Protein Kinase C Isoforms Alpha Research Articles

Characterization of vascular endothelial growth factor's effect on the activation of protein kinase C, its isoforms, and endothelial cell growth.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen which mediates its effects by binding to tyrosine kinase receptors. We have characterized the VEGF-activated intracellular signal transduction pathway in bovine aortic endothelial cells and correlated this to its mitogenic effects. VEGF induced concentration- and time-dependent increases in protein kinase C (PKC) activation with a maximum of 2.2-fold above the basal level at 5 x 10(-10) M within 10 min as measured both by in situ and translocation assays. Immunoblotting analysis of PKC isoforms in cytosolic and membrane fractions indicated that after VEGF stimulation the content of Ca(2+)-sensitive PKC isoforms (alpha and betaII) was increased in the membrane fractions, whereas no changes were observed for PKC isoforms delta and epsilon. The stimulation of PKC activity by VEGF was preceded by the activation of phospholipase Cgamma (PLCgamma). This was demonstrated by parallel increases in PLCgamma tyrosine phosphorylation, [3H]inositol phosphate production, and [3H]arachidonic acid-labeled diacylglycerol formation in bovine aortic endothelial cells. In addition, VEGF increased phosphatidylinositol 3-kinase activity 2.1-fold which was inhibited by wortmannin, a phosphatidylinositol 3-kinase inhibitor, without decreasing the VEGF-induced increase in PKC activity or endothelial cell growth. Interestingly, genistein, a tyrosine kinase inhibitor, and GFX or H-7, PKC inhibitors, abolished both VEGF-induced PKC activation and endothelial cell proliferation. VEGF's mitogenic effect was inhibited by a PKC isoform beta-selective inhibitor, LY333531, in a concentration-dependent manner. In contrast, antisense PKC-alpha oligonucleotides enhanced VEGF-stimulated cell growth with a simultaneous decrease of 70% in PKC-alpha protein content. Thus, VEGF appears to mediate its mitogenic effects partly through the activation of the PLCgamma and PKC pathway, involving predominately PKC-beta isoform activation in endothelial cells.

Differentiation of pancreatic ductal carcinoma cells associated with selective expression of protein kinase C isoforms.

The signal transduction pathways important in regulating the growth and differentiation of malignant cells are poorly understood. Recent evidence has implicated activation of the protein kinase C (PKC) family of signaling proteins in pancreatic carcinoma during cytokine-induced cytostasis and differentiation. A human pancreatic adenocarcinoma (HPAC) cell line was exposed to tumor necrosis factor-alpha (TNF-alpha; 40 ng/ml) for 6 days. Cytostasis and viability were confirmed by daily MTT [(3(4,5)-dimethyl-thiazol-2-yl) 2,5-diphenyl-tetrazolium bromide] and trypan exclusion assay. Protein fractions were isolated daily and subjected to immunoblot analysis for the normal (terminally differentiated) pancreatic ductal cell marker carbonic anhydrase II (CA II) as well as specific PKC isoforms (alpha, beta, gamma, eta, and zeta). Growth arrest occurred in HPAC cells after exposure to TNF-alpha for 48 h, with viability maintained above 90% throughout the 6-day time course. CA II immunoreactivity was not detected in untreated controls but appeared after 2 days of TNF-alpha exposure, peaking on day 6. Concurrently, TNF-alpha induced the selective downregulation of PKC-alpha, whereas PKC-gamma levels increased. PKC-beta and PKC-eta immunoreactivity did not change. The atypical PKC-zeta isoform developed a doublet banding pattern in response to TNF-alpha, although overall PKC-zeta levels did not change. TNF-alpha-induced growth arrest and differentiation in HPAC cells is associated with the selective downregulation of PKC-alpha and upregulation of PKC-gamma.

Stimulation of mitogen-activated protein kinase by gonadotropin-releasing hormone: evidence for the involvement of protein kinase C.

