Constitutive Protein Kinase C Research Articles

Changes in body mass alone explain almost all of the variance in the serum sodium concentrations during prolonged exercise. Has commercial influence impeded scientific endeavour?

In 1991, we provided definitive evidence that exercise-associated hyponatraemia (EAH) is caused by abnormal fluid retention in those who overdrink during prolonged exercise, but this finding was ignored. Instead, in...

Constitutive PKC phosphorylation of the D1 dopamine receptor inhibits receptor signaling

The D1 dopamine (DA) receptor (D1) is phosphorylated by protein kinase C (PKC), yet the role of PKC in D1 signaling is undefined. We investigated PKC effects on the D1 using transiently transfected cells. Activation of PKC by phorbol esters (PE) increased D1 phosphorylation. Co‐transfection of the D1 with PKC isoforms α, βI, γ, δ, ε, and λ resulted in greater D1 phosphorylation, whereas PKC isoforms μ, ν, η, and ζ were without effect. Activation of an M1 muscarinic receptor resulted in increased D1 phosphorylation, indicating heterologous phosphorylation by PKC. PKC effects on D1 signaling were evaluated using cAMP assays. Surprisingly, both activation and inhibition of PKC were found to potentiate DA‐stimulated cAMP accumulation. The potentiation is specific to D1 activation, since PE‐treatment had no effect on forskolin‐stimulated cAMP levels. To focus on D1‐specific PKC effects, we performed site‐directed mutagenesis of the D1 in order to identify the PKC phosphorylation sites. Five sites were identified: S259, S397, S398, S417, and S421. Simultaneous mutation of these sites eliminated PKC phosphorylation of the D1 resulting in increased G protein coupling and cAMP accumulation which was no longer potentiated by PKC inhibitors. These data suggest that constitutive PKC phosphorylation of the D1 regulates the signaling tone of the receptor.

Probing the Control Elements of the CYP1A1 Switching Module in H4IIE Hepatoma Cells

Previous research from our laboratory has shown a switch-like response to PCB 126 mediated CYP1A1 induction in primary rat hepatocytes and in H4IIE rat hepatoma cells. On a single cell level, cells appear to be either "on" or "off" for CYP1A1 induction at a given dose; some cells never respond to PCB 126. These cells represent a non-responding population. Cells that are switched "on" by PCB 126 display varying levels of induction, much like the dimmer on a light switch. The goal of the present research is to begin to uncover the mechanism for this switch-like response to CYP1A1 induction in H4IIE rat hepatoma cells. The AhR pathway is modulated by multiple co-activators and by phosphorylation. This research focuses on the phosphorylation cascades initiated by PCB 126 and the role they play in CYP1A1 induction. Our research reveals a likely role for protein kinase C (PKC) in this switch response. Inhibition of PKC by H-7 dramatically reduced the percent of cells that express CYP1A1 in response to PCB 126 treatment, as determined by flow cytometry. The effect of H-7 was concentration dependent, decreasing the number of cells expressing CYP1A1 rather than decreasing the level of CYP1A1 in all cells. This finding provides further evidence for the switch-like behavior of CYP1A1 induction and implicates PKC in this response to PCB126. The protein kinase inhibitor, HA-1004, had only a minor effect on CYP1A1 induction. A high-throughput immunoblot screen for 40 proteins revealed the regulation of several proteins/phosphoproteins by PCB 126. Most importantly, two proteins containing phosphoserine/phoshothreonine residues were increased by PCB126 treatment. However, PKC translocation studies and activity studies failed to verify that PCB126 activates PKC. It is possible that constitutive PKC activity is sufficient to maintain phosphorylation of critical components of the AhR pathway. Immunoblotting studies showed that MAP kinases ERK and JNK are not activated by PCB 126 in H4IIE cells and the ERK inhibitor U0126 did not impair CYP1A1 induction. Additional studies are planned to further investigate the role of PKC in the switch-like response to PCB 126.

