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

The cytoplasmic form of protein kinase C (PKC) is inactive, probably because the pseudosubstrate region in its regulatory domain blocks the substrate-binding site in its kinase domain. Calcium ions cause a translocation to the membrane: maximum activation requires a negative lipid such as phosphatidylserine (PS) and the neutral lipid diacylglycerol (DAG) but the mechanism by which PS and DAG activate PKC is unknown. Pseudosubstrate region 19-36 of PKC-beta has six basic and one acidic amino acids and region 19-29 has five basic and no acidic amino acids. Since any binding of basic residues in the pseudosubstrate region to acidic lipids in the membrane should stabilize the active form of PKC, we studied how peptides with amino acids equivalent to residues 19-36 and 19-29 of PKC-beta bound to phospholipid vesicles. We made equilibrium dialysis, filtration, and electrophoretic mobility measurements. The fraction of bound peptide is a steep sigmoidal function of the mol fraction of negative lipid in the membrane, as predicted from a simple theoretical model that assumes the basic residues provide identical independent binding sites. The proportionality constant between the number of bound peptides/area and the concentration of peptide in the bulk aqueous phase is 1 micron for a membrane with 25% negative lipid formed in 0.1 M KCl. Equivalently, the association constant of the peptide with the membrane is 10(4) M-1, or the net binding energy is 6 kcal/mol. Thus the interaction of basic residues in the pseudosubstrate region with acidic lipids in the membrane could provide 6 kcal/mol free energy towards stabilizing the active form of PKC.
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PMID:Peptides that mimic the pseudosubstrate region of protein kinase C bind to acidic lipids in membranes. 188 33

The appearance of the biphasic insulin secretory response several days after birth suggests that maturation of a critical step in stimulus-secretion coupling occurs during the early neonatal period. To clarify the role of protein kinase C (PKC) during this time, we examined the pancreatic islets of adult, 3-day neonatal, and 19-day fetal rats for the presence of different PKC isoenzymes. Western-blot analysis of islet extracts showed the presence of PKC isoforms in both adult and neonatal tissues. Immunocytochemistry of adult islets revealed a differential expression in islet cell types. PKC-alpha was found only in beta-cells, PKC-gamma in alpha-cells, and PKC-epsilon in delta-cells and vascular walls. Immunoreactivity for PKC-beta was not detected in any cell type. All three isoenzymes were also present in neonatal islets; however, in contrast to adult tissue, immunoreactivity for either PKC-alpha or PKC-gamma was present in relatively few cells. There was no apparent immunoreactivity for PKC-alpha or PKC-gamma in fetal islets, although these tissues contained strong staining for insulin and glucagon. These data show that three of the PKC isoforms are restricted to a particular islet cell type, where they may play a unique role in the secretion of a specific hormone. Moreover, our results demonstrate that these enzymes, especially PKC-alpha, appear during the early neonatal period. This age-dependent expression may be linked to the development of the biphasic insulin release response.
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PMID:Age-dependent expression of protein kinase C isoforms in rat islets. 193 8

We have studied the expression of mRNA encoding all known protein kinase C (PKC) isozymes (alpha, beta, gamma, delta, epsilon, zeta, and eta) in murine tumor cell lines that exemplify hemopoietic cells arrested at different stages of development as well as in normal hemopoietic cells. We demonstrate that some of the isozymes, PKC-alpha, -beta, and -eta, are differentially expressed in different lineages. PKC-alpha and -beta generally are not detectable in myeloid cell lines, where PKC-delta is the predominant isoform. Both PKC-alpha and -beta are abundant in most T and B lymphocytic lines, but steady state levels of PKC-beta mRNA are lowest in plasma cell tumors, which exemplify the terminally differentiated B lymphocyte. In contrast, the levels of PKC-alpha mRNA remain high in plasma cell tumors, and a novel, 2.5-kb PKC-alpha mRNA gains prominence. PKC-eta mRNA is the major PKC isoform expressed in T lymphocytes, but it also is highly abundant in some myeloid lines. PKC-delta is expressed at high levels in all the lines we studied, whereas PKC-epsilon and -zeta are found in most cells but only at rather low levels. Analysis of myeloid clones derived from bipotential B lineage progenitor cell lines suggests that the B cell phenotype is associated with the expression of PKC-alpha. The close correlation of protein levels with mRNA levels indicates that PKC expression in hemopoietic cells is mainly regulated at the level of mRNA. The lineage- and differentiation stage-specific patterns of PKC-isozyme expression presented here suggest the involvement of specific PKC isozymes in differentiation as well as lineage determination of hemopoietic cells.
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PMID:Expression of protein kinase C genes in hemopoietic cells is cell-type- and B cell-differentiation stage specific. 194 Mar 80

Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of murine erythroleukemia cells (MELC). Commitment, the irreversible initiation of the program of terminal-cell differentiation, is first detected in HMBA-sensitive DS19-SC9 MELC in culture after 10 to 12 h of exposure to HMBA. Vincristine (VC)-resistant MELC derived from the DS19-SC9 MELC line display increased sensitivity to HMBA and become committed with little or no latent period. In the present study, we showed that the MELC line R1, which is resistant to HMBA-mediated differentiation, became sensitive to inducer if selected for a low level of VC resistance (less than 10 ng of VC per ml). Four independently derived VC-resistant cell lines from HMBA-resistant R1 cells, designated R1[VCR]a to R1[VCR]d, acquired sensitivity to HMBA and the accelerated kinetics of commitment that are characteristic of VC-resistant MELC derived from the parental DS19-SC9 cells. The calcium channel blocker verapamil suppresses the VC resistance of R1[VCR] cells but does not alter the accelerated response to HMBA. In R1[VCR] cells there was no detectable increase in the level of the 140-kilodalton P-glycoprotein. Transient inhibition of protein synthesis during the latent period delays inducer-mediated commitment of VC-sensitive DS19-SC9 MELC but does not alter the accelerated commitment kinetics of R1[VCR]a cells. Previously, we have reported evidence that protein kinase C beta (PKC beta) plays a role in HMBA-induced MELC differentiation and that compared with DS19-SC9 cells, R1 cells have a relatively low level and R1[VCR]a cells have a high level of PKC beta. These findings suggest that (i) acquisition of VC resistance overcomes the block acquired by R1 cells to HMBA-mediated differentiation; (ii) the accelerated kinetics of HMBA-induced commitment of VC-resistant MELC is not dependent on the verapamil-sensitive transport channel that is responsible, at least in part, for resistance to VC; (iii) in VC-resistant MELC, there is constitutive expression or accumulation of a protein required for HMBA-induced differentiation; and (iv) an elevated level of PKC beta activity may play a role in the altered response of R1[VCR] and other VC-resistant MELC to HMBA.
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PMID:Conversion of differentiation inducer resistance to differentiation inducer sensitivity in erythroleukemia cells. 197 44

Iron-transferrin (FeTF) is an essential growth factor required for proliferation of lymphoid cells. FeTF activates protein kinase C (PKC) in the lymphoblastoid T-cell line, CCRF-CEM. We have treated CEM cells with human FeTF, then examined levels of PKC mRNA by hybridization analysis using cDNA probes specific for alpha-, beta-, and gamma-PKC subspecies. CEM cell mRNA hybridized with the beta-subspecies probe but not with probes for alpha- or gamma-subspecies. After exposure to FeTF an increase in PKC-beta mRNA was detectable at 10 minutes, peaked at 12 hours, and was sustained for 72 hours. Nuclear transcription assays demonstrated that rates of PKC-beta mRNA transcription were increased in FeTF-treated cells. By contrast, steady state levels of PKC-beta mRNA did not increase after treatment of cells with apotransferrin or gallium TF. Similarly, treatment with soluble iron as ferric ammonium citrate did not increase steady state levels of PKC-beta mRNA, despite producing a marked increase in cellular ferritin content. Ferritin increased from a baseline value of 63 ng/10(6) cells to 98 and 100 ng/10(6) cells in CEM cells treated for 1 hour with ferric ammonium citrate or FeTF, respectively. FeTF did not increase cytoplasmic-free calcium in CEM cells loaded with fura-2, indicating that binding of FeTF to transferrin receptors did not open membrane Ca2+ channels or release intracellular Ca2+. In addition, pretreatment of cells with desferrioxamine, but not ferrioxamine, blocked the FeTF-induced increase in PKC-beta transcripts. Therefore, iron as FeTF (not soluble iron or nonferric TF) stimulates transcription of the CEM cell PKC-beta gene. Transcriptional rate of the PKC-beta gene does not correlate with cellular iron content as judged by ferritin measurements. Furthermore, the requirement for FeTF does not appear to reflect activation of a classic agonist pathway as judged by stable cellular Ca2+. These data suggest that delivery of iron by FeTF to one or more specific cellular compartments may stimulate PKC-beta gene transcription in CEM cells.
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PMID:Induction of protein kinase C mRNA in cultured lymphoblastoid T cells by iron-transferrin but not by soluble iron. 200 52

