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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)

We have examined the expression and responses to activation, of novel/atypical protein kinase C (PKC) isoforms epsilon, zeta, and delta in the T cell lymphoma cell line K-4. The effects of 1-h phorbol 12-myristate 13-acetate (PMA) and OKT3 activation of K-4 cells on PKC isoform distribution were examined. In addition, the effects of PMA-mediated down-regulation on the expression of PKC epsilon and zeta were determined using high concentrations of PMA over 24- and 48-h time periods in these cells. PKC zeta expression was not altered by incubation of K-4 cells with up to 200 ng/ml PMA over a 24- or 48-h period. PKC epsilon was down-regulated in a concentration-dependent manner by PMA after both 24- and 48-h of activation. Expression of PKC epsilon was not completely depressed, however, even at the highest concentration of the phorbol ester after 48-h incubation with PMA. The presence of PKC epsilon, zeta, and delta was confirmed by immunohistochemistry with distinct patterns of expression observed. PMA-induced PKC activation for a 1-h period resulted in a translocation of PKC delta from resting cytoplasmic/nuclear staining to a cytoplasmic aggregate. Following 1-h activation through the T cell receptor-associated complex CD3, PKC delta translocated from a peri-nuclear/cytoplasmic compartment to a putative cytoskeletal location in K-4 cells. This translocation was time dependent and redistributed to a cytoplasmic aggregate prior to the cytoskeleton. Similarly, following 1-h activation through the T cell receptor, PKC zeta redistributed directly to what is possibly a cytoskeletal cell compartment. The cytoplasmic distribution of PKC zeta was unaltered following activation with PMA over a 1-h time period. There was no apparent redistribution of PKC epsilon cytoplasmic staining pattern following a 1-h direct or indirect activation. These results underline the differences in individual PKC isoform distribution, and responses to different stimuli, thereby providing additional evidence for the use of discrete PKC isoform signaling pathways in T cells. Furthermore, this data underlines the differences in PMA-mediated PKC activation and activation through the T cell receptor.
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PMID:Regulation of non-classical protein kinase C isoenzymes in a human T cell line. 784 22

A function for protein kinase C-zeta (PKC-zeta), a member of the phorbol ester nonresponsive atypical protein kinase C subfamily, in modulating differentiation was examined in the leukemic U937 cell. Transfected U937 cells stably overexpressing PKC-zeta displayed a longer doubling time, lower saturation density at confluency, and an increase in adherence to plastic as compared to control cells. PKC-zeta cells expressed a more differentiated phenotype as assessed by changes in morphology, surface antigen expression, and lysosomal enzyme activities and were distinct from parental U937 cells stimulated to differentiate by exposure to phorbol esters. In contrast to parental U937 cells, PKC-zeta cells constitutively expressed mRNA transcripts for c-jun and a low mobility AP-1 binding activity. Thus, PKC-zeta overexpression stimulates a type of phenotypic differentiation that differs significantly from maturation occurring upon activation of other PKC subfamilies induced by phorbol ester treatment. Increased expression of the c-jun protooncogene and an increase in AP-1 binding activity in PKC-zeta cells provides a potential mechanism for explaining the altered differentiation status of this cell.
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PMID:Overexpression of protein kinase C-zeta stimulates leukemic cell differentiation. 784 21

To elucidate the role of protein kinase C (PKC) in nerve growth factor (NGF)-induced differentiation, PMA downregulation of pheochromocytoma (PC12) cells was undertaken. Prolonged treatment (2 d) of PC12 cells with PMA (1 microM) resulted in depleting the cells of alpha, beta, delta, and epsilon-PKC isoforms, but had no effect on the expression of the atypical PKC isoform zeta. PC12 cells, which expressed only PKC zeta, were evaluated for their responses to NGF. Removal of the PMA-sensitive PKC isoforms enhanced the ability of NGF to promote neurite extension. Both the percentage cells with neurites and length of neurites were increased in the PMA-treated cells, whereas no effect was observed on the number of neurites per cell or branching of individual neurites. In addition, PMA downregulation resulted in an increase in the incorporation of 3H-thymidine without any significant effect on the expression of c-fos. Addition of NGF to PC12 cells depleted of the PMA-sensitive PKC isoforms resulted in the activation of PKC zeta (Wooten et al., 1994). To test whether the transient activation of PKC zeta is a necessary component of the neuritogenetic pathway, antisense oligonucleotide strategy was utilized to remove this particular PKC isoform. The addition of a 20-bp antisense oligonucleotide directed against the 5' coding sequence of PKC zeta attenuated NGF-induced neurite outgrowth in PC12 cells lacking PMA-sensitive PKC isoforms. Sense oligonucleotide directed at the same site was without effect on NGF responses. These data indicate that PKC zeta comprises a portion of the NGF pathway and underscores the importance of this isoform in neuronal differentiation. Moreover, these findings demonstrate that the PMA-insensitive pathway, which was previously characterized as PKC-independent, and the neurite induction pathway are synonymous and mediated by PKC zeta.
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PMID:Nerve growth factor-induced differentiation of PC12 cells employs the PMA-insensitive protein kinase C-zeta isoform. 785 79