GnRH regulates gonadotropin biosynthesis and secretion. Multiple intracellular signaling pathways are activated by GnRH, including phosphoinositol turnover, release of intracellular calcium and influx of extracellular calcium, and activation of protein kinase C (PKC), among others. In this study, we investigated whether GnRH stimulates mitogen-activated protein kinase (MAPKs), and whether this pathway plays a role in the transcriptional activation of the gonadotropin alpha-gene. In alpha T3-1 gonadotrope cells, treatment with GnRH caused 4- to 5-fold induction of MAPK activity. Stimulation of MAPK activity was detected within 5 min of GnRH treatment and persisted for 60 min. MAPK activation by GnRH was also seen in primary cultures of rat pituitary cells. Treatment with phorbol 12-myristate 13-acetate (TPA) caused 4- to 5-fold induction of MAPK activity in alpha T3-1 cells. Pretreatment with TPA, however, decreased both GnRH- and TPA-induced MAPK activation, suggesting that PKC is involved in GnRH-mediated activation of MAPK. Western blot analyses of PKC isoforms alpha and epsilon confirmed that they were depleted by chronic treatment with TPA, whereas MAPK protein levels were unaffected. Because transcriptional stimulation of the glycoprotein hormone alpha-gene by GnRH is also inhibited by PKC depletion, additional experiments were performed to explore a potential role for MAPK in alpha-gene expression. Cotransfection of a dominant negative inhibitors of MAPK isoforms (ERK1 and ERK2) suppressed basal expression of the alpha-promoter by 60%, but had less effect on the extent of GnRH stimulation in alpha T3-1 cells. These experiments indicate that GnRH stimulates MAPK activity, probably through a pathway involving PKC. Although PKC depletion inhibits both MAPK- and GnRH-stimulated alpha-gene transcription, pathways other than MAPK are also likely to be involved in mediating the transcriptional effects of GnRH.

Protein kinase C beta from Friend erythroleukemia cells is associated with chromatin and DNA.

Certain protein kinase C (PKC) isotypes are localized to the nucleus during cellular proliferation in murine erythroid cells, as well as in human promyelocytic leukemia and erythroleukemia cells. Because the structure of these PKC isotypes contains a conserved cysteine-rich region that contains the zinc finger DNA binding motif, we tested the hypothesis that selected PKC isotypes found in Friend erythroleukemia cells can bind to DNA. Cell lysates from murine Friend erythroleukemia cells, which express alpha, beta I, and beta II PKC, expressed greater amounts of the beta isoforms than the alpha isoform of PKC in their nuclei, and PKC beta I was found in the chromatin of these cells. Lysates of these cells were tested for their ability to bind to a DNA-cellulose column. Bound proteins were eluted with a step gradient of increasing KCl concentrations, and eluant fractions were then subjected to immunoblot analysis using isotype-specific antibodies to the alpha and beta I isotypes of PKC. DNA binding was detected for the PKC beta I isotype, which is present in the nucleus, but not for the more abundant PKC alpha isotype, which resides primarily in the cytoplasm. These results demonstrate that PKC can associate with DNA, and that this association is isotype specific in Friend erythroleukemia cells.

Different GABAB‐Mediated Effects on Protein Kinase C Activity and Immunoreactivity in Neonatal and Adult Rat Hippocampal Slices

The effects of GABA on protein kinase C (PKC) were investigated in rat hippocampal slices at various postnatal ages [postnatal day (P) 1-P60]. At P4, GABA (300 microM) induced a rapid (in 1-2 min) 40-50% increase of PKC activity in the membrane fraction and a decrease in the cytosol. These effects were mediated by GABAB receptors because (a) they were neither blocked by 10 microM bicuculline nor reproduced by 10 microM isoguvacine and (b) they were mimicked by the GABAB agonist baclofen (3-30 microM), an effect fully antagonized by the GABAB antagonist 2-hydroxysaclofen (10 microM). A baclofen-induced increased PKC activity in the membrane fraction was only present during the early postnatal period (P1-P14); it was associated with a translocation from the cytosol to the membrane of the immunoreactivity of some PKC isoforms (alpha-, beta-, and epsilon-PKCs). In contrast, after P21, PKC activity and alpha-, beta-, epsilon-, and gamma-PKC immunoreactivities were decreased by baclofen in the membrane fraction and increased in the cytosol. These results suggest that the stimulation of GABAB receptors differentially modulates PKC activity via distinct second messenger pathways in developing and mature hippocampi.

