Protein kinase C activation potentiates the rapid secretion of the amyloid precursor protein from rat cortical synaptosomes

In this study we have used the presynaptic-rich rat cerebrocortical synaptosomal preparation to investigate the proteolytic cleavage of the amyloid precursor protein (AbetaPP) by the alpha-secretase pathway within the betaA4 domain to generate a soluble secreted N-terminal fragment (AbetaPPs). AbetaPP was detected in crude cortical synaptosomal membranes, although at a lower density than that observed in whole-tissue homogenates. Protein kinase C (PKC) activation induced a translocation of the conventional PKC isoform beta1 and novel PKCepsilon from cytosol to membrane fractions, but there was no alteration in the proportion of AbetaPP associated with the Triton-soluble and -insoluble fractions. AbetaPPs was constitutively secreted from cortical synaptosomes, with this secretion being enhanced significantly by the direct activation of PKC with phorbol ester. The PKC-induced secretion of AbetaPPs was only partially blocked by the PKC inhibitor GF109203X (2.5 microM), whereas the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein was significantly inhibited by GF109203X. The differential sensitivities of the MARCKS phosphorylation and AbetaPPs secretion to GF109203X may imply that different PKC isoforms are involved in these two events in the synaptosomal system. These findings strongly suggest that the alpha-secretase activity leading to the secretion of AbetaPPs can occur at the level of the presynaptic terminal.     

Protein kinase C regulates Fas (CD95/APO-1) expression

Fas (CD95/APO-1) is a transmembrane protein of the TNF/neuron growth factor receptor family. Ligation of Fas by specific Abs or Fas ligand (FasL/CD95 ligand) induces rapid apoptotic cell death in a variety of cell types. Despite progress in understanding the death signals transduced from Fas, very little is known with regard to the mechanisms by which Fas expression is regulated. Using our previously established murine T cell hybridoma model A1.1, we show that specific protein kinase C (PKC) inhibitors could block activation-induced Fas expression and apoptosis. The activation of PKC with PMA or 1-oleoyl-2-acetyl-sn-glycerol could mimic the TCR signal by inducing the expression of Fas but not FasL. PKC-dependent Fas expression was also observed in several murine and human tumor cell lines. Since the inhibition of Ca2+ redistribution by an inhibitor of intracellular Ca2+ mobilization, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride, inhibited TCR-induced FasL but not Fas, the expression of Fas appears to be independent of Ca2+ mobilization. Significantly, expression of the newly identified Fas-regulatory gene, TDAG51, was found to be dependent upon the activity of PKC. PKC activation only induced Fas expression in cells expressing wild-type TDAG51. Thus, Fas expression is likely mediated by PKC through TDAG51.     

The effects of morphine on the release of noradrenaline from the mouse vas deferens

Electrical field stimulation of the mouse vas deferens (TO and C57/BL strains) caused the release of noradrenaline into the bathing medium. 2 Phenoxybenzamine (30 muM) or phentolamine (36 muM) plus cocaine (13 muM) caused a considerable increase in the noradrenaline output. 3 In the vasa deferentia from TO mice the output per pulse of noradrenaline was constant at frequencies of stimulation from 0.5 to 15 Hz whereas in the vasa deferentia from C57/BL mice the output per pulse of noradrenaline increased two-fold from 1.5 to 15 Hz. 4 Morphine (2 muM) inhibited the contractions of the vasa deferentia from TO mice. This effect was greater at low (0.1-1 Hz) than at high (10 Hz) frequencies of stimulation. Morphine (2 muM) did not inhibit the response of the tissue to exogenous noradrenaline. 5 Morphine (1 muM) reduced the noradrenaline output from the vasa deferentia of TO mice stimulated at 1.5 Hz but did not reduce the noradrenaline output at 15 Hz. At 1.5 Hz the reduction of noradrenaline output was reversed by naloxone (0.05 muM). 6 Morphine (5 muM) did not inhibit the uptake of [3H]-noradrenaline into the vasa deferentia from TO mice. 7 Only in high concentrations (ID50 30.88 muM) did morphine inhibit the contractions of the vasa deferentia from C57/BL mice. 8 Normorphine (100 muM) did not reduce the noradrenaline output from vasa deferentia of C57/BL mice.     

