Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

As indicated by direct evidence, obtained by altering the cell-membrane permeability for Ca2+ in murine erythroleukaemia (MEL) cells, calpain is the triggering factor which connects fluctuations of the intracellular Ca2+ concentrations to the decay of protein kinase C (PKC), as well as to the kinetics of cell differentiation induced by hexamethylenebisacetamide. Cell exposure to verapamil caused a profound decrease in the rate of PKC down-regulation and a slower initial rate of accumulation of mature erythroid cells, whereas addition of the Ca2+ ionophore A23187 produced opposite effects. The high susceptibility of PKC-delta to calpain degradation, at concentrations of Ca2+ much lower than those required for degradation of the other PKC isoforms, may be explained by the finding that this kinase isoform is predominantly associated with the cell membrane. The different cellular localizations, as well as the different susceptibilities to calpain digestion, further support the hypothesis that in MEL cells the various PKC isoforms play distinct biological functions that are critical for the maintenance of the undifferentiated state of the cell and for its commitment to terminal erythroid differentiation.
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PMID:Changes in calcium influx affect the differentiation of murine erythroleukaemia cells. 782 42

Phosphorylation of calpain II (or its inhibitor) by the catalytic subunit of cyclic AMP-dependent protein kinase (A-PK), cyclic GMP-dependent protein kinase (G-PK), and protein kinase C (PK-C) was analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Among these protein kinases, the catalytic subunit of A-PK exhibited the strongest phosphorylations of both calpain II and its inhibitor. Arachidonic acid and staurosporine effectively inhibited phosphorylation regardless the type of kinase tested. Despite its lack of effect on the phosphorylation of calpain II by the catalytic subunit of A-PK, sphingosine moderately enhanced the phosphorylation of calpain II by G-PK. Other agents, including phosphatidylethanolamine, phosphatidylinositol and 1, 2-dioleoyl-sn-glycerol, had no significant effect.
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PMID:Regulation of the phosphorylation of calpain II and its inhibitor. 784 69

Platelet actin binding protein (ABP) as isolated from human platelets exists in at least four phosphorylated forms which we have designated ABP-0, ABP-1, ABP-2, and ABP-3 whose phosphate content ranges from 18 (ABP-0) to 40 (ABP-3) moles Pi/mole ABP. These forms differ in their resistance to calpain cleavage and ability to cross-link F-actin with ABP-3 being the best in each of these properties. Attempts to phosphorylate ABP-1, two or three with protein kinase C (PKC) were unsuccessful except if the proteins were pretreated with Escherichia coli alkaline phosphatase. All of the forms could be phosphorylated with cAMP-dependent kinase (PKA) and subsequent resistance to calpain cleavage conferred. Phosphorylation/dephosphorylation of ABP may be an important regulatory mechanism by which the cytoskeletal architecture is stabilized or transformed.
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PMID:Existence of multiple phosphorylated forms of human platelet actin binding protein. 786 35

Calpain has been identified as the intracellular proteinase that catalyzes the selective down-regulation of protein kinase C (PKC) isoforms, occurring in the early stages of commitment to terminal erythroid differentiation of murine erythroleukemia (MEL) cells induced by hexamethylenebisacetamide. This conclusion has been reached through direct experiments performed with two MEL cell clones, one characterized by a high and the other by a low rate of differentiation. In both cell types, introduction of an anti-calpain antibody resulted in a significant delay in the onset of down-regulation of PKC isoforms, and in an increase in the latent period that precedes differentiation. Both cell lines also displayed reduced rates of PKC decay and accumulation of mature erythroid cells. Furthermore, in the fast-responding clone, calpastatin, the natural calpain-inhibitor protein, was found to be almost completely absent, resulting in activation and expression of proteolytic activity of calpain even at micromolar concentrations of Ca2+, a condition not sufficient to trigger calpain activation in the slowly responding clone which contains high levels of calpastatin. The fast-responding MEL cell clone, enriched with calpastatin, displayed a lower rate of cell differentiation, with a kinetics almost identical to that observed following introduction of the anti-calpain antibody. It is proposed that Ca(2+)-dependent proteolysis plays a crucial role for the progress of MEL cell differentiation through the specific degradation of PKC isozymes.
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PMID:Modulation of the intracellular Ca(2+)-dependent proteolytic system is critically correlated with the kinetics of differentiation of murine erythroleukemia cells. 792 35