Role of protein kinase C alpha in the induction of carcinoembryonic antigen by transforming growth factor beta 1.

Previous studies showed that transforming growth factor beta 1 (TGF beta 1) regulates the expression of carcinoembryonic antigen (CEA) and CEA-cross-reactive glycoproteins (CEA-GLYs) in human colon carcinoma cells through a signal-transducing pathway associated with protein kinase C (PKC) (Chakrabarty, J. Cell. Physiol., 1992, 152:494-499). In this study we determined the role of the PKC alpha isoform in the regulation of CEA and CEA-GLYs expression by TGF beta 1. Expression of PKC alpha antisense RNA, through transfection experiments with an antisense PKC alpha expression vector, resulted in down-modulation of PKC alpha RNA and protein expression. TGF beta 1 was unable to stimulate the expression and secretion of CEA in cells in which the expression of PKC alpha protein was substantially reduced. The ability of TGF beta 1 to stimulate the expression of the 95- and 55-kDa CEA-GLYs, however, was not affected. We therefore conclude that TGF beta 1 regulates the secretion and expression of CEA through a signal-transducing pathway associated with PKC alpha. TGF beta 1 may also regulate the expression of CEA-GLYs through signal-transducing pathways associated with other PKC isoforms.

Effects of insulin on the translocation of protein kinase C-θ and other protein kinase C isoforms in rat skeletal muscles

Protein kinase C (PKC)-theta is a newly recognized major PKC isoform in skeletal muscle. In this study we found that insulin provoked rapid biphasic increases in membrane-associated immunoreactive PKC-theta, as well as PKC-alpha, PKC-beta and PKC-epsilon, in rat soleus muscles incubated in vitro. Effects of insulin on PKC isoforms in the soleus were comparable in magnitude with those of phorbol esters. Increases in membrane-associated PKC-theta, PKC-alpha, PKC-beta and PKC-epsilon were also observed in rat gastrocnemius muscles after insulin treatment in vivo. Our findings suggest that PKC-theta, like other diacylglycerol-sensitive PKC isoforms (alpha, beta and epsilon), may play a role in insulin action in skeletal muscles.

High glucose concentrations and protein kinase C isoforms in vascular smooth muscle cells

High extracellular glucose activates protein kinase C (PKC), a family of kinases vital to intracellular signaling. However, which PKC isoforms are involved and where in the cell they operate is unclear. We tested the hypothesis that only those PKC isoforms binding to diacylglycerol (DAG) are activated by high glucose. We also reasoned that the isoforms would translocate to different parts of the cell, where they presumably serve different functions. The PKC isoforms alpha, beta, delta, epsilon, and zeta were studied. Twenty mM glucose caused an increase in total PKC activity at six hours, which was maintained at 24 hours. High glucose decreased the angiotensin II-induced calcium signal. This effect was reversed by preincubating the cells with the PKC inhibitor staurosporine. Glucose induced a translocation of all PKC isoforms except PKC zeta by Western blot. Confocal microscopy showed that PKC alpha, beta, and epsilon were translocated into the nucleus. PKC delta showed strong association with cytoskeletal structures. The effects were sustained at 24 hours for PKC isoform beta and to a lesser extent for PKC delta and epsilon, but not for PKC alpha. Thus, PKC isoforms differ in their propensity to be activated by high glucose. Those isoforms binding to DAG are activated. Both cytoskeletal and nuclear signaling may be involved.

