Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The key roles of the excitatory neurotransmitter glutamate and its second messengers, nitric oxide (NO) and cGMP, in long-term potentiation and neural plasticity are well documented. However, complex functions such as memory are likely to require long term changes in synaptic efficacy which require gene expression and protein synthesis. Here we demonstrate that the glutamate receptor agonist, N-methyl-D-aspartic acid (NMDA), nitric oxide (NO) and cGMP each repress expression of the gonadotropin-releasing hormone (GnRH) gene in the hypothalamic cell line, GT1. This repression is dependent upon signals from NMDA receptors activating NO synthase to synthesize NO. In turn NO induces guanylyl cyclase to synthesize cGMP, activating cGMP- dependent protein kinase. Repression requires elevation of calcium because it only occurs in the presence of calcium ionophore or with release of intracellular calcium. Repression also requires protein synthesis. Activation of this pathway specifically represses expression of a reporter gene containing the regulatory region of the GnRH gene in transfected GT1 cells, indicating that repression occurs at the transcriptional level. Furthermore the target for transcriptional repression is a 300 bp neuron-specific enhancer found 1.5 kb upstream of the GnRH gene which is sufficient to confer repression to a heterologous promoter. Thus the NMDA/NO/cGMP neurotransmitter signal transduction pathway controls not only synaptic function but also neuron-specific gene expression.
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PMID:NMDA and nitric oxide act through the cGMP signal transduction pathway to repress hypothalamic gonadotropin-releasing hormone gene expression. 859 37

The p53 tumour suppressor protein is a potent transcription factor which plays a central role in the defence of cells against DNA damage and the propagation of malignant clones. We have previously shown that phosphorylation of serine 386 in mouse p53 by the growth- associated protein kinase, casein kinase II (CKII), plays an important role in the ability of p53 to block the proliferation of drug-resistant colonies. In this paper we show that blocking phosphorylation of serine 386 through an alanine substitution, or placing a constitutive negative charge at this position in the form of aspartate, had no significant influence on p53-dependent transcriptional activation of a promoter containing 13 copies of a p53 consensus binding sequence, or of the p21WAF1 promoter which is a natural target for p53. In contrast, the alanine mutant showed a weak reduction in the ability of p53 to repress expression from the c-fos promoter, which is a target for p53-dependent repression in vivo. Strikingly, when the repression of the SV40 early promoter was examined, a reduction in the repression capacity of up to 5-fold was observed. Moreover, repression of the SV40 promoter could be partially restored by aspartic acid substitution at the phosphorylation site. These data indicate that phosphorylation at a specific C-terminal site can selectively regulate p53-dependent repression, but not transactivation.
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PMID:Phosphorylation of p53 at the casein kinase II site selectively regulates p53-dependent transcriptional repression but not transactivation. 860 47

Although Rafs play a central role in signal transduction, the mechanism(s) by which they become activated is poorly understood. Raf-1 activation is dependent on the protein's ability to bind Ras, but Ras binding is insufficient to activate Raf-1 tyrosine phosphorylation to this Ras-induced activation, in the absence of an over-expressed tyrosine kinase. We demonstrate that Raf-1 purified form Sf9 cells coinfected with baculovirus Ras but not Src could be inactivated by protein tyrosine phosphatase PTP-1B. 14-3-3 and Hsp90 proteins blocked both the tyrosine dephosphorylation and inactivation of Raf-1, suggesting that Raf-1 activity is phosphotyrosine dependent. In Ras-transformed NIH 3T3 cells, a minority of Raf-1 protein was membrane associated, but essentially all Raf-1 activity and Raf-1 phosphotyrosine fractionated with plasma membranes. Thus, the tyrosine-phosphorylated and active pool of Raf-1 constitute a membrane-localized subfraction which could also be inactivated with PTP-1B. By contrast, B-Raf has aspartic acid residues at positions homologous to those of the phosphorylated tyrosines (at 340 and 341) of Raf-1 and displays a high basal level of activity. B-Raf was not detectably tyrosine phosphorylated, membrane localized, or further activated upon Ras transformation, even though B-Raf has been shown to bind to Ras in vitro. We conclude that tyrosine phosphorylation is an essential component of the mechanism by which Ras activates Raf-1 kinase activity and that steady-state activated Ras is insufficient to activate B-Raf in vivo.
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PMID:Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation. 862 47

Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors activate muscle gene expression by binding an A/T-rich DNA sequence in the control regions of muscle-specific genes. There are four MEF2 factors in vertebrates, MEF2A-D, which share homology in an amino-terminal MADS domain and an adjacent region known as the MEF2 domain, that together mediate DNA binding and dimerization. We show that serine 59 located between the MADS and MEF2 domains of MEF2C is phosphorylated in vivo and can be phosphorylated in vitro by casein kinase-II (CKII). Phosphorylation of this site enhanced the DNA binding and transcriptional activity of MEF2C by increasing its DNA binding activity 5-fold. In vivo 32P labeling experiments showed that serine 59 is the only phosphorylation site in the MADS and MEF2 domains. Mutagenesis of this serine to an aspartic acid resulted in an increase in DNA binding and transcriptional activity of MEF2C comparable to that observed when this site was phosphorylated, suggesting that phosphorylation augments DNA binding activity by introducing negative charge. This phosphorylation site, which corresponds to a CKII recognition site, is conserved in all known MEF2 factors in organisms ranging from flies to humans, consistent with its importance for the functions of MEF2C.
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PMID:Phosphorylation of the MADS-Box transcription factor MEF2C enhances its DNA binding activity. 866 3

IkappaBalpha is a phosphoprotein that sequesters the NF-kappaB/Rel transcription factors in the cytoplasm by physical association. Following induction by a wide variety of agents, IkappaBalpha is further phosphorylated and degraded, allowing NF-kappaB/Rel proteins to translocate to the nucleus and induce transcription. We have previously reported that the constitutive phosphorylation site resides in the C-terminal PEST region of IkappaBalpha and is phosphorylated by casein kinase II (CKII). Here we show that serine 293 is the preferred CKII phosphorylation site. Additionally, we show compensatory phosphorylation by CKII at neighboring serine and threonine residues. Thus, only when all five of the serine and threonine residues in the C-terminal region of IkappaBalpha are converted to alanine (MutF), is constitutive phosphorylation abolished. Finally, we show that constitutive phosphorylation is required for efficient degradation of free IkappaBalpha, in that unassociated Mutf has a half-life two times longer than wild-type IkappaBalpha. A serine residue alone at position 293, as well as aspartic acid at this position, can revert the Mutf phenotype. Therefore, the constitutive CKII phosphorylation site is an integral part of the PEST region of IkappaBalpha, and this phosphorylation is required for rapid proteolysis of the unassociated protein.
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PMID:Constitutive phosphorylation of IkappaBalpha by casein kinase II occurs preferentially at serine 293: requirement for degradation of free IkappaBalpha. 866 71

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.
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PMID:Negative regulation of Raf-1 by phosphorylation of serine 621. 881 53

Serine 657 in protein kinase C-alpha (PKCalpha) is a site of phosphorylation on expression of the recombinant protein in mammalian cells. To define the function of this phosphorylation, PKCalpha species with mutations of this site were investigated. The alanine mutant, S657A PKCalpha, displayed slow phosphate accumulation in pulse-chase experiments, indicating a rate-limiting role in the initial phase of phosphorylation. Consistent with this, the aspartic acid mutant, S657D PKCalpha, showed an increased rate of phosphate accumulation. Both the S657D and S657A PKCalpha mutants were slow to accumulate as fully phosphorylated forms during a second phase of phosphorylation. This latter property is shown to correlate with an increased phosphatase sensitivity and decreased protein kinase activity for these two PKCalpha mutants. It is further shown that once fully phosphorylated, the S657D PKCalpha mutant displays WT PKCalpha properties with respect to thermal stability and phosphatase sensitivity in vitro and in vivo; in contrast, the S657A PKCalpha mutant remains sensitive. The properties of the Ser-657 site PKCalpha mutants define functional roles for this phosphorylation in both the accumulation of phosphate on PKCalpha as well as in its agonist-induced dephosphorylation. These results are discussed in the context of a working model of PKCalpha behavior, providing insight into the workings of other kinases with equivalent sites of phosphorylation.
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PMID:Phosphorylation of protein kinase C-alpha on serine 657 controls the accumulation of active enzyme and contributes to its phosphatase-resistant state. 901 3