Protein Kinase C βII Plays an Essential Role in Dendritic Cell Differentiation and Autoregulates Its Own Expression

Dendritic cells (DC) arise from a diverse group of hematopoietic progenitors and have marked phenotypic and functional heterogeneity. The signal transduction pathways that regulate the ability of progenitors to undergo DC differentiation, as well as the specific characteristics of the resulting DC, are only beginning to be characterized. We have found previously that activation of protein kinase C (PKC) by cytokines or phorbol esters drives normal human CD34(+) hematopoietic progenitors and myeloid leukemic blasts (KG1, K562 cell lines, and primary patient blasts) to differentiate into DC. We now report that PKC activation is also required for cytokine-driven DC differentiation from monocytes. Of the cPKC isoforms, only PKC-betaII was consistently activated by DC differentiation-inducing stimuli in normal and leukemic progenitors. Transfection of PKC-betaII into the differentiation-resistant KG1a subline restored the ability to undergo DC differentiation in a signal strength-dependent fashion as follows: 1) by development of characteristic morphology; 2) the up-regulation of DC surface markers; 3) the induction of expression of the NFkappaB family member Rel B; and 4) the potent ability to stimulate allo-T cells. Most unexpectedly, the restoration of PKC-betaII signaling in KG1a was not directly due to overexpression of the transfected classical PKC (alpha, betaII, or gamma) but rather through induction of endogenous PKC-beta gene expression by the transfected classical PKC. The mechanism of this positive autoregulation involves up-regulation of PKC-beta promoter activity by constitutive PKC signaling. These findings indicate that the regulation of PKC-betaII expression and signaling play critical roles in mediating progenitor to DC differentiation.

Constitutively and autonomously active protein kinase C associated with 14-3-3 zeta in the rodent brain.

Persistent activation of protein kinase C (PKC) is required for the expression of synaptic plasticity in the brain. There are several mechanisms proposed that can lead to the prolonged activation of PKC. These include long lasting production of lipid activators (diacylglycerol and fatty acid) through mitogen-activated protein (MAP) kinase pathway, and a modification of PKC by reactive oxygen species. In nerve growth factor (NGF)-differentiated PC12 cells, we found that constitutive and autonomous Ca2+-independent PKC activity is associated with 14-3-3 zeta. Because PKC and 14-3-3 zeta are both involved in synaptic plasticity and learning and memory, we examined whether PKC interacts with 14-3-3 zeta in the brain and whether the PKC/14-3-3 zeta complex has autonomous activity. Here we show that three subclasses of PKC, Ca2+-dependent classical PKC, Ca2+-independent novel PKC, and Ca2+-independent and diacylglycerol-insensitive atypical PKC, all interact with 14-3-3 zeta in the rodent brain. The pool size of 14-3-3 zeta bound form of PKC is small (1-4% of each PKC isoform), but they show constitutive and autonomous activity. Our study indicates that the binding of PKC with 14-3-3 zeta is at least in part independent of phosphorylation of PKC and that the C1 domain of PKC is involved in the binding. As both molecules are enriched in synaptic locus, the constitutive PKC activity and its interaction with 14-3-3 zeta could be a mechanism for the persistent PKC activation in the brain.

Phosphorylation of GluR4 AMPA-type glutamate receptor subunit by protein kinase C in cultured retina amacrine neurons.

We have previously reported that the activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors is potentiated by protein kinase C (PKC) in cultured chick retina amacrine neurons, and that constitutive PKC activity is necessary for basal AMPA receptor activity (Carvalho et al., 1998). In this study, we evaluated the phosphorylation of the GluR4 subunit, which is very abundant in cultured amacrine neurons, to correlate it with the effects of PKC on AMPA receptor activity in these cells. 32P-labelling of GluR4 increased upon AMPA receptor stimulation or cell treatment with phorbol 12-myristate 13-acetate (PMA) before stimulating with kainate. By contrast, phosphorylation of GluR4 was not changed when PKC was inhibited by treating the cells with the selective PKC inhibitor GF 109203X before stimulation with kainate. We conclude that GluR4 is phosphorylated upon PKC activation and/or stimulation of AMPA receptors in cultured amacrine cells. Additionally, AMPA receptor activation with kainate in cultured chick amacrine cells leads to translocation of conventional and novel PKC isoforms to the cell membrane, suggesting that PKC could be activated upon AMPA receptor stimulation in these cells.

Induction of a Specific Olfactory Memory Leads to a Long-Lasting Activation of Protein Kinase C in the Antennal Lobe of the Honeybee

In this study we investigated the role of protein kinase C (PKC) in associative learning of Apis mellifera. Changes in PKC activity induced by olfactory conditioning were measured in the antennal lobes, a brain structure involved in associative learning. Multiple conditioning trials inducing a memory different from that induced by a single conditioning trial specifically cause an increase in PKC activity. This increase begins 1 hr after conditioning, lasts up to 3 d, and is attributable to an increased level of constitutive PKC. The increased level of constitutive PKC consists of an early proteolysis-dependent phase and a late phase that requires RNA and protein synthesis. Inhibition of the pathways resulting in constitutive PKC selectively impairs distinct phases of multiple-trial induced memory. The inhibition of the proteolytic mechanism has an instant effect on an early phase of multiple-trial induced memory but does not affect acquisition and the late phase of memory. Blocking of the transient PKC activation during conditioning does not affect the induction of memory formation. Thus, the constitutive PKC in the antennal lobe seems to contribute to the early phase of memory that is induced by multiple-trial conditioning.