Redistribution of protein kinase C isoforms in association with vascular hypertrophy of rat aorta.

Freshly enzymatically isolated cells from the aorta of a rat model of vascular hypertrophy were used to investigate the role of protein kinase C (PKC) isoforms during a physiologically relevant growth response. With the combination of immunofluorescence with digital imaging microscopy, PKC isoforms alpha, delta, and zeta were found to be present in single cells from control and hypertrophied rat aortas. The alpha- and zeta-isoforms were distributed in the cytoplasm of control cells; however, in hypertrophied cells, sustained translocations of alpha-PKC to the surface membrane and zeta-PKC to the intranuclear area were seen. delta-PKC was concentrated in the perinuclear area in control cells but appeared to translocate to a more diffuse localization in the cytosol of hypertrophied cells. Staining of mitochondria with rhodamine 123 indicated some similarity in the spatial distribution compared with that of delta-PKC. In control cells, translocation of isoforms alpha and delta was activated by phenylephrine or 12-deoxyphorbol 13-isobutyrate 20-acetate. Agonist stimulation produced translocation of no isoforms in the hypertrophied cells. These results indicate that isoform-specific spatial distribution and translocation of PKC occur in association with the growth response of vascular hypertrophy.

12(S)-HETE enhancement of prostate tumor cell invasion: selective role of PKC alpha.

Prostate carcinoma has become the second most fatal cancer in American men. In Dunning R3327 rat prostate adenocarcinoma cells, elevated invasiveness positively correlates with metastatic potential. However, the mechanism(s) responsible for regulation of tumor cell motility and invasion is poorly understood. We have reported that a lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], augments tumor cell metastatic potential through activation of protein kinase C (PKC). We proposed to determine the effect of 12(S)-HETE on the motility and invasion of low-metastatic rat prostate AT2.1 tumor cells and the effect of 12(S)-HETE activation of specific PKC isoform(s) in these processes. The motility of AT2.1 cells was determined by the colloidal gold phagokinetic track assay and the invasiveness measured as their ability to invade through basement membrane Matrigel-coated filters. Expression of PKC isoforms was determined by Western blotting of the whole cell lysate with isoform-specific anti-PKC antibodies. Cytosol and membrane fractions were prepared and the subcellular distribution of PKC was analyzed by Western blotting and activity assay. The effect of 12(S)-HETE on cell proliferation was examined. Data were analyzed for significance of difference with the two-sampled, two-sided Student's t test. 12(S)-HETE increased the motility and invasion of AT2.1 cells, and this 12(S)-HETE-increased motility and invasion were inhibited by a selective PKC inhibitor, calphostin C, as well as a Ca2 chelator, bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/tetra(acetoxy-methyl)ester. AT2.1 cells expressed the PKC isoforms alpha and delta, and 12(S)-HETE increased the membrane association of PKC alpha but not delta. Further, the motility and invasion of AT2.1 cells were increased by thymelea toxin, a selective activator of PKC alpha over PKC delta. 12(S)-HETE augments the invasiveness of AT2.1 cells via selective activation of PKC alpha. 12(S)-HETE modulation of PKC alpha invasiveness may be an important mechanism of action for the regulation of the invasive potential of rat prostate carcinoma cells, and the 12-lipoxygenase enzyme and/or PKC alpha may serve as key targets for the development of anti-invasive agents useful for combating the spread of prostate cancer.

Invasive human pituitary tumors express a point-mutated alpha-protein kinase-C.