We have examined the role of protein kinase C (PKC)-beta II and its functional relationship to inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and intracellular Ca2+ in the contraction of smooth muscle cells from the rabbit internal and sphincter (IAS). PKC-beta (0.1-100 U/ml) and Ins(1,4,5)P3 (10(-9) to 10(-6) M) caused concentration-dependent contraction of IAS smooth muscle cells permeabilized by saponin. The combination of threshold concentrations of Ins(1,4,5)P3 (10(-9) M) and PKC (0.1 U/ml) was more than additive, causing near maximal shortening (28.2 +/- 2.1% decrease in cell length from control). The response to high concentrations of Ins(1,4,5)P3 and PKC used in combination was not greater than the response to either agent alone. The calmodulin antagonist W-7 (10(-9) M) inhibited the maximal contraction induced by Ins(1,4,5)P3 but not contraction caused by PKC, whereas the PKC antagonist H-7 (10(-6) M) inhibited the maximal contraction induced by PKC but not contraction caused by Ins(1,4,5)P3. Threshold doses of the ionophores A23187 (10(-9) M) and ionomycin (0.2 ng/ml) caused little contraction by themselves, but they potentiated the response elicited by a threshold concentration of PKC (0.1 U/ml), inducing maximal contraction. Preincubation of IAS cells with 4 mM Sr2+, which inhibits the release of intracellular Ca2+, abolished the potentiating effect of Ins(1,4,5)P3 and calcium ionophores on PKC, but the calmodulin antagonist W-7 did not. These data suggest that the contractile effect of maximally effective doses of PKC is independent of the effects of Ins(1,4,5)P3. At submaximal concentrations, however, PKC-dependent contraction is potentiated by Ins(1,4,5)P3 or by ionophore-mediated release of intracellular Ca2+ without requiring calmodulin activation.
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PMID:Regulation of smooth muscle contraction in rabbit internal anal sphincter by protein kinase C and Ins(1,4,5)P3. 201 29

The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms.
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PMID:Characterization of calcium-independent forms of protein kinase C-beta in phorbol ester-treated rabbit platelets. 202 87

The localization of protein kinase C-beta-like immunoreactivity (PKC-beta-LI) was studied in the rat dorsal root ganglion (DRG) using an antibody specific for a peptide sequence common to the beta 1- and beta 2-subtypes. PKC-beta-LI was seen in 45% of neuronal cell bodies and in nerve fibers, which were mostly myelinated. The PKC-beta-LI-containing cell bodies had a diameter significantly larger than the unlabeled cell bodies. The results suggest that PKC-beta is a PKC subtype involved in cell surface signal transduction in the subpopulation of large DRG neurons.
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PMID:Localization of protein kinase C-beta-like immunoreactivity in the rat dorsal root ganglion. 215 99

In this study we examined the effects of insulin on protein kinase C (PKC) activity in cultured fetal chick neurons. PKC activity, measured as 32P incorporation into histone H1 in the presence of calcium (500 microM), phosphatidylserine (100 micrograms/ml), and diolein (3.3 micrograms/ml) minus the incorporation in the presence of calcium alone, was detected in neuronal cytosolic (207 +/- 33 pmol/min/mg) and membrane (33 +/- 8 pmol/min/mg) fractions. Insulin added to intact neurons increased the activity of PKC in both cytosolic and membrane fractions by about 40%. Neurons preincubated with cycloheximide (10 micrograms/ml) 30 min prior to insulin treatment showed the same degree of stimulation of PKC activity by insulin. The activation of PKC was maximal within 5-10 min of insulin exposure and was sustained for at least 60 min. Insulin stimulated PKC in a dose-dependent manner, with a maximal response obtained at 100 ng/ml. Addition of phosphatidylserine and diolein to neuronal cell extracts resulted in the phosphorylation of four major cytosolic proteins (70, 57, 18, and 16 kDa) and one major membrane protein (75 kDa). Phosphorylation of all five proteins was increased 2-fold in extracts from insulin-treated neurons. Immunoblot analysis of whole cell extracts using antibodies against PKC-alpha, PKC-beta, PKC-gamma, PKC-delta, and PKC-epsilon revealed that cultured fetal chick neurons contained only one of these PKC isoforms, the epsilon-isoform. The enzyme was mostly cytosolic. Insulin had no effect on either the amount of distribution of PKC-epsilon in cultured neurons but induced a small change in the mobility of PKC-epsilon on sodium dodecyl sulfate-polyacrylamide gels. When assay conditions were designed to measure specifically the activity of PKC-epsilon, using a synthetic peptide substrate in the absence of calcium, activity was 50 +/- 12% higher in insulin-treated cells (p less than 0.005). PKC activity in control and insulin treated-neurons was almost completely inhibited when assays included a peptide identical to the pseudo-substrate binding site of PKC-epsilon. We conclude that PKC-epsilon is the major PKC isoform present in cultured fetal chick neurons. Insulin stimulates PKC-epsilon activity by a mechanism that does not involve translocation of the enzyme from cytosol to membrane.
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PMID:Insulin stimulates the activity of a novel protein kinase C, PKC-epsilon, in cultured fetal chick neurons. 220 68

The isoform pattern of protein kinase C (PKC) was examined in wild-type and Adriamycin-resistant (HL-60/AR) HL-60 leukemia cells. Analyses were carried out by immunoblotting with mouse monoclonal antibodies against PKC-alpha and PKC-beta and a rabbit polyclonal antibody against the variable (V3) region of PKC-gamma. HL-60/AR cells contained an equivalent level of PKC-alpha and a lower amount of PKC-beta than HL-60 cells. In contrast, only HL-60/AR cells contained PKC-gamma. These results indicate that the regulation of this family of isoenzymes is altered in drug-resistant cells.
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PMID:Protein kinase C-gamma is present in adriamycin resistant HL-60 leukemia cells. 230 37


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