Breast cancer cell invasiveness: correlation with protein kinase C activity and differential regulation by phorbol ester in estrogen receptor-positive and -negative cells.

Increased protein kinase C (PKC) activity in malignant breast tissue and in most aggressive breast cancer cell lines has suggested a possible role of PKC in breast carcinogenesis and tumor progression. We have investigated here the involvement of PKC in the in vitro invasiveness and motility of several breast cancer cell lines. Modulation of PKC activity by treatment with a phorbol ester (TPA), drastically increased the invasiveness of 2 estrogen receptor-positive (ER+) lines (MCF7 and ZR 75.1), whereas it markedly decreased the invasiveness of 2 ER- cell lines (MDA-MB-231 and MDA-MB-435). A PKC inhibitor (H7) reversed the TPA effects in MCF7 cells, whereas it mimicked TPA action in MDA-MB-231 cells. All of these effects of TPA also were observed to a similar extent for cell chemotaxis, and they were not dependent on protein neo-synthesis. In parallel, short TPA treatment induced cell spreading and microtubule organization in MCF7 cells and inverse morphological changes in MDA-MB-231 cells. In ER+ cells, constitutive PKC activity and PKCalpha expression were very low as compared to ER- cells, and this correlated with the invasive potential of the cells. The opposed effects of TPA in ER+ and ER- cells could be due to the abnormal TPA regulation of PKCalpha observed in ER- cells.

Phosphorylation by Protein Kinase C Is Required For Acute Activation of Cystic Fibrosis Transmembrane Conductance Regulator by Protein Kinase A

Protein kinase A (PKA) stimulates Cl secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR), a tightly regulated Cl- channel in the apical membrane of many secretory epithelia. The CFTR channel is also modulated by protein kinase C (PKC), but the regulatory mechanisms are poorly understood. Here we present evidence that PKA-mediated phosphorylation alone is not a sufficient stimulus to open the CFTR chloride channel in the presence of MgATP; constitutive PKC phosphorylation is essential for acute activation of CFTR by PKA. When patches were excised from transfected Chinese hamster ovary cells, CFTR responses to PKA became progressively smaller with time and eventually disappeared. This decline in PKA responsiveness did not occur in the presence of exogenous PKC and was reversed by the addition of PKC to channels that had become refractory to PKA. PKC enhanced PKA stimulation of open probability without increasing the number of functional channels. Short-term pretreatment of cells with the PKC inhibitor chelerythrine (1 microM) reduced the channel activity that could be elicited by forskolin in cell-attached patches. Moreover, in whole cell patches, acute stimulation of CFTR currents by chlorophenylthio-cAMP was abolished by two chemically unrelated PKC inhibitors, although an abrupt, partial activation was observed after a delay of >15 min. Modulation by PKC was most pronounced when basal PKC phosphorylation was reduced by briefly preincubating cells with chelerythrine. Constitutive PKC phosphorylation in unstimulated cells permits the maximum elevation of open probability by PKA to reach a level that is approximately 60% of that attained during in vitro exposure to both kinases. Differences in basal PKC activity may contribute to the variable cAMP responsiveness of CFTR channels in different cell types.

Effect of phorbol 12, 13-dibutyrate on ligand binding, enzyme activity and translocation of protein kinase C isoforms in the alpha T3-1 gonadotrope-derived cell line.