Protein kinase-C (PKC) is a ubiquitous eukaryotic kinase that plays a key role in transmembrane signaling and influences important cellular processes, such as proliferation. Increases in its activity and expression have been demonstrated in adenomatous human pituitaries, with protein expression being the highest in invasive tumors (1). Moreover, in these same invasive tumors, the mean increase in expression (8.9-fold) does not correlate with the mean increase in activity (2.6-fold), suggesting a dysfunction in PKC in these tumors. Here, we show that the PKC alpha-isoform (alpha PKC) is overexpressed in human pituitary tumors. The complete sequencing of the PKC cDNA from four invasive tumors has revealed a point mutation that is absent in the noninvasive tumors analyzed. The point mutation is located at position 294 of the protein, in the V3 region, leading to a substitution of a negatively charged aspartic acid by an apolar glycine. Thus, not only is alpha PKC overexpressed in human pituitary tumors, but it is also structurally altered in the invasive subpopulation of these tumors.

Conventional protein kinase C isoforms are not essential for cellular proliferation of a T cell lymphoma line.

The role of conventional protein kinase C (PKC) isoforms in the proliferation of a T cell lymphoma line was investigated, PKC isozyme-deficient cell lines were generated from HuT 78 which expresses PKC isoforms alpha, beta, epsilon and zeta. The first of these, K-4, expresses PKC alpha, epsilon and zeta but not beta. L-2, a mutant of K-4, expresses PKC alpha and zeta but expresses neither PKC alpha, beta, gamma, epsilon nor zeta when cultured continuously in PMA (L-2/PMA). All four cell lines continued to proliferate although at reduced rates in the presence of the PKC inhibitors staurosporine and H-7. K-4 and HuT 78 undergo growth arrest when activated with PMA (rate of proliferation decreases by 76 and 95%, respectively). While PMA-induced growth arrest occurs in L-2 cells which possess PKC alpha and zeta, PMA-induced growth arrest does not occur in L-2/PMA which is deficient in these isoforms. As these cell lines continue to proliferate in the presence of PKC inhibitors and since proliferation occurs in the absence of PKC isoforms (L-2/PMA), the results suggest that neither conventional PKC nor the epsilon and zeta isoforms are essential for proliferation of these cells. These data further suggest that PKC alpha and/or zeta may be involved in PMA-induced growth arrest.

Down-Regulation of Human Protein Kinase C a Is Associated With Terminal Neutrophil Differentiation

We have established an RNase protection method to quantify the expression of mRNA for the human protein kinase C (PK-C) isoforms alpha, beta 1, beta 2, and gamma. This was used to investigate whether each isoform is differentially expressed during the differentiation of hematopoietic cells. Myeloid and lymphoid cells express PK-C alpha, beta 1, and beta 2 mRNAs in various proportions. PK-C gamma mRNA was detected in human brain, but not in hematopoietic cells. PK-C alpha mRNA decreases as HL-60 cells mature to a neutrophil phenotype in response to retinoic acid, but its abundance does not change during monocytic differentiation in response to vitamin D3. PK-C alpha mRNA and protein were undetectable in peripheral blood neutrophils, but are present in monocytes. The mRNAs for PK-C beta 1 and beta 2 isoforms increase during HL-60 differentiation and are expressed in both neutrophils and monocytes. Therefore, the PK-C alpha isoform is specifically down-regulated during human neutrophil terminal differentiation. These data suggest that mature neutrophil functions do not require the PK-C alpha isoform.

Down-regulation of human protein kinase C alpha is associated with terminal neutrophil differentiation

We have established an RNase protection method to quantify the expression of mRNA for the human protein kinase C (PK-C) isoforms alpha, beta 1, beta 2, and gamma. This was used to investigate whether each isoform is differentially expressed during the differentiation of hematopoietic cells. Myeloid and lymphoid cells express PK-C alpha, beta 1, and beta 2 mRNAs in various proportions. PK-C gamma mRNA was detected in human brain, but not in hematopoietic cells. PK-C alpha mRNA decreases as HL-60 cells mature to a neutrophil phenotype in response to retinoic acid, but its abundance does not change during monocytic differentiation in response to vitamin D3. PK-C alpha mRNA and protein were undetectable in peripheral blood neutrophils, but are present in monocytes. The mRNAs for PK-C beta 1 and beta 2 isoforms increase during HL-60 differentiation and are expressed in both neutrophils and monocytes. Therefore, the PK-C alpha isoform is specifically down-regulated during human neutrophil terminal differentiation. These data suggest that mature neutrophil functions do not require the PK-C alpha isoform.

