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:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of cAMP-dependent protein kinase (cAMP-PK) phosphorylation on the degradation of the microtubule-associated protein tau by calpain were studied. Purified bovine brain tau that had been phosphorylated by cAMP-PK had a slower migration pattern on sodium dodecyl sulfate-polyacrylamide gels and a more acidic, less heterogeneous pattern on two-dimensional, nonequilibrium pH gradient electrophoresis (NEPHGE) gels compared with untreated tau. Phosphorylation of tau by cAMP-PK significantly inhibited its proteolysis by calpain compared with untreated tau. To our knowledge this is the first demonstration that phosphorylation of tau by a specific kinase results in increased resistance to hydrolysis by calpain. Tau dephosphorylated by alkaline phosphatase migrated more rapidly on sodium dodecyl sulfate-polyacrylamide gels and also showed an altered two-dimensional NEPHGE pattern. Dephosphorylation of tau had no effect on its susceptibility to calpain proteolysis, indicating that regulation of the susceptibility to calpain hydrolysis is due to the phosphorylation of a specific site(s). These results suggest a role for phosphorylation in regulating the degradation of tau. Abnormal phosphorylation could result in a protease-resistant tau population which may contribute to the formation of paired helical filaments in Alzheimer's disease.
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PMID:Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain. 173 Jul 2

To identify the protein kinase that is responsible for catalyzing phosphorylation of actin-binding protein (ABP) in platelets, we have examined the effects of protein kinase C and cAMP-dependent protein kinase on this process. We found that purified platelet protein kinase C from platelets was unable to phosphorylate ABP in vitro. However, a crude platelet kinase preparation phosphorylated ABP in the presence of cAMP, but not in the presence of Ca2+/phosphatidylserine. Fresh platelet plasma membranes incubated with [gamma-32P]ATP phosphorylated ABP in the presence of cAMP and the process was blocked by a cAMP-dependent protein kinase inhibitor; ABP phosphorylation induced by prostaglandin E1 (PGE1) appeared to be reduced by the subsequent addition of thrombin. These results strongly suggest that in situ ABP is phosphorylated by activated cAMP-dependent protein kinase when platelet function is inhibited by PGE1. Furthermore, in the PGE1-treated platelets, ABP was proteolyzed at a slower rate than in control platelets when they were lysed with Triton in the absence of EGTA. Partially purified ABP was proteolyzed by calpain in vitro at a slower rate as well. It was demonstrated that ABP from PGE1-treated platelets recovered its sensitivity to calpain after ABP was incubated with a protein phosphatase that had been purified from platelets. We postulate that ABP is stabilized against proteolysis in response to cAMP-elevating agents and that this blocks cytoskeleton reorganization.
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PMID:In situ phosphorylation of platelet actin-binding protein by cAMP-dependent protein kinase stabilizes it against proteolysis by calpain. 254 93

Hormone responsiveness is mediated by signal transduction mechanisms involving second messengers, such as cAMP and Ca2+, which regulate reversible changes in the phosphorylation state of proteins. During senescence individuals frequently exhibit a diminished responsiveness to hormones. We examined changes in enzymes involved in protein phosphorylation reactions that might account for this decreased adaptiveness in old mice, and observed the following post-maturational changes: (1) cAMP-dependent protein kinase (Pk-A) specific activity decreased in spleen cytosol and in the particulate fractions of lung, spleen and liver of 24-month-old mice as compared to 2-month-old mice. Splenic cytosolic Pk-A activity decreased by 18 months of age, while particulate activity decreased by 6 months; (2) The amount of 8-N3-[32P]cAMP, a photoaffinity analog of cAMP, incorporated into Pk-A regulatory (R)-subunits from spleen and liver particulate fractions decreased, while photolabeling of R-subunit degradative products with this analog in heart and spleen cytosol increased. (3) Age-dependent increases in membrane-associated protease activities were found in all organs, along with a decrease in cytosolic lung calpain activity. These proteolytic changes may account for the enhanced R-subunit degradation and decreased Pk-A activities observed during senescence. (4) Age-dependent alterations in Ca2+/phospholipid-dependent protein kinase (Pk-C) are organ specific: lung, liver, brain, and heart demonstrate no change in Pk-C activity, while spleen exhibits decreased activity. We hypothesize that these age-dependent alterations in kinase and proteolytic activities may be in part responsible for changes in cellular response to hormonal stimulation, differentiation signals, and antigen responsiveness during senescence.
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PMID:Age-dependent changes in murine protein kinase and protease enzymes. 285 80