A selective PKC inhibitor, UCN-01, was shown to exhibit anti-tumor activity in vitro and in vivo. We investigated UCN-01 with respect to isozyme-specific PKC inhibition using purified recombinant or rabbit brain PKC isozymes, cPKC alpha, beta and gamma, nPKC delta, epsilon and eta, and a PKC zeta. Of the PKC isozymes examined, cPKC alpha was inhibited by UCN-01 most effectively (Ki = 0.44 nM), suggesting cPKC alpha is the prime candidate for the physiological target of UCN-01. The Ki values of UCN-01 estimated from Dixon plots for cPKC isozymes are approximately 1 nM, whereas the Ki values for nPKC isozymes are about 20 nM. Moreover, the Ki value for aPKC zeta is 3.8 microM. Thus, UCN-01 discriminates between PKC subfamilies. In addition, the inhibitory effects of staurosporine, H7, and calphostin C on aPKC zeta were examined and compared with those for cPKC alpha.
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PMID:UCN-01, an anti-tumor drug, is a selective inhibitor of the conventional PKC subfamily. 786 10

At subnanomolar concentrations, vasoactive intestinal peptide (VIP) can act as an astroglial mitogen and as a secretagogue for neurotrophic substances released from glia (Brenneman et al.: J Neurosci Res 25:386-394, 1990). Here we report that treatment with subnanomolar (0.1 nM) VIP, that does not produce an increase in intracellular cAMP levels, induced the translocation of protein kinase C (PKC) from the cytoplasm to the nucleus in neonatal cortical astrocytes, as revealed by immunohistochemistry, Western blot analysis, and measurements of the enzyme activity. Western blot analysis of subcellular fractions, using PKC isotype-specific antisera, showed PKC alpha as well as the two novel PKC isotypes, delta and zeta immunoreactivities, whereas PKC beta or gamma immunoreactivities were not detected. PKC alpha was associated predominantly with the cytosolic compartment, while PKC delta was found in the plasma membrane and in nuclear fractions. In contrast, PKC zeta was distributed ubiquitously within the major subcellular fractions. Treatment of the cells with 0.1 nM VIP caused a marked increase in nuclear PKC alpha and, to a lesser extent, PKC delta and PKC zeta immunoreactivities. Western blot analysis showed that a low (1 nM) concentration of phorbol, 12-myristate, 13 acetate also caused the subcellular redistribution of PKC immunoreactivities from the cytoplasm to the nuclear fraction, similar to VIP treatment. Exposure of astrocytes to high concentrations (1 microM) of phorbol, 12-myristate, 13 acetate resulted in the down-regulation of PKC alpha and PKC delta, while distribution of PKC zeta immunoreactivities were only slightly altered.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Subnanomolar concentration of VIP induces the nuclear translocation of protein kinase C in neonatal rat cortical astrocytes. 788 16

Protein, mRNA and activity levels of the calcium-independent protein kinase C (nPKC) isoenzymes were examined in NG108-15 cells. Western blot analyses reveal that proliferating NG 108-15 cells express the delta, epsilon, and eta, but not the theta species. The atypical species PKC zeta was also detected. Differentiation of these cells with dibutyryl cAMP was associated with increase in the levels of PKC epsilon, with no significant changes in its steady-state mRNA levels. The levels of the other isoforms were not altered by the differentiated state. Similarly, no changes in nPKC activity were discerned in either the soluble or particulate fractions when histone or other proteins were used as substrates. These data suggest that the PKC epsilon isoform may be important for the production and maintenance of the differentiated state in NG 108-15 cells.
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PMID:Expression and regulation of calcium-independent protein kinase C in NG 108-15 cell differentiation. 794 90

Rat lymphoblasts are arrested in the G1 phase of the cell cycle and can be promoted to proceed up to the S phase, when they are stimulated by phorbol ester. In this work, we have studied some details of the phorbol 12,13-dibutyrate (PBu2)-stimulated proliferation. We show that in response to PBu2 at least four different protein kinase C (PKC) isoforms translocate to the membrane. A specific PKC zeta antibody recognizes two bands of 75 and 82 kDa. These two activities are separated using a Mono Q chromatography and we show that p75 is the classical PKC zeta isoform, while p82 might be a related isoform which is PBu2 sensitive. Our data show that there is a correlation between the ability of PBu2 to promote mitogenesis and to activate ERK2 kinase, suggesting that ERK2 kinase might be the limiting step of the process. We also show that ERK kinase activation precedes Raf-1 kinase hyperphosphorylation, suggesting that Raf-1 kinase activation is not required for ERK kinase activation. This idea was checked using a Raf-1 kinase antisense (AS) oligonucleotide. The results obtained with the Raf-1 AS oligonucleotide indicate that this serine/threonine kinase is dispensable for ERK kinase activation, but needed for the PBu2 mitogenic signaling even as late as 7 h after the delivery of the signal.
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PMID:Raf-1 and ERK2 kinases are required for phorbol 12,13-dibutyrate-stimulated proliferation of rat lymphoblasts. ERK2 activation precedes Raf-1 hyperphosphorylation. 795 67