Proliferation of human and mouse astrocytes in vitro: signalling through the protein kinase C pathway

While several mitogens for astrocytes have been described, the signal transduction pathway(s) that mediates their proliferative effect remains unclear; in this report, a major role for the protein kinase C (PKC) system is suggested by several lines of evidence. Firstly, biologically active phorbol esters, 4 beta-phorbol-12,13-dibutyrate and phorbol-12-myristate-13-acetate, increase the proliferation of astrocytes as determined by [3H]thymidine incorporation or bromodeoxyuridine immunofluorescence; this effect is not reproduced by a phorbol ester that binds to PKC but does not activate it (4 alpha-phorbol-12,13-didecanoate). Secondly, 2 relatively selective inhibitors of PKC, H7 and staurosporine, attenuate the basal rate of proliferation of astrocytes in concentrations that were not cytotoxic to cells. Thirdly, mitogen-enhanced proliferation of astrocytes can be blocked by PKC inhibitors; this is observed for all astrocyte mitogens tested. Fourthly, measurements of PKC enzyme activity in astrocytes in response to serum-mitogenic factors, or to staurosporine, revealed a statistically significant correlation with proliferation rate. The mediation by PKC is not dependent on species- or age factors, since neonatal mouse or adult human astrocytes gave comparable results. The results have relevance to normal development and reactive gliosis post-injury, 2 conditions where astrocytes undergo proliferation, and to glioma growth.     

Congenital absence of the vas deferens: incomplete penetrance of cystic fibrosis gene mutations

PURPOSE: We evaluated the penetrance of cystic fibrosis gene mutations for the clinical phenotype of congenital bilateral absence of the vas deferens. MATERIALS AND METHODS: We retrospectively reviewed the fertility status of 244 brothers of 105 men with congenital bilateral absence of the vas deferens. Testing for the most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and for haplotype analysis of the intron 8/polythymidine segment was recommended for all men with congenital bilateral absence of the vas deferens. RESULTS: Of 244 brothers of men with congenital bilateral absence of the vas deferens 131 were eligible for assessment of fertility. Of the 131 evaluable brothers only 7 (5%) were found to have congenital bilateral absence of the vas deferens. This prevalence is 5 times lower than that predicted for congenital bilateral absence of the vas deferens (25%) based on the autosomal recessive inheritance pattern seen in classical cystic fibrosis. For couples in which the man has congenital bilateral absence of the vas deferens and the female partner tests negative for standard CFTR gene mutations including 5T analysis, the maximum risk of having a child with congenital bilateral absence of the vas deferens is less than 1.0%. CONCLUSIONS: Our data are consistent with incomplete penetrance for the congenital bilateral absence of the vas deferens phenotype after inheritance of cystic fibrosis gene mutations, even after adjusting for environmental factors and wolffian anomalies. Incomplete penetrance may account for a low prevalence of congenital bilateral absence of the vas deferens in the population and may lower the risk of having a child with congenital bilateral absence of the vas deferens for couples undergoing sperm retrieval and assisted reproductive techniques.     

Protein kinase C regulates chondrogenesis of mesenchymes via mitogen-activated protein kinase signaling

