Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The activity of the eukaryotic elongation factor 2 (eEF-2)-specific Ca(2+)- and calmodulin-dependent protein kinase III (CaM PK III) is regulated by phosphorylation. The kinase can be inactivated by treatment with alkaline phosphatase and subsequently reactivated by endogenous protein kinase. This kinase can be substituted for by the catalytic subunit of cAMP-dependent protein kinase but not by casein kinase II. The purified kinase preparation contains only one protein as judged by gel electrophoresis. This protein has a molecular mass of approximately 90 kDa and an isoelectric point of 5.2. Reactivation of the eEF-2 kinase is associated with the phosphorylation of this protein. The amino acid sequence obtained from the 90-kDa protein reveals substantial homology with that of murine heat shock protein 86 (HSP 86) a member of the HSP 90-family. Conventional preparations of HSP 90 contain an inactive eEF-2 kinase that could be activated after dephosphorylation and phosphorylation by the catalytic subunit of cAMP-dependent protein kinase.
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PMID:Phosphorylation regulates the activity of the eEF-2-specific Ca(2+)- and calmodulin-dependent protein kinase III. 188 75

The Mr = 38,300 polypeptide of the purified recombinant rat DNA polymerase beta served as an excellent substrate for protein kinase C (PKC) in vitro but not for the catalytic subunit of cAMP-dependent protein kinase. The phosphorylation by PKC resulted in inactivation of DNA polymerase beta activity, and recovery was achieved by dephosphorylation with alkaline phosphatase. Since the phosphorylated DNA polymerase beta was retained with use of a single-stranded DNA-cellulose column, inactivation might occur at a site different from that for the DNA binding. Amino acid sequence analysis of the phosphopeptides revealed that the phosphorylated sites were 2 serine residues at positions 44 and 55 from the NH2 terminus, either or both of which might be involved in the catalytic activity of DNA polymerase beta. Thus, the inactivation of the DNA repair enzyme, DNA polymerase beta, by PKC may be an important process in the modification of DNA metabolism in the nucleus through signal transduction processes.
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PMID:Inactivation of DNA polymerase beta by in vitro phosphorylation with protein kinase C. 204 Jun 2

Studies involving 32P labeling and wet ashing of isolated dynein reveal that isolated dynein contains approximately 6 mol of phosphate predominantly distributed over four polypeptides of molecular masses of 78, 76, 47, and 23 kDa. Dynein must, therefore, be phosphorylated to at least this extent in vivo. The catalytic subunit of cAMP-dependent protein kinase and an axonemal cAMP-dependent protein kinase contaminating the dynein preparation can further phosphorylate dynein in vitro. Each kinase can place up to 0.5 mol of phosphate on native dynein polypeptides of molecular masses of 78 and 34 kDa. Removal of two of the phosphates on isolated dynein by either acid or alkaline phosphatase results in a 28% decrease in the specific activity of dynein in the presence or absence of microtubules. Selective attenuation of the microtubule-activated ATPase, but not the uncoupled free dynein ATPase, would be indicative of a regulatory function of the phosphates. The in vivo regulation of the dynein ATPase by the two phosphates accessible to acid or alkaline phosphatase is therefore subject to question. Other phosphates on dynein must be examined for their effect on the microtubule-dynein cross-bridge cycle and motility before phosphorylation can definitively be established as a mode of dynein regulation.
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PMID:Phosphorylation of Tetrahymena 22 S dynein. 214 71