We have previously shown that at least five linked genes are co-amplified and overexpressed in the multi-drug resistant (MDR) Chinese hamster ovary cell line CHRC5. We show here that one of these genes (class 4) codes for a small phosphorylated, cytosolic protein, sorcin/V19, known to be overproduced by many MDR cell lines. The class 4 gene codes for a nested set of mRNAs, varying in size between 1000 and 2500 nucleotides. Sequence analysis of complementary DNAs shows that these mRNAs encode a protein of 198 amino acids. The identity of this protein with sorcin was established by comparison with the amino acid sequence of two peptides from mouse sorcin. Hamster sorcin is a 22-kd protein with four 'E-F hand' structures typical of calcium-binding sites and it has substantial homology with the light chain of calpain. Two of the calcium-binding sites contain putative recognition sites for cAMP-dependent protein kinase. These may account for the known phosphorylation of sorcin. The unknown function of sorcin might therefore be controlled by both calcium and cAMP levels. The contribution of sorcin to multidrug resistance, if any, remains to be tested.
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PMID:A 22-kd protein (sorcin/V19) encoded by an amplified gene in multidrug-resistant cells, is homologous to the calcium-binding light chain of calpain. 302 74

The antioxidant, butylated hydroxytoluene (BHT), causes lung toxicity in mice followed by regenerative repair, and can also modulate the development of carcinogen-induced lung adenomas. We are investigating changes in pulmonary biochemistry following BHT treatment in order to understand the mechanisms of BHT-induced pulmonary regenerative repair. BHT administration lowered cytosolic Ca2+-activated neutral protease (calpain) activity, increased the activity of the endogenous calpain inhibitor, calpastatin, increased the extent of photoincorporation of 8-N3-[32P]cAMP into a Mr 37,000 proteolytic product derived from cAMP-dependent protein kinase regulatory (R) subunits, and increased membrane-associated protease activity. All of these changes were dependent on the BHT dosage; the altered proteolytic activities occurred at a dose lower than that which caused observable lung toxicity as assessed by the lung weight/body weight ratio. Decreased cytosolic calpain activity was detectable within 1 day after BHT administration, was lowest at 4-7 days, and had not returned to control levels by Day 21, a time when normal lung morphology had been regained. The decrease in calpain activity cannot fully be accounted for by increased calpastatin activity; upon separation of these proteins by DEAE chromatography, the amount of calpain activity from BHT-treated mice remained lower than the corresponding peak from control mice. Increased photolabeling of the Mr 37,000 protein began at 1 day and continued to increase up to 4 days after BHT. All of the cytosolic changes preceded the increased particulate proteolytic activity by 1-2 days. R-subunits which have dissociated from their catalytic subunits are more susceptible to degradation by calpain, but BHT treatment did not enhance subunit dissociation as determined by the elution profile of 8-N3-[32P]cAMP-labeled R-subunits following DEAE chromatography. A large percentage of the particulate protease activity was inhibited by calpastatin, leupeptin, and E-64, all of which are known to inhibit calpain activity; this suggested that calpain accounted for most of this activity. Changes in the activities of proteases which catalyze limited proteolysis reactions may play an important role in the repair of acute lung injury.
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PMID:Changes in pulmonary calpain activity following treatment of mice with butylated hydroxytoluene. 303 20

Three enzymes relevant to signal transduction were compared in replicating, quiescent and senescent human diploid fibroblasts (HDF). These were Ca(2+)-dependent thiol protease (calpain), cAMP-dependent protein kinase (Pk-A), and calcium/phospholipid-dependent protein kinase C (Pk-C). The amounts of these enzymes in quiescent HDF were slightly greater or the same as in replicating HDF. In contrast, senescent HDF exhibited higher Pk-C, Pk-A and proteolytic activities than did either replicating or quiescent cells. While the elevated protein kinase activities could be accounted for by the larger size of senescent cells relative to younger cells, the increased calpain activity exceeded this size differential. Immunoblotting studies with antisera to both Pk-C and calpain demonstrated increased enzyme concentrations in parallel with the increased activities. Photolabeling cell extracts with an analog of cAMP, 8-N3-[32P]cAMP, provides an estimate of Pk-A concentration. By this criterion, senescent HDF have more Pk-A molecules than do young cells that are either replicating or quiescent. Only the type I isozyme of Pk-A (Pk-A-I) was observed in any of these cells. Photolabeling with 8-N3-[32P]cAMP demonstrated more degradative fragments of the Pk-A regulatory subunit (RI) in senescent cells also. This is a logical consequence of the increased calpain activity in senescent cells, since RI is a substrate for calpain. These results imply that senescent cells do not fail to enter S phase owing to inadequate concentrations of Pk-A or Pk-C. Rather, the increased quantities of these enzymes in senescent cells may reflect aberrations elsewhere along signal transduction pathways that coordinate cell size with cell proliferation.
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PMID:Serine/threonine protein kinases and calcium-dependent protease in senescent IMR-90 fibroblasts. 811 16