A possible regulatory mechanism of protein kinase C (PKC) in the chondrogenesis of chick limb bud mesenchymes has been investigated. Inhibition or down-regulation of PKC resulted in the activation of a mitogen-activated protein kinase subtype Erk-1 and the inhibition of chondrogenesis. On the other hand, inhibition of Erk-1 with PD98059 enhanced chondrogenesis and relieved PKC-induced blockage of chondrogenesis. Erk-1 inhibition, however, did not affect expression and subcellular distribution of PKC isoforms expressed in mesenchymes nor cell proliferation. The results suggest that PKC regulates chondrogenesis by modulating Erk-1 activity. Inhibition or depletion of PKC inhibited proliferation of chondrogenic competent cells, and Erk-1 inhibition did not affect PKC modulation of cell proliferation. However, PKC-induced modulation of expression of cell adhesion molecules involved in precartilage condensation was reversed by the inhibition of Erk-1. Expression of N-cadherin was detected at the early period of chondrogenesis. Inhibition or depletion of PKC induced sustained expression of N-cadherin, and Erk-1 inhibition blocked the effects of PKC modulation. The expression of integrin alpha5 beta1 and fibronectin was found to be increased transiently during chondrogenesis. Depletion or inhibition of PKC caused a continuous increase of the expression of these molecules throughout the culture period, and Erk-1 inhibition abolished the modulating effects of PKC. Because reduction of the examined cell adhesion molecule expression is a prerequisite for the progression of chondrogenesis after cell condensation, our results indicate that PKC regulates chondrogenesis by modulating expression of these molecules via Erk-1 signaling.     

Determination of the specific substrate sequence motifs of protein kinase C isozymes

Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (alpha, betaI, betaII, gamma, delta, epsilon, eta, mu, and zeta) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position +1 carboxyl-terminal of the phosphorylated Ser and basic residues at position -3. All isozymes, except PKC mu, selected peptides with basic amino acids at positions -6, -4, and -2. PKC alpha, -betaI, -betaII, -gamma, and -eta selected peptides with basic amino acid at positions +2, +3, and +4, but PKC delta, -epsilon, -zeta, and -mu preferred peptides with hydrophobic amino acid at these positions. At position -5, the selectivity was quite different among the various isozymes; PKC alpha, -gamma, and -delta selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKC mu showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position -3 to +2 for PKC alpha, -betaI, -betaII, -gamma, -delta, and -eta were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKC alpha, -betaI,-delta, -zeta, and -mu were prepared and used for the determination of Km and Vmax for these isozymes. As judged by Vmax/Km values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft.     

Involvement of sigma-receptors in the increase in contraction of mouse vas deferens induced by exogenous ATP

The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (mtGPD) plays an important role in the regulation of insulin secretion and has been postulated as a candidate responsible for the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM) in humans as well as in rodent models of NIDDM. Recent molecular genetic studies of the Goto-Kakizaki (GK) rat model of NIDDM have identified loci linked to NIDDM. To elucidate whether rat mtGPD might play a role in the pathogenesis of NIDDM, the rat mtGPD gene (Gpd2) was cloned, and a genetic marker for Gpd2 was developed. The gene mapped to the region of rat chromosome 3 that contains a region linked to NIDDM in the GK rat. Fluorescence in situ hybridization was also carried out to verify the map position.     

Activation of guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase in rat vas deferens and distal colon is not accompanied by inhibition of contraction

There is good evidence that in vascular smooth muscle, the relaxant effects of sodium nitroprusside (SNP) are mediated by increases in cGMP levels and activation of cGMP-dependent protein kinase (PKG). However, in rat vas deferens and rat distal colon, cGMP-elevating agents such as SNP and atrial natriuretic factor (ANF) have been shown to elevate cGMP without inducing relaxation. The lack of relaxation might be explained by either lack of activation of PKG by these agents or low levels of PKG in these tissues. The object of the present study was to investigate these possibilities by simultaneously monitoring cGMP levels, PKG activity and contractility in isolated strips of rat vas deferens, rat proximal colon and distal colon exposed to high concentrations of SNP or ANF. Verification of the specificity of the assay for PKG was obtained using MonoQ chromatography to resolve soluble smooth muscle extracts, followed by immunoblotting with a PKG-specific antibody to identify the kinase. In rat vas deferens, 5 mM SNP increased cGMP levels (14-fold) and PKG activity ratios (3.4-fold) but did not inhibit phenylephrine-induced contractions. In both rat proximal and rat distal colon, 100 nM ANF significantly elevated cGMP levels and PKG activity ratios, but only in the proximal colon was inhibition of spontaneous contractions observed. Total PKG activity was much lower (approximately 16 pmol PO4/min/mg protein) in rat vas deferens, which was not relaxed by SNP, than in rabbit aorta (approximately 148 pmol PO4/min/mg), which was relaxed. However, in the rat proximal colon, despite low PKG levels (approximately 11 pmole/min/mg), ANF did inhibit contractions. Thus the inability of the cGMP-elevating agents SNP and ANF to inhibit contractions in rat vas deferens and rat distal colon cannot be explained by either of the possibilities suggested above.     

Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1

Phosphorylation sites in members of the protein kinase A (PKA), PKG, and PKC kinase subfamily are conserved. Thus, the PKB kinase PDK1 may be responsible for the phosphorylation of PKC isotypes. PDK1 phosphorylated the activation loop sites of PKCzeta and PKCdelta in vitro and in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner in vivo in human embryonic kidney (293) cells. All members of the PKC family tested formed complexes with PDK1. PDK1-dependent phosphorylation of PKCdelta in vitro was stimulated by combined PKC and PDK1 activators. The activation loop phosphorylation of PKCdelta in response to serum stimulation of cells was PI 3-kinase-dependent and was enhanced by PDK1 coexpression.     

Differential activation of protein kinase C delta and epsilon gene expression by gonadotropin-releasing hormone in alphaT3-1 cells. Autoregulation by protein kinase C

The effect of gonadotropin-releasing hormone (GnRH) upon protein kinase C (PKC) delta and PKCepsilon gene expression was investigated in the gonadotroph-derived alphaT3-1 cell line. Stimulation of the cells with a stable analog [D-Trp6]GnRH (GnRH-A) resulted in a rapid elevation of PKCepsilon mRNA levels (1 h), while PKCdelta mRNA levels were elevated only after 24 h of incubation. The rapid elevation of PKCepsilon mRNA by GnRH-A was blocked by pretreatment with a GnRH antagonist or actinomycin D. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca2+ ionophore ionomycin, mimicked the rapid effect of GnRH-A upon PKCepsilon mRNA elevation. Additionally, the rapid stimulatory effect of GnRH-A was blocked by the selective PKC inhibitor GF109203X, by TPA-mediated down-regulation of endogenous PKC, or by Ca2+ removal. Interestingly, serum-starvation (24 h) advanced the stimulation of PKCdelta mRNA levels by GnRH-A and the effect could be detected at 1 h of incubation. The rapid effect of GnRH-A upon PKCdelta mRNA levels in serum-starved cells was mimicked by TPA, but not by ionomycin, and was abolished by down-regulation of PKC or by Ca2+ removal. Preactivation of alphaT3-1 cells with GnRH-A for 1 h followed by removal of ligand and serum resulted in elevation of PKCdelta mRNA levels after 24 h of incubation. Western blot analysis revealed that GnRH-A and TPA stimulated (within 5 min) the activation and some degradation of PKCdelta and PKCepsilon. We conclude that Ca2+ and PKC are involved in GnRH-A elevation of PKCdelta and PKCepsilon mRNA levels, with Ca2+ being necessary but not sufficient, while PKC is both necessary and sufficient to mediate the GnRH-A response. A serum factor masks PKCdelta but not PKCepsilon mRNA elevation by GnRH-A, and its removal exposes preactivation of PKCdelta mRNA by GnRH-A which can be memorized for 24 h. PKCdelta and PKCepsilon gene expression evoked by GnRH-A is autoregulated by PKC, and both isotypes might participate in the neurohormone action.     

Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1)

BACKGROUND: Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop. RESULTS: This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC betaII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC alpha and betaII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme. CONCLUSIONS: We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.     