Dehydroepiandrosterone (DHEA) treatment is effective in preventing or delaying the onset of various genetic and induced disorders of mice and rats. Associated with the beneficial therapeutic effects exerted by action of this steroid is the development of hepatomegaly. To determine whether the changes associated with hepatomegaly also involve alterations in activities of tissue enzymes, we evaluated the effects of DHEA (0.45% in food, w/w) on hepatic protein kinases, phosphatases, and lipogenic enzymes in mice of various strains. The rates of fatty acid and cholesterol syntheses also were evaluated. DHEA administration resulted in profound changes in the sodium dodecylsulfate-polyacrylamide gel electrophoresis patterns of endogenous radiophosphorylated proteins obtained by incubation of liver homogenates with (gamma-32P]ATP. These changes were dependent upon the medium used for homogenization. Thus, when homogenates of liver tissue of DHEA-treated mice were prepared in Tris buffer containing sucrose (0.25 M) there was a marked decrease in phosphorylation of the proteins of relative molecular weight approximately 116,000 (Mr approximately 116,000), approximately 82,000, approximately 80,000, approximately 58,000, approximately 56,000, approximately 48,000, approximately 34,000, and approximately 31,000 compared with controls. With liver homogenates of DHEA-treated mice prepared in Tris buffer alone, there was a marked increase in phosphorylation of the proteins of Mr approximately 70,000, approximately 49,000, approximately 34,000, approximately 31,000, and 28,000 compared with controls. Moreover, the specific activity of kinases for endogenous protein acceptors in liver of control mice was higher than that in liver of DHEA-treated animals. The specific activities of casein kinase, cAMP-dependent protein kinase, and cGMP-dependent protein kinase remained unchanged with DHEA treatment, but the specific activity of histone kinase was increased approximately 30%. Long-term administration of DHEA also was associated with increases in the specific activities of liver AMPase and GTPase (approximately two times), but not of other nucleotidases, alkaline phosphatase, acid phosphatase, glucose-6-phosphatase, or phosphotyrosine phosphatase. The activity of hepatic NADP-linked malic enzyme was increased significantly (two to three times) by DHEA treatment of female mice of three different strains, but was unchanged in male C57BL/6 mice. The specific activities of hepatic glucose-6-phosphate dehydrogenase, NADP-linked isocitrate dehydrogenase, and ATP-citrate lyase were not affected significantly by DHEA treatment of mice. The rate of hepatic lipogenesis, determined by incorporation of tritium from 3H2O into fatty acids, was decreased approximately 70% in DHEA-treated mice, while the rate of cholesterol synthesis was increased approximately 44% compared with controls.
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PMID:Dehydroepiandrosterone feeding and protein phosphorylation, phosphatases, and lipogenic enzymes in mouse liver. 215 82

Phosphatidylinositol 4-phosphate (PIP) kinase (E.C. 2.7.1.68) has been purified about 1200-fold from rat liver plasma membranes, taking advantage of affinity chromatography on quercetin-Sepharose as a novel step. The purified PIP kinase showed no contamination by the following enzyme activities: phosphatidylinositol (PI) kinase (EC 2.7.1.67), protein kinase C (EC 2.7.1.-), diacylglycerol kinase (EC 2.7.1.-), phospholipase C (EC 3.1.4.11), protein-tyrosine kinase (EC 2.7.1.112), alkaline phosphatase (EC 3.1.3.1), triphosphoinositide phosphomonoesterase (EC 3.1.3.36), adenylate kinase (EC 2.7.4.3) and cAMP-dependent protein kinase (EC 2.7.1.37). The liver membrane enzyme requires high Mg2+ concentrations with a KM value of 10 mM. Ca2+ or Mn2+ could replace Mg2+ to a certain, though small, extent. Apparent KM values with respect to PIP and ATP were 10 and 65 microM, respectively. GTP was slightly utilized by the kinase as phosphate donor while CTP was not. Quercetin inhibited the enzyme with Ki = 34 microM. Extending our previous observations (Urumow, T. and Wieland, O.H. (1986) FEBS Lett. 207, 253-257 and Urumow, T. and Wieland, O.H. (1988) Biochim. Biophys. Acta 972, 232-238) [gamma S]pppG still stimulated the PIP kinase in extracts of solubilized liver membranes. 20-40% (NH4)2SO4 precipitation of the membrane extracts yielded a fraction that contained the bulk of enzyme activity but did not respond to stimulation by [gamma S]pppG any longer. This was restored by recombination with a protein fraction collected at 40-70% (NH4)2SO4 saturation, presumably containing a GTP binding protein and/or some other factor separated from the PIP kinase. In the reconstituted system [gamma S]pppG stimulated PIP kinase in a concentration dependent manner with maximal activation at 5 microM. This effect was not mimicked by [gamma S]pppA and was blocked by [beta S]ppG. These results strongly support our view that in liver membranes PIP kinase is regulated by a G-protein.
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PMID:Purification and partial characterization of phosphatidylinositol-4-phosphate kinase from rat liver plasma membranes. Further evidence for a stimulatory G-protein. 215 97