The carboxyl group of an aspartic acid in the active site of the serine-specific protein kinase, cAMP-dependent protein kinase, is poised near the hydroxyl proton of a peptide substrate in the X-ray crystallographic structure (Madhusudan et al., 1994), suggesting that this residue may act as a general-base catalyst in the phosphoryl transfer reaction. Indeed, several proposals have been made in this regard. We measured the pre-steady-state kinetics in this enzyme using a rapid quench flow technique to understand the role of this putative base. The phosphorylation of the peptide substrate, GRTGRRNSI, by cAMP-dependent protein kinase exhibited "burst" kinetics consistent with a mechanism in which the peptide is phosphorylated rapidly (154 s(-1)) and the product(s) is (are) released slowly (16 s(-1)). The replacement of Mg2+ with Mn2+ leads to a 13-fold reduction in this observed "burst" rate constant, suggesting that this transient is limited either by the phosphoryl transfer step or by a metal ion-dependent conformational change step. The influence of deuterium oxide on the pre-steady-state kinetics was monitored in the presence of both divalent metal ions, and no solvent isotope effect was measured on either "burst" phase. A large solvent isotope effect is observed on k(cat) in the presence of either metal ion, and a proton inventory analysis in the presence of Mg2+ indicates that two or more protons are transferred in the product release step. Finally, no pH dependence is observed on the "burst" rate constant using either Mg2+ or Mn2+ over the pH range of 6-9. The combined data do not support a mechanism involving a general-base catalyst whose pK(a) is greater than 5 or less than 10 if the "burst" phase is cleanly limited by the phosphoryl transfer step. If the "burst" phase is limited by a metal ion-dependent conformational change step, the measurement of the phosphoryl transfer step is obscured, and the participation of a base catalyst is indeterminate.
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PMID:Is there a catalytic base in the active site of cAMP-dependent protein kinase? 906 28

Rhodostomin (RHO) from Agkistrodon rhodostoma venom, consisting of 68 amino acids with an arginine-glycine-aspartic acid (RGD) sequence and 12 cysteine residues, is a potent inhibitor of platelet aggregation. We previously demonstrated that cell culture plates coated with the bacterially produced fusion protein of glutathione S-transferase-RHO [GST-RHO(RGD)] can facilitate human hepatoma cell attachment via intergrin interaction within 15 min. In this study, we further characterized the effect of RHO fusion protein on platelet cells by creating two other related fusion proteins, GST-RHO(RGE) and GST-(PS)RHO. The former was a single amino acid-substituted mutant, in which the aspartic acid residue of RGD was replaced by glutamic acid, and the latter was an insertion mutant, in which a pentapeptide of protein kinase A phosphorylation site was inserted between GST and RHO. These two mutant proteins together with a wild-type of GST-RHO(RGD) and native form of RHO were used to study effects on the inhibition of ADP-induced platelet aggregation. Results indicated that GST-RHO(RGD) inhibited platelet aggregation as potently as the native RHO, while the two other mutants were inactive. Furthermore, when unactivated platelet cells attached on the GST-RHO(RGD)-coated plate, they became a flattened pancake shape. From the results of facilitation of cell attachment on fusion protein-coated plates, we concluded that: (1) the GST-RHO(RGD) fusion protein is equally functional in inhibition of platelet aggregation and facilitation of cell attachment, which is through the interaction of RGD and integrins on the cell membrane; (2) the GST-RHO(RGE) mutant protein is unable to bind with integrins and results in loss of function; (3) the insertion mutant of GST-(PS)RHO may disrupt a proper conformation of RHO and also results in loss of function; (4) the bacterially produced fusion protein GST-RHO(RGD) can be properly used as an antithrombotic agent and an extracellular matrix.
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PMID:Glutathione S-transferase-rhodostomin fusion protein inhibits platelet aggregation and induces platelet shape change. 908 May 76

As one of the first steps to elucidate the relationship between the structure and function of CTP:phosphocholine cytidylytransferase (EC 2.7.7.15) in plants, the cytidylyltransferase cDNA of Arabidopsis thaliana was cloned and characterized. The A. thaliana cytidylyltransferase cDNA is 1447 bp long and contains an open reading frame of 993 bp coding for a protein of 331 amino acids. The deduced structure of the enzyme was composed of three main regions; the catalytic domain in the N-terminal half, the hydrophilic C-terminal region and the amphipathic domain in the middle. The catalytic domain region was relatively well conserved among different organisms, showing 76 and 72% homology with the rat and yeast protein sequences, respectively. The hydropathy profile revealed that the C-terminal non-catalytic portion of the protein was very hydrophilic, highly enriched in negatively charged aspartic acid and glutamic acid residues. In the region between the catalytic domain and the C-terminal region, there was an amphipathic alpha-helical domain, which was believed to bind the membrane surface in the active formation. Unlike animal counterparts, there was only one potential site of phosphorylation by protein kinase C and none by Ca2+/calmodulin protein kinase II in the C-terminal region. The identity of cytidylyltransferase cDNA was verified by successful transformation of a yeast mutant defective in the enzyme activity, using an expression vector inserted with the A. thaliana cytidylyltransferase cDNA. This was further confirmed by in vivo analysis of the enzyme reaction product after labeling the yeast transformants with radioactive phosphocholine. Southern analysis indicated the presence of a single copy of the citidylyltransferase gene in A. thaliana.
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PMID:Cloning of CTP:phosphocholine cytidylyltransferase cDNA from Arabidopsis thaliana. 908 66


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