Two versions of the calmodulin binding domain of the plasma membrane Ca2+ ATPase, a 24-amino acid peptide, C24W (Q-I-L-W-F-R-G-L-N-R-I-Q-T-Q-I-R-V-V-N-A-F-R-S-S-NH2), and the corresponding phosphothreonine containing peptide, C24W-P (Q-I-L-W-F-R-G-L-N-R-I-Q-T(phospho)-Q-I-R-V-V-N-A-F-R-S-S-NH2), were synthesized. They were used to investigate the effect of threonine phosphorylation by protein kinase C on the binding of calmodulin by the calmodulin binding domain and on the inhibitory role of the domain on the activity of the Ca2+ pump. The phosphopeptide C24W-P was obtained after global phosphorylation of the free Thr side chain on the protected resin bound peptide. The phosphorylated calmodulin binding domain failed to bind calmodulin; this was shown by gel shift experiments, by fluorescence energy transfer studies and by competition experiments against calmodulin stimulation of the pump. The inhibition of the Ca2+ pump activity by the calmodulin binding domain in the absence of calmodulin was also affected by the phosphorylation of the threonine; the inhibition of the fully active calpain-truncated pump by the phosphothreonine containing peptide was lower than that by the unphosphorylated synthetic domain.
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PMID:Phosphorylation of the calmodulin binding domain of the plasma membrane Ca2+ pump by protein kinase C reduces its interaction with calmodulin and with its pump receptor site. 792 86

Although myristoylated alanine-rich C kinase substrate (MARCKS), has been employed as an indicator for the activation of protein kinase C (PKC) in intact cells, little is known about its specificity for PKC family members. To address this question, we partially purified human MARCKS from baculovirus-infected cells and compared the kinetic parameters for phosphorylation by PKC isozymes, conventional PKC alpha (cPKC alpha), novel PKC delta (nPKC delta), nPKC epsilon, and atypical PKC zeta (apKC zeta), all of which are distributed in a wide variety of cells. cPKC alpha, nPKC delta, and nPKC epsilon efficiently phosphorylated intact MARCKS protein in vitro. The affinity of MARCKS for cPKC alpha, nPKC delta, and nPKC epsilon was extremely high and decreased in the order alpha > delta > epsilon with Km values of 10.7, 20.7, and 29.8 nM, respectively. The rate of phosphorylation also decreased in the same order. In contrast, a PKC zeta did not phosphorylate MARCKS efficiently, and we were unable to estimate the kinetic parameters. These results suggest that cPKC alpha, nPKC delta, and nPKC epsilon but not a PKC zeta are enzymes that phosphorylate MARCKS in response to PKC activators in intact cells. The structural requirements of MARCKS for efficient phosphorylation by these PKC members were then examined using a peptide that surrounds the phosphorylation site of MARCKS (peptide MARCKS). Interestingly, intact MARCKS showed a 90-150 times lower rate of phosphorylation by PKCs compared with peptide MARCKS, whereas the former showed a 40-180 times higher affinity for these PKC members. This implies that intact MARCKS protein retains a very high affinity for PKC with the sacrifice of its phospho-accepting activity. The structural requirements of PKC were then examined using a calpain-cleaved active fragment of nPKC delta. MARCKS was phosphorylated by the active catalytic fragment as efficiently as by intact nPKC delta, indicating that the kinase domain is sufficient for the high affinity interaction with intact MARCKS. However, gel overlay assay revealed that both intact nPKC delta and its regulatory domain bind to MARCKS, suggesting that both the kinase and regulatory domains of nPKC delta are involved in the high affinity interaction with intact MARCKS protein.
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PMID:Specificity of the high affinity interaction of protein kinase C with a physiological substrate, myristoylated alanine-rich protein kinase C substrate. 798 36

The platelet membrane is lined by a membrane skeleton, which in turn appears to be associated with underlying cytoplasmic actin filaments. Glycoprotein IIb-IIIa appears to associate with the membrane skeleton in unstimulated platelets. Upon platelet activation, unidentified intracellular signals cause GP IIb-IIIa to become competent to bind adhesive ligand. We suggest that the membrane skeleton may play a role in allowing this inside-out signaling. Signaling molecules that appear to associate with the membrane skeleton in unstimulated platelets include pp60c-src, pp62c-yes, and GAP. Preliminary evidence suggests that components of the membrane skeleton may become phosphorylated on tyrosine residues prior to GP IIb-IIIa-ligand interactions. Once GP IIb-IIIa binds adhesive ligand in a platelet aggregate, there is signaling in the opposite direction. One consequence of the outside-in transmembrane signaling is that the membrane skeleton becomes more tightly associated with the underlying actin filaments as focal contact-like structures form. Proteins that accumulate in these focal contact-like structures with a time course identical to that of GP IIb-IIIa and in a GP IIb-IIIa-dependent manner include talin, vinculin, and spectrin. Signaling molecules that accumulate in the focal contact-like structures include pp60c-src, pp62c-yes, phosphoinositide 3-kinase, and protein kinase C. These are potential candidates for the enzymes that mediate the ligand-induced transmembrane signaling. Another enzyme involved in the ligand-induced signaling is calpain. This enzyme is activated as a consequence of ligand-GP IIb-IIIa interactions and cleaves components of the membrane skeleton. Future experiments will be needed to identify other signaling enzymes activated as a consequence of GP IIb-IIIa interactions and to determine which ones are responsible for inducing the cytoskeletal reorganizations that occur in platelets and other cells when integrins bind their adhesive ligands.
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PMID:Transmembrane signaling across the platelet integrin glycoprotein IIb-IIIa. 801 93