Constitutive presence of a catalytic fragment of protein kinase C epsilon in a small cell lung carcinoma cell line.

Protein kinase C (PKC) has been implicated in a variety of cellular responses such as proliferation, differentiation, and secretion. We assessed the role of PKC in the mitogenic effects of gastrin-releasing peptide (in a small cell lung cancer (SCLC) cell line. Using antisera that specifically recognize the PKC isoforms alpha, beta, gamma, delta, and epsilon, we determined that PKC epsilon is the major isoform in the SCLC cell line NCI-N417, followed by PKC alpha and delta. In addition to the 90-kDa PKC epsilon, our anti-PKC epsilon antiserum specifically detected a 40-kDa immunoreactive protein. Treatment of the cells with either 20 nM phorbol myristate acetate or 50 nM GRP enhanced significantly the level of the 40-kDa protein in a time-dependent (1-8 h), cycloheximide-sensitive fashion. Subcellular fractionation revealed that 90% of PKC epsilon was in particulate form, while the 40-kDa immunoreactive protein was cytosolic. To test the hypothesis that the 40-kDa soluble protein represented a catalytically independent PKC epsilon fragment, cytosolic extracts were assayed for kinase activity. 45-50% of the activity was apparent in the absence of the PKC activators phosphatidylserine and diacylglycerol. This effector-independent kinase activity was further purified by affinity chromatography using a synthetic peptide corresponding to the pseudosubstrate region of PKC epsilon (ERMRPRKRQGAVRRRV) coupled to Sepharose. The partially purified protein, recognized by the anti-PKC epsilon antiserum, exhibited histone kinase activity with kinetics similar to those of the tryptically generated catalytic fragment of brain PKC epsilon. This activity was inhibited by staurosporine (IC50 = 1 x 10(-8) M) and by the pseudosubstrate inhibitor peptide (IC50 = 7.7 x 10(-8) M). The SCLC kinase and the brain PKC epsilon catalytic fragment were similar as indicated by the relative sizes of the PKC epsilon immunoreactive peptides generated with protease V8 from Staphylococcus aureus (Mr approximately 37,000, 34,000, 28,000, 26,000, and 25,000). Taken together, we conclude that a variant SCLC cell line expresses a constitutively active catalytic fragment of PKC epsilon. Regulation by 12-O-tetradecanoyl-13-acetate or GRP via de novo protein synthesis suggests a novel mechanism of control of PKC diversity with implications for small cell lung cancer and possibly other malignancies.

Differential activation of protein kinase C alpha is associated with arachidonate release in Madin-Darby canine kidney cells.

The heterogeneity of the protein kinase C (PKC) gene family strongly suggests that different isoforms may have distinct functions in mediating signal transduction. However, there is very little direct evidence for this. PKC has been implicated in arachidonate (AA) release in many cell types. We sought to investigate whether bradykinin- and phorbol ester-stimulated AA release in Madin-Darby canine kidney (MDCK) cells was correlated with differential activation of PKC isoforms. Using phorbol esters to (i) activate the enzyme and (ii) to down-regulate it, we report that differential activation (translocation) of PKC alpha is associated with AA release in MDCK cells and that specific down-regulation of PKC alpha is associated with a loss of AA release in response to stimulation with dioctanoylglycerol and phorbol ester. We also demonstrate that bradykinin-stimulated AA release was associated with differential activation of PKC alpha and was inhibited in PKC alpha down-regulated cells. Thus, we conclude that the PKC alpha isoform is likely to be responsible for mediating AA release in these cells.