We have examined the neonatal developmental expression of protein kinase C subspecies (PKCs) in rat brain, pituitary glands and cells by enzymatic activity assays, immunohistochemistry and Western blot analysis with type-specific antibodies. A very large increase (455%) was noticed in brain PKC activity during the first week of life with the particulate fraction (22% of total enzyme activity on day 1) increasing dramatically (900%) during the first week to 50% of enzyme activity. In contrast, the pituitary gland showed high activity on day 1 that decreased progressively to reach the lowest levels at 1 year of age. Paradoxically, the number of pituitary cells immunolabeled for PKC increases as a function of age. Western blot analysis showed only small changes in PKC alpha, PKC beta and PKC epsilon when brains from 6-day-old and 3-month-old female rats were compared, whereas PKC tau and PKC delta increased markedly during this period. On the other hand, brain PKC zeta decreased between 6 days and 3 months of age. Western blot analysis showed no major changes in pituitary PKC alpha, PKC beta and PKC zeta when 6-day-old and 3-month-old female rats were compared, while PKC tau was not detected. The major band of pituitary PKC delta (76 kDa) decreased markedly between 6 days and 3 months of age whereas the minor band (68 kDa) did not change.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Developmental expression of protein kinase C subspecies in rat brain-pituitary axis. 795 91

Two immunoreactive proteins (75 and 80 kDa) were detected in rat brain and rabbit aorta using a polyclonal peptide-directed antibody to the C terminus of the zeta isoenzyme of protein kinase C (PKC). The 75-kDa protein resembled authentic PKC zeta; it was recovered in cytosolic fractions prepared in the presence or absence of Ca2+. The 80-kDa protein, however, bound to the particulate fraction in a Ca(2+)-dependent and reversible manner, but phosphorylated a synthetic peptide derived from the autoinhibitory domain of PKC epsilon in a Ca(2+)-independent but phospholipid- and diacylglycerol-dependent manner. Purification of the 80-kDa protein from rat brain, which separated two PKC zeta-immunoreactive proteins of 81.3 and 79.4 kDa, was achieved by EGTA extraction of the particulate fraction followed by chromatography on DEAE-Sephacel, phenyl-Sepharose, and hydroxylapatite. The 81.3-kDa protein copurified with PKC alpha, and the 79.4-kDa protein copurified with PKC beta and PKC gamma. PKC alpha, -beta and -gamma isoenzymes separated by hydroxylapatite chromatography all cross-reacted with anti-PKC zeta. Using recombinant PKC isoenzymes, however, anti-PKC zeta was shown to recognize rPKC alpha, rPKC beta 1, and rPKC beta II but not rPKC gamma. The regulatory properties of these isoenzymes differed from each other and depended on the particular substrate. PKC alpha was found to separate into two peaks on hydroxylapatite chromatography. The first peak exhibited regulatory properties characteristic of PKC alpha, whereas the second peak (PKC alpha') exhibited constitutive activity. PKC alpha' appears to be derived from PKC alpha by irreversible oxidative modification.
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PMID:Identification and characterization of protein kinase C zeta-immunoreactive proteins. 796 99

Arachidonic acid release and prostaglandin production are stimulated by both phorbol esters and growth factors in various cell types. Whereas phorbol esters activate and transmit a signal via protein kinase C, this pathway is not necessarily involved in growth factor signal transduction. We investigated the involvement of protein kinase C in the pathway of arachidonic acid metabolism by CHO cells transfected with full-length EGF receptor (CHOwt). Two isoforms of protein kinase C were identified in CHOwt cells, alpha and zeta. On downregulation, the parallel loss of phorbol ester-stimulated arachidonic acid release and the alpha-isoform suggests a possible involvement of this isoform in phospholipase A2 activation in these cells. In addition, we propose that the zeta-isoform may be separately involved in prostaglandin production as residual phorbol ester-stimulation of PGE production occurs in downregulated cells where PKC zeta is the sole remaining isoform. EGF stimulation of arachidonic acid release, as a measure of phospholipase A2 activation, and subsequent prostaglandin production are unaffected by inhibition of protein kinase C in CHOwt cells. Indeed one such inhibitor, staurosporine, augmented the EGF effect. These results suggest that PKC is not required for EGF activation of phospholipase A2 in these cells.
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PMID:The role of protein kinase C in arachidonic acid release and prostaglandin E production from CHO cells transfected with EGF receptors. 798 Dec 36


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