BALB/cBy (BALB) and CXB H mice responded similarly to a single intraperitoneal injection of butylated hydroxytoluene (BHT). This transient pneumotoxicity was characterized by an elevated lung weight and alveolar damage. These changes were accompanied by alterations in the calcium second messenger pathway, namely, two- to five-fold decreases in the activities and pulmonary content of protein kinase C alpha (PKC alpha) and calcium-dependent protease isozyme II (calpain II). BALB and CXB H mice varied in their responsiveness to a chronic (4-6 weekly injections) BHT regimen. CXB H mice became tolerant to BHT and all of the above parameters had returned to control values when examined after the last injection. Chronic BHT administration also failed to enhance lung tumor multiplicity in CXB H mice when the first BHT injection was preceded by the carcinogen, urethane. In contrast, the additional BHT doses potentiated the pathological and biochemical alterations in BALB mice above that found with acute treatment. This included a chronic inflammatory response characterized by the presence of activated macrophages, and a lung tumor multiplicity that was 3-fold greater than in BALB mice receiving urethane alone. One BHT injection increased calpain II mRNA in both strains (1.5- to 3-fold); mRNA declined following multiple BHT injections in BALB mice, but remained elevated in CXB H mice. Studies with the cytochrome P450 inhibitor, piperonyl butoxide, indicated that metabolism of BHT was required for both its acute and chronic effects. We conclude the following: (i) Differences between inbred mice in their susceptibility to chronic pneumotoxicant exposure may exist even when they respond similarly to an acute exposure of the same agent; (ii) A chronic inflammatory state involving a high concentration of activated macrophages may play an important role in tumor enhancement by a non-carcinogen; (iii) The protein contents of PKC alpha and calpain II decrease during BHT-induced lung damage.
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PMID:Strain-related differences in the pneumotoxic effects of chronically administered butylated hydroxytoluene on protein kinase C and calpain. 802 39

Proteolytic cleavage of protein kinase C (PKC) under cell-free conditions generates a co-factor independent, free catalytic subunit (PKM). However, the difficulty in visualizing PKM in intact cells has generated controversy regarding its physiological relevance. In the present study, treatment of SH-SY-5Y cells with 2-O-tetradecanoylphorbol 13-acetate resulted in complete down-regulation of PKC within 24 h without detection of PKM. By contrast, low levels of PKM were transiently detected following ionophore-mediated calcium influx under conditions which induced no detectable PKC loss. PKM was not detected during rapid cell-free degradation of partially purified SH-SY-5Y PKC alpha by purified human brain mM calpain. However, when the kinetics of PKC degradation were slowed by lowering levels of calpain, PKM was transiently detected. PKM was also only transiently observed following calpain-mediated degradation of purified rat brain PKC alpha. Densitometric analyses indicated that, once formed, PKM was degraded approximately 10 times faster than PKC. These data provide an explanation as to why PKM is difficult to observe in situ, and indicate that PKM should not be considered as an 'unregulated' kinase, since its persistence is apparently strictly regulated by proteolysis.
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PMID:Degradation of protein kinase C alpha and its free catalytic subunit, protein kinase M, in intact human neuroblastoma cells and under cell-free conditions. Evidence that PKM is degraded by mM calpain-mediated proteolysis at a faster rate than PKC. 807 May 69

The proteolysis of the 32P-labeled holoenzyme of cyclic AMP-dependent protein kinase (A-PKII:DEAE, peak II fraction) was analysed by SDS-polyacrylamide gel electrophoresis and autoradiography. The contaminants of the A-PKII and calpain II apparently did not interfere with the accuracy of this highly sensitive analysis. Phosphorylation of calpain II by the catalytic subunit of cyclic AMP-dependent protein kinase (A-PK) greatly enhanced the proteolysis of A-PKII, whereas phosphorylation by protein kinase C (PK-C) or cyclic GMP-dependent protein kinase (G-PK) slightly altered the proteolysis.
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PMID:Modulation of the activity of calpain II by phosphorylation--changes in the proteolysis of cyclic AMP-dependent protein kinase (peak II, DEAE). 819 24


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