Mouse L929 cells were used to study the mechanism of cAMP induction of alkaline phosphatase (AP) activity. Following treatment with 200 microM 8-chlorophenylthio-cAMP (CPT-cAMP), alkaline phosphatase enzyme activity was observed to increase 80-fold after 24 h. The CPT-cAMP dose response of the alkaline phosphatase enzyme activity correlated well with the CPT-cAMP activation of cAMP-dependent protein kinase in L cells. A cDNA clone for the alkaline phosphatase was isolated and used to demonstrate a 10-fold increase in alkaline phosphatase mRNA levels after a 24-h treatment of L cells with CPT-cAMP. Increased mRNA levels were first detected 4-6 h, after CPT-cAMP treatment, and the level of alkaline phosphatase mRNA decreased rapidly after removal of CPT-cAMP. In vitro nuclear transcription studies showed that a 3-fold increase in alkaline phosphatase gene transcription was detectable 6 h after CPT treatment, and this increase was blocked by cycloheximide. In order to determine if the catalytic (C) subunit of cAMP-dependent protein kinase was able to mediate the induction of AP, L cells were transfected with expression vectors containing the metallothionein promoter and coding for the C alpha isoform of the catalytic subunit of cAMP-dependent protein kinase or for a catalytic subunit in which lysine 72 had been mutated to methionine (C alpha K72M). Zinc treatment of stably transfected cells expressing the wild-type C subunit showed an increase in protein kinase activity and an increase in AP activity. Zinc treatment of cells containing the mutant C subunit expression vector produced an increase in the amount of a protein which was recognized by C subunit antibodies on Western blots, but these cells showed no increase in protein kinase activity or in AP activity. We conclude that the C subunit is sufficient for transcriptional induction of the AP gene and that the phosphotransferase activity of the C subunit is required for this induction.
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PMID:Induction of alkaline phosphatase in mouse L cells by overexpression of the catalytic subunit of cAMP-dependent protein kinase. 216 96

The present studies examine the effects of in vivo and in situ progesterone treatment in the regulation of site-specific phosphorylation of the chicken oviduct progesterone receptor (PR). By gas-phase protein sequencing we have identified three hormonally regulated phosphorylation sites: Ser-211, Ser-260, and Ser-530. We determined phosphorylation stoichiometries by analyzing the amounts of phosphorylated and dephosphorylated serine at each site. Stoichiometries of sites 211 and 260 were about 20% under basal conditions and increased 1.5-2-fold by in situ progesterone treatment. Site 530 was virtually absent under basal conditions and induced to greater than 33% by in situ progesterone treatment. We tested several protein kinases for phosphorylation of the PR in vitro on these sites or peptides containing these sites. We found that the catalytic subunit of cAMP-dependent protein kinase mimicked the in vivo, hormone-induced altered mobility of PRs in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the in vivo and in vitro alterations were reversed by alkaline phosphatase. Finally, we showed that cAMP-dependent protein kinase phosphorylated Ser-528.
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PMID:Hormonal regulation and identification of chicken progesterone receptor phosphorylation sites. 239 63

The role of phosphorylation/dephosphorylation in the regulation of CTP:phosphocholine cytidylyltransferase activity was investigated. Incubation of post mitochondrial supernatant with cAMP-dependent protein kinase (50 units) led to an increased (28%) recovery of the cytidylyltransferase in the cytosolic fraction, while incubation with an intestinal alkaline phosphatase (20 units) led to an increased (61%) recovery in the microsomal fraction. When pure cytidylyltransferase was incubated with washed microsomes in the presence of cAMP-dependent protein kinase (133 units), the enzyme associated with the supernatant fraction increased (3.12 +/- 0.02 to 3.77 +/- 0.03 nmol/min/ml) while that of the microsomal fraction decreased (1.36 +/- 0.01 to 0.56 +/- 0.05 nmol/min/ml) by 2.5-fold. The increase in the cytidylyltransferase activity in the supernatant corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (40 units) decreased the cytidylyltransferase activity in the supernatant (3.61 +/- 0.08 to 2.88 +/- 0.07 nmol/min/ml) while the activity in the microsomal fraction increased (0.56 +/- 0.08 to 1.16 +/- 0.06 nmol/min/ml) by 2-fold. The decrease in the cytidylyltransferase activity in the supernatant corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of cytidylyltransferase with phosphatidylcholine vesicles in the presence of cAMP-dependent protein kinase (110 units) decreased the cytidylyltransferase activity by 30%. The decrease in cytidylyltransferase activity corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (20 units) resulted in a 41% increase in the cytidylyltransferase activity. The increase in cytidylyltransferase activity corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of the cytidylyltransferase with [gamma-32P] ATP and cAMP-dependent protein kinase led to incorporation of 32P into the serine residues of cytidylyltransferase. If the cytidylyltransferase were preincubated with alkaline phosphatase prior to incubation with cAMP-dependent protein kinase, 2-fold more 32P (0.2 mol P/mol cytidylyltransferase) was incorporated into the cytidylyltransferase. Collectively, this data is in agreement with a role for reversible phosphorylation in the regulation of cytidylyltransferase.
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PMID:CTP:phosphocholine cytidylyltransferase is a substrate for cAMP-dependent protein kinase in vitro. 253 19

