Phosphorylation of CTP Synthetase from Saccharomyces cerevisiae by Protein Kinase C

Yang, Weng‐Lang; Carman, George M.

doi:10.1074/jbc.270.25.14983

Cited by 69 publications

(79 citation statements)

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“…In addition, the kinetic analysis of the purified mutant enzymes revealed that the S36A and S354A mutations caused a decrease in the V max of the reaction. These results were consistent with previous data showing that protein kinase C phosphorylation stimulates the activity of the pure wild type CTP synthetase (33,35). Interestingly, the CTP synthetase activity in cells bearing the S330A mutant enzyme was elevated, and kinetic analysis of the purified enzyme showed that the S330A mutation caused an elevation in the V max of the reaction.…”

Section: Discussionsupporting

confidence: 82%

“…Regulation of this enzyme is critical because the product of its reaction CTP is essential for the synthesis of nucleic acids and membrane phospholipids (18). One of the mechanisms by which CTP synthetase activity is regulated in S. cerevisiae is by phosphorylation via protein kinase C (33,35). Protein kinase C is a lipid-dependent protein kinase required for S. cerevisiae cell cycle (74 -78) and plays a role maintaining cell wall integrity (79).…”

Section: Discussionmentioning

confidence: 99%

“…In vitro studies show that CTP synthetase is a substrate for protein kinases A (34) and C (33,35). These phosphorylations result in the stimulation of CTP synthetase activity by a mechanism that increases catalytic turnover (33)(34)(35). In addition, phosphorylation facilitates the nucleotide-dependent tetramerization of the enzyme (29) and causes a decrease in the sensitivity of the enzyme to inhibition by CTP (34,35).…”

Section: ) (30)mentioning

confidence: 99%

“…The URA7-encoded CTP synthetase is phosphorylated on multiple serine residues in vivo (33). In vitro studies show that CTP synthetase is a substrate for protein kinases A (34) and C (33,35). These phosphorylations result in the stimulation of CTP synthetase activity by a mechanism that increases catalytic turnover (33)(34)(35).…”

Section: ) (30)mentioning

confidence: 99%

Section: ) (30)mentioning

confidence: 99%

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Phosphorylation of CTP Synthetase on Ser36, Ser330, Ser354, and Ser454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces cerevisiae

Park

O'Brien

Carman

2003

Journal of Biological Chemistry

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The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinase C. We examined the hypothesis that Ser 36 , Ser 330 , Ser 354 , and Ser 454 , contained in a protein kinase C sequence motif in CTP synthetase, were target sites for the kinase. Synthetic peptides containing a phosphorylation motif at these serine residues served as substrates for protein kinase C in vitro. Ser 3 Ala (S36A, S330A, S354A, and S454A) mutations in CTP synthetase were constructed by site-directed mutagenesis and expressed normally in a ura7 ura8 double mutant that lacks CTP synthetase activity. The CTP synthetase activity in extracts from cells bearing the S36A, S354A, and S454A mutant enzymes was reduced when compared with cells bearing the wild type enzyme. Kinetic analysis of purified mutant enzymes showed that the S36A and S354A mutations caused a decrease in the V max of the reaction. This regulation could be attributed in part by the effects phosphorylation has on the nucleotide-dependent oligomerization of CTP synthetase. In contrast, CTP synthetase activity in cells bearing the S330A mutant enzyme was elevated, and kinetic analysis of purified enzyme showed that the S330A mutation caused an elevation in the V max of the reaction. In vitro data indicated that phosphorylation of CTP synthetase at Ser 330 affected the phosphorylation of the enzyme at another site. The phosphorylation of CTP synthetase at Ser 36 , Ser 330 , Ser 354 , and Ser 454 residues was physiologically relevant. Cells bearing the S36A, S354A, and S454A mutations had reduced CTP levels, whereas cells with the S330A mutation had elevated CTP levels. The alterations in CTP levels correlated with the regulatory effects CTP has on the pathways responsible for the synthesis of the membrane phospholipid phosphatidylcholine.In the yeast Saccharomyces cerevisiae CTP synthetase catalyzes the ATP-dependent transfer of the amide nitrogen of glutamine to the C-4 position of UTP to form CTP (1, 2). GTP stimulates the reaction by accelerating the formation of a covalent glutaminyl enzyme catalytic intermediate (2-5). URA7 (6) and URA8 (7) are duplicate genes that code for CTP synthetase in S. cerevisiae. The yeast CTP synthetase enzymes (6, 7) contain a conserved glutamine amide transfer domain (see Fig. 1) common to CTP synthetases from other organisms (8 -16). The URA7-encoded CTP synthetase is more abundant than the URA8-encoded enzyme (17) and is responsible for the majority of the CTP synthesized in vivo (7). Neither the URA7 nor the URA8 gene is essential as long as cells possess one functional CTP synthetase gene (6, 7). CTP synthetase is an indispensable enzyme because its reaction product CTP is essential for the synthesis of nucleic acids and membrane phospholipids (18). The importance of understanding the regulation of CTP synthetase is emphasized by the fact that unregulated levels of its activity is a common property of various cancers in humans (19 -26).The yeast CTP synthetase is regulated by genetic and biochemical m...