In this study, the phosphorylation, calpain hydrolysis and tubulin binding of three recombinant human tau isoforms were examined. The three isoforms used in these studies were tau with three (T3) or four (T4) tandemly repeated tubulin binding domains located in the carboxy-terminal half of the molecule; and tau with four-tandem repeats and a 58-amino acid insert in the amino terminus (T4L). Both cAMP-dependent protein kinase (cAMP-PK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) readily phosphorylated the three human tau isoforms, although cAMP-PK phosphorylated them to a significantly greater extent than CaMKII. Phosphorylation of T3, T4 and T4L by cAMP-PK or CaMKII resulted in the slowed migration of the protein bands on sodium dodecyl sulfate-polyacrylamide gels and a shift of the isoelectric variants to more acidic positions on two-dimensional non-equilibrium pH gradient electrophoresis gels compared with controls. However, the phosphorylation-induced changes in the electrophoretic migration of the tau isoforms were unique for each kinase. Two-dimensional phosphopeptide maps and sequential phosphorylation experiments indicate that cAMP-PK phosphorylates sites in the human tau isoforms that are phosphorylated by CaMKII, as well as unique sites that are not phosphorylated by CaMKII. T3, T4 and T4L were hydrolyzed similarly by calpain; however, the calpain proteolysis of the recombinant tau isoforms was significantly faster than the proteolysis of human or bovine tau. Phosphorylation of the isoforms by either cAMP-PK or CaMKII did not alter the rate or extent of calpain proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation, calpain proteolysis and tubulin binding of recombinant human tau isoforms. 838 12

The effects of cAMP-dependent protein kinase (cAMP-PK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation on the calpain-mediated degradation of microtubule-associated protein 2 (MAP-2) were studied. Both cAMP-PK and CaMKII readily phosphorylated MAP-2. However, cAMP-PK phosphorylated MAP-2 to a significantly greater extent than did CaMKII (4.5 mol 32P/mol MAP-2 and 1.4 mol 32P/mol MAP-2, respectively). Phosphorylation of MAP-2 by cAMP-PK, but not by CaMKII, significantly inhibited the calpain-induced hydrolysis of MAP-2. These results demonstrate that the phosphorylation of sites on the MAP-2 molecule accessible to cAMP-PK, but not to CaMKII, result in increased resistance to calpain proteolysis.
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PMID:Calpain-mediated proteolysis of microtubule-associated protein 2 (MAP-2) is inhibited by phosphorylation by cAMP-dependent protein kinase, but not by Ca2+/calmodulin-dependent protein kinase II. 839 Oct 85

Stimulation of secretion from rat alveolar epithelial type II cells by the beta-adrenergic agonist terbutaline activates cAMP-dependent protein kinase (PKA). The same secretagogue also activates endogenous protease calpain in type II cells. In this study, we investigated the effect of calpain activation on PKA and its phosphorylation activity in stimulated type II cells. Type II cells were either pretreated with cell-permeable calpain specific inhibitor (N-acetyl-leucyl-leucyl-methioninal) or untreated, and subsequently stimulated with terbutaline. Stimulus-induced phosphorylation activity was assayed using the PKA-specific substrate Kemptide. Maximum PKA activity was observed within 1-3 min of stimulation. Peak activity of the untreated cells was 20-25% higher and longer than that of the inhibitor-treated cells. The stimulus-induced phosphorylation activity of both cell groups was suppressable by PKA-specific inhibitor. Concomitant photoaffinity labeling with radioactive 8-azido-cAMP revealed that a 39 kDa proteolytic fragment was generated in response to stimulation by terbutaline. Stimulus-induced activation of PKA resulted in the phosphorylation of two endogenous proteins, p112 and p47. Phosphorylation of p112 and p47 was modulated in cells pretreated with calpain inhibitor or in the presence of PKA inhibitor. Aggregate results indicate that stimulus-induced proteolysis of pKA occurs in type II cells, suggesting that limited proteolysis of PKA by endogenous calpain may convert an initial transient signal to sustained and augumented phosphorylation activity for secretion.
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PMID:Secretagogue-induced proteolysis of cAMP-dependent protein kinase in intact rat alveolar epithelial type II cells. 863 Mar 29

A middle region of human endothelial actin-binding protein (ABP) was subcloned and expressed in the pT7-7/E. coli BL21 (DE3) system. As predicted by the amino acid sequence this 71 kD truncated protein (residues 1717-2360) contained a calpain cleavage site and two of the three presumptive cAMP-dependent protein kinase phosphorylation sites. This peptide fragment comprised all the elements needed to confer stability against calpain proteolysis to ABP after PKA phosphorylation.
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PMID:Expression in Escherichia coli, phosphorylation with cAMP-dependent protein kinase and proteolysis by calpain of a 71-kDa domain of human endothelial actin binding protein. 912 21


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