptide cleavage site, uncovered partial amino-terminal acetylation, and detected Phosphorylation at four serine (Ser)/Thr residues, two of which were previously unknown in maize. In vitro experiments indicated that Thr-527 and Ser-528, but not Thr-309 and Ser-506, are targets of PDRP. Modeling suggests that the two hydrogen bonds between the highly conserved residues Ser-528 and glycine-525 are required for PDRP-mediated Phosphorylation of the active-site Thr-527 of PPDK. Taken together, our results suggest that the regulation of maize plastid PPDK isoform (C4PPDK) activity is much more complex than previously reported. These diverse regulatory pathways may work alone or in combination to fine-tune C4PPDK activity in response to changes in lighting.
Kathryn L Ball - One of the best experts on this subject based on the ideXlab platform.
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Reversible Phosphorylation at the c terminal regulatory domain of p21waf1 cip1 modulates proliferating cell nuclear antigen binding
Journal of Biological Chemistry, 2000Co-Authors: Mary T Scott, Nick Morrice, Kathryn L BallAbstract:The p53-inducible gene product p21WAF1/CIP1 plays a critical role in regulating the rate of tumor incidence, and identifying mechanisms of its post-translational regulation will define key pathways that link growth control to p21-dependent tumor suppression. A eukaryotic cell model system has been developed to determine whether protein kinase signaling pathways that phosphorylate human p21 exist in vivo and whether such pathways regulate the binding of p21 to one of its key target proteins, proliferating cell nuclear antigen (PCNA). Although human p21 expressed in Sf9 cells is able to form a complex with human PCNA, the inclusion of cell-permeable phosphatase inhibitors renders p21 protein inactive for PCNA binding. The treatment of this inactive isoform of p21 with alkaline phosphatase restores its binding to PCNA, suggesting that p21 expressed inSf9 cells is subject to Reversible Phosphorylation at a key regulatory site(s). A biochemical approach was subsequently used to map the Phosphorylation sites within p21, whose modification in vitro can inhibit p21-PCNA complex formation, to the C-terminal domain at residues Thr145 or Ser146. A phospho-specific antibody was developed that only bound to full-length p21 protein after Phosphorylation in vitro at Ser146, and this reagent was further used to demonstrate that the inactive isoform of p21 recovered fromSf9 cells treated with phosphatase inhibitors had been phosphorylated in vivo at Ser146. These data identify the first Phosphorylation site within the C-terminal regulatory domain of p21 whose modification in vivomodulates p21-PCNA interactions and define a eukaryotic cell model that can be used to study post-translational signaling pathways that regulate p21.
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Reversible Phosphorylation at the c terminal regulatory domain of p21 waf1 cip1 modulates proliferating cell nuclear antigen binding
Journal of Biological Chemistry, 2000Co-Authors: Mary T Scott, Nick Morrice, Kathryn L BallAbstract:Abstract The p53-inducible gene product p21WAF1/CIP1 plays a critical role in regulating the rate of tumor incidence, and identifying mechanisms of its post-translational regulation will define key pathways that link growth control to p21-dependent tumor suppression. A eukaryotic cell model system has been developed to determine whether protein kinase signaling pathways that phosphorylate human p21 exist in vivo and whether such pathways regulate the binding of p21 to one of its key target proteins, proliferating cell nuclear antigen (PCNA). Although human p21 expressed in Sf9 cells is able to form a complex with human PCNA, the inclusion of cell-permeable phosphatase inhibitors renders p21 protein inactive for PCNA binding. The treatment of this inactive isoform of p21 with alkaline phosphatase restores its binding to PCNA, suggesting that p21 expressed inSf9 cells is subject to Reversible Phosphorylation at a key regulatory site(s). A biochemical approach was subsequently used to map the Phosphorylation sites within p21, whose modification in vitro can inhibit p21-PCNA complex formation, to the C-terminal domain at residues Thr145 or Ser146. A phospho-specific antibody was developed that only bound to full-length p21 protein after Phosphorylation in vitro at Ser146, and this reagent was further used to demonstrate that the inactive isoform of p21 recovered fromSf9 cells treated with phosphatase inhibitors had been phosphorylated in vivo at Ser146. These data identify the first Phosphorylation site within the C-terminal regulatory domain of p21 whose modification in vivomodulates p21-PCNA interactions and define a eukaryotic cell model that can be used to study post-translational signaling pathways that regulate p21.