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Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Section: ) (30)mentioning

confidence: 99%

Section: ) (30)mentioning

confidence: 99%

Section: ) (30)mentioning

confidence: 99%

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Phosphorylation of CTP Synthetase on Ser36, Ser330, Ser354, and Ser454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces cerevisiae

Park

O'Brien

Carman

2003

Journal of Biological Chemistry

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Novel miniproteins engineered by the transfer of active sites to small natural scaffolds

et al. 1998

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Small multidisulfide‐containing proteins are attractive structural templates to produce a biologically active conformation that mimics the binding surface of natural large proteins. In particular, the structural motif that is evolutionary conserved in all scorpion toxins has a small size (30–40 amino acid residues), a great structural stability, and high permissiveness for sequence mutation. This motif is composed of a β‐sheet and an α‐helix bridged in the interior core by three disulfides. We have used this motif successfully to transfer within its β‐sheet new functional sites, including the curaremimetic loop of a snake neurotoxin and the CDR2‐like site of human CD4. Accumulated evidence indicated that the two miniproteins produced, the curaremimetic miniprotein and the CD4 mimetic, contain the α/β fold that is characteristic of the scaffold used and bind respectively to the acetylcholine receptor and to the envelope gp120 of HIV‐1. Furthermore, the latter was shown to prevent viral infection of lymphocytes. These examples illustrate that, by the transfer of active sites to small and stable natural scaffolds, it is possible to engineer miniproteins reproducing, in part, the function of much larger proteins. Such miniproteins may be of great utility as tools in structure‐function studies and as leads in drug design. © 1998 John Wiley & Sons, Inc. Biopoly 47: 93–100, 1998

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Substrate Recognition

Brocklehurst

Gul

Pickersgill

2009

Amino Acids, Peptides and Proteins in Organic Chemistry

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Specific molecular recognition processes by which molecules recognize and discriminate between closely related partners are involved in essentially all aspects of biological function, ranging in complexity from enzyme-inhibitor and enzymesubstrate systems to phenomena such as gene expression, the immune system, and synaptic transmission, and these are important considerations in drug design [1]. This chapter is concerned with general concepts exemplified by a selection of relatively well-defined enzyme-substrate and, by extension, enzyme-inhibitor systems. The original concept of recognition by complementarity in the enzymesubstrate adsorptive (Michaelis) complex was suggested by Emil Fischer in 1894 using his well-known lock-and-key analogy [2]. It is now generally accepted that this has now been superseded by complementarity in the reaction transition state, based on the ideas reported by J.B.S. Haldane [3] in 1930 and elaborated by Linus Pauling [4] in 1946. The catalytic potential of enzymes derives from stabilization in the transition state relative to any stabilization in the Michaelis complex. The latter is anticatalytic in that the free energy expended in stabilizing the adsorptive complex offsets the transition-state stabilization energy (e.g., [5]).Valuable chapters and books on enzyme catalysis and inhibition are available (e.g., [5][6][7][8][9][10][11]) and detailed mechanistic studies on many individual enzymes are described in Sinnot [12]. Macromolecular catalysis includes also catalysis by catalytic antibodies [13] and by synthetic polymers [14], including molecularly imprinted polymers [15]. The contributions of selections of various types of chemical catalysis such as acid-base, nucleophilic, and electrophilic catalysis [5,6,8] are almost always insufficient to account for the high catalytic efficiency of enzymes. A particularly important additional contribution is the entropic advantage that derives from the fact that enzyme catalysis occurs within an enzyme-substrate complex. The complex is essentially a single molecular species and the catalytic act involves one or more unimolecular steps during which there is no loss of translational or rotational entropy

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Phosphorylation of CTP Synthetase from Saccharomyces cerevisiae by Protein Kinase C

Cited by 69 publications

References 38 publications

Phosphorylation of CTP Synthetase on Ser36, Ser330, Ser354, and Ser454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces cerevisiae

Phosphorylation of CTP Synthetase on Ser36, Ser330, Ser354, and Ser454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces cerevisiae

Novel miniproteins engineered by the transfer of active sites to small natural scaffolds

Substrate Recognition

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