We have examined the potential for using calf uterine progesterone receptor (PR) as a substrate for phosphorylation by cAMP-dependent protein kinase (cAMP-PK), PR was found to interact with anti-PR monoclonal antibody alpha PR6 (Sullivan et al., 1986), which was to immunopurify the receptor. Protein staining of the purified preparation revealed the presence of two major bands corresponding to 114 kDa and 90 kDa peptides; only 114 kDa peptide could be photoaffinity-labeled with R5020. The 90 kDa peptide co-migrated with 90 kDa heat shock protein (hsp-90) precipitated by anti-hsp-90 monoclonal antibody AC88 (Riehl et al., 1985). Incubation of the immunopurified protein-A-Sepharose-adsorbed PR with the catalytic subunit of cAMP-PK in the presence of gamma-[32P]ATP and divalent cations resulted in a Mg++-dependent incorporation of 32P-radioactivity into both the 114 kDa and the hsp-90 peptides. Small 32P-incorporation was also seen in the 114 kDa peptide in the presence of Mn++. A 60 degrees C preincubation of immunopurified PR increased the extent of phosphorylation of the hsp-90 peptide. A pretreatment with alkaline phosphatase reduced the ability of PR to act as a substrate while the steroid occupancy of PR appeared to enhance the phosphorylation of the 114 kDa peptide. The differential cation requirement for the phosphorylation of 114 kDa and hsp-90 peptides and a selective hormone-dependent increase in the phosphorylation of the 114 kDa peptide suggest a possible role of phosphorylation in mediating progesterone action in the calf uterus.
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PMID:Phosphorylation of calf uterine progesterone receptor by cAMP-dependent protein kinase. 254 44

Stathmin is a ubiquitous soluble protein (Mr approximately 19,000, pI approximately 6.2-5.5) whose phosphorylation is associated with the intracellular mechanisms involved in the regulations of cell differentiation and functions by extracellular effectors. Its purification from rat brain and the preparation of specific antibodies allowed us to identify a set of immunologically related unphosphorylated (N1, N2) and phosphorylated (P1, P2a, P2b, P3) proteins of decreasing isoelectric points. All these proteins yielded identical silver-stained or 32P-radioactive peptide maps with the protease V8 from Staphylococcus aureus, indicating that they are also structurally related. In vitro phosphorylation with the exogenous catalytic subunit of the cAMP-dependent protein kinase, as well as dephosphorylation with alkaline phosphatase, indicated that P1, P2, and P3 derived from N1 and N2 by progressive phosphorylation. Phosphorylation of individual proteins extracted from semi-preparative two-dimensional polyacrylamide gels demonstrated the existence of two distinct isoforms of stathmin, alpha and beta: N1 and N2 are their respective unphosphorylated forms (alpha O and beta O), whereas proteins P1-P3 could be resolved as at least three increasingly phosphorylated forms of both alpha and beta stathmin (alpha 1, alpha 2, alpha(3) and beta 1, beta 2, beta(3]. In intact pituitary GH4C1 cells, hormones like thyrotropin-releasing hormone and vasoactive intestinal peptide induced a similar conversion from N1 and N2 to P1, P2, and P3. The phosphorylation of both alpha and beta isoforms of stathmin is therefore a physiologically significant response to specific extracellular regulatory agents. In conclusion, stathmin represents a family of at least two distinct protein isoforms, whose respective phosphorylation and expression might play a role in its likely function as an intracellular relay of various converging extracellular signals.
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PMID:Identification of two distinct isoforms of stathmin and characterization of their respective phosphorylated forms. 272 86


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