Protein kinase C isotypes in human erythroleukemia (K562) cell proliferation and differentiation. Evidence that beta II protein kinase C is required for proliferation

The human erythroleukemia (K562) cell line undergoes megakaryocytic differentiation and cessation of proliferation when treated with phorbol myristate acetate (PMA). To investigate the role of individual protein kinase C (PKC) isotypes in these events, we have assessed PKC isotype expression during leukemic proliferation and PMA-induced differentiation. Immunoblot analysis using isotype-specific antibodies demonstrates that proliferating K562 cells express the alpha, beta II, and zeta PKC isotypes. PMA-induced differentiation and cytostasis lead to a decrease in beta II PKC and increases in alpha and zeta PKC levels. The role of the alpha and beta II PKC isotypes was further assessed in cells overexpressing these isotypes. K562 cells overexpressing human alpha PKC grew more slowly and were more sensitive to the cytostatic effects of PMA than control cells, whereas cells overexpressing beta II PKC were less sensitive to PMA. PMA-induced cytostasis is reversed upon removal of PMA. Resumption of proliferation is accompanied by reexpression of beta II PKC to near control levels, whereas alpha and zeta PKC levels remain elevated for several days after removal of PMA. Proliferation of PMA-withdrawn cells can be partially inhibited by antisense beta II PKC oligodeoxyribonucleotide. Growth inhibition is dose-dependent, specific for beta II PKC-directed antisense oligonucleotide, and associated with significant inhibition of beta II PKC levels indicating that beta II PKC is essential for K562 cell proliferation. Sodium butyrate, which unlike PMA induces megakaryocytic differentiation without cytostasis, causes increases in both alpha and beta II PKC levels. These data demonstrate that beta II PKC is required for K562 cell proliferation, whereas alpha PKC is involved in megakaryocytic differentiation.     

A screen for upstream components of the yeast protein kinase C signal transduction pathway identifies the product of the SLG1 gene

We employed the constitutive BCK1-20 allele of the gene for the MAP kinase kinase kinase (MAP-KKK) in the yeast Pkc signal transduction pathway to develop a genetic screen for mutants in genes encoding upstream components. Transposon mutagenesis yielded a mutant that was completely dependent on the active allele in the absence of osmotic stabilization. The transposon had integrated at the yeast SLG1 (HCS77) locus. This gene encodes a putative membrane protein. Haploid slg1 deletion strains are sensitive to caffeine, as expected for mutants in the Pkc pathway, as well as a variety of other drugs. The response to elevated temperatures and the dependence on osmotic stabilization depends on the genetic background. Thus, in the strain used for mutagenesis, disruption of SLG1 causes the cells to become non-viable in the absence of osmotic stabilization at both 30 degrees C and 37 degrees C. In a different genetic background this phenotype was not observed. Sensitivity of the haploid deletion mutants to caffeine can be partially suppressed by overexpression of genes for other components of the Pkc pathway, such as PKC1, SLT2, ROM2, and STE20. In addition, a SLG1-lacZ reporter construct shows higher expression in the presence of caffeine or magnesium chloride in a wild-type diploid background.     

Involvement of MAP kinase-independent protein kinase C signaling pathway in the EGF-induced p21(WAF1/Cip1) expression and growth inhibition of A431 cells

Previous studies have revealed that the growth inhibition of A431 cells overexpressing epidermal growth factor (EGF) receptors by a high concentration of EGF is mainly due to the expression of cycline dependent kinase (CDK) inhibitor p21(WAF1/Cip1). However, the signal transduction mechanism from the activated EGF receptor to the induction of p21(WAF1/Cip1) gene is still poorly understood. We investigated which signaling pathway plays an important role in p21(WAF1/Cip1) expression and growth inhibition by using specific inhibitors of the signaling molecules. A broad PKC inhibitor, PKC delta inhibitor, but not the conventional PKC inhibitor suppressed the EGF-induced p21(WAF1/Cip1) expression and the growth inhibition of A431 cells. These inhibitors did not alter either the activation of EGF receptor or the stimulation of MAP kinase at detectable levels. Furthermore, we found that the induction of p21(WAF1/Cip1) at the early phase (within 12 hr after stimulation) by a high concentration of EGF was independent of the MAP kinase activation by using dominant negative Ras. These results suggest that PKC, especially PKC delta plays a crucial role in the EGF-induced p21(WAF1/Cip1) expression, resulting in the growth inhibition of A431 cells.