The effect of incubating alpha T3-1 cells with phorbol 12,13-dibutyrate (PDBu) on the protein kinase C (PKC) isoform content (predominantly alpha, epsilon and zeta isoforms) was assessed by immunoblotting, enzyme activity assay and [3H]PDBu binding. After exposure to PDBu for 17 h the immunoreactivity detected for both PKC alpha and PKC epsilon had disappeared from cytosol and had increased slightly in membranes. Immunoreactivity for PKC zeta was present as two bands in cytosol; after PDBu treatment both bands decreased in intensity, the higher molecular weight band more than the lower. The lower molecular weight band corresponded with a component of constitutive PKC activity eluting from DEAE cellulose that was defined by inhibition of basal activity with GF 109203X or H7. Investigation of very short treatment times with PDBu using binding, immunoblot and activity measurements (in the presence/absence of Ca2+) indicated that translocation of PKC alpha and epsilon was very rapid-detectable by 10 sec, maximal within minutes. Reduction of these isoforms in membranes took much longer, and was not apparent up to 150 min. The immunoblot data for PKC zeta in cytosol showed no detectable effect of PDBu treatment on the low molecular weight band up to 150 min although it was reduced at 17 h. Translocation of the upper band was detectable at 10 sec but this band may have resulted from cross-reaction with other PKC isoforms. The constitutive activity and low molecular weight ("authentic') PKC zeta immunoreactivity were partially affected after long exposure only, suggesting an action of PDBu on PKC zeta secondary to activation of the other PKC isoforms. An endogenous receptor agonist, luteinising hormone-releasing hormone (LHRH), was also used to assess by immunoblotting, translocation of the PKC isoforms. Although all the isoforms did translocate from cytosol to membrane fractions, they did so with distinctly different time courses: PKC epsilon moved more rapidly than PKC zeta which appeared to translocate more quickly than PKC alpha. After downregulation of the responsive PKC isoforms with PDBu, the remaining PKC zeta was not translocated by LHRH.

Human Cancer Cells Exhibit Protein Kinase C-dependent c-erbB-2 Transmodulation That Correlates with Phosphatase Sensitivity and Kinase Activity

The c-erbB-2 receptor tyrosine kinase is often overexpressed in human tumors, but the functional implications of this phenotype remain unclear. We previously used phosphorylation-specific antibodies to define major differences in c-erbB-2 tyrosine kinase activity between overexpressing human tumor cell lines (Epstein, R. J., Druker, B. J., Roberts, T. M., and Stiles, C. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10435-10439). Here we extend this approach to define the relationship between c-erbB-2 tyrosine phosphorylation and protein kinase C (PKC)-dependent transmodulation. Phosphorylation-specific antibodies to the juxtamembrane PKC site Thr686 recognize tyrosine-dephosphorylated wild-type c-erbB-2 following G8/DHFR 3T3 cell treatment with PKC agonists. B104-1-1 cells transformed by activated c-erbB-2 express a subset of tyrosine-phosphorylated receptors that are homologously phosphorylated on Thr686, indicating that Thr686 phosphorylation alone is insufficient to abrogate receptor tyrosine phosphorylation. Similarly, the c-erbB-2-overexpressing human cancer cell lines SK-Ov-3 and BT-474 express constitutively Thr686-phosphorylated receptors. SK-Ov-3 cells express predominantly kinase-inactive c-erbB-2 that is heavily Thr686-phosphorylated, indicating that Thr686 phosphorylation in this line is heterologous in origin. In contrast, BT-474 cells express constitutively autophosphorylated c-erbB-2 despite Thr686 phosphorylation. These results indicate that Thr686 phosphorylation does not directly abolish c-erbB-2 activity and suggest that such phosphorylation reflects constitutive PKC activity induced by either receptor-activating mutations or heterologous growth factors. The latter possibility suggests in turn that c-erbB-2 interacts in an as yet undefined way with heterologous growth factor receptors in human tumor cells.

Selective responses (actin polymerization, shape changes, locomotion, pinocytosis) to the PKC inhibitor Ro 31-8220 suggest that PKC discriminately regulates functions of human blood lymphocytes

The results suggest that protein kinase C (PKC) plays a pivotal role in the control of F-actin levels, locomotion, pinocytosis, and cell shape in lymphocytes. The PKC inhibitor Ro 31-8220 elicits a high proportion of polarized (ED50 = 1.5 x 10(-6) M) and locomoting cells and reduces the relative amount of F-actin (by 29% at 10(-5) M) in initially resting cells. Phorbol myristate acetate (PMA) counterbalances the polarizing effect of Ro 31-8220. This indicates that the spherical shape and the F-actin content of resting cells are maintained by constitutive PKC activity. PMA-induced increases in fluid pinocytosis, F-actin content, and formation of nonpolar cells with surface protrusion are suppressed by Ro 31-8220 (IC50 = 2-4 x 10(-7) M). Spherical cells and, at higher concentrations (ED50 = 3.3 x 10(-6) M), polarized cells are formed instead. As a result, lymphocyte function switches from fluid pinocytosis to cell polarity and locomotion. The data indicate that PKC is instrumental in selectively switching lymphocyte function between resting state, locomotor activity, and fluid pinocytosis. Ro 31-8220 is extremely potent in stimulating lymphocyte polarity and locomotion (B and T cells). It acts faster and/or produces a higher proportion of polarized lymphocytes than other available agonists. It may thus be used as a tool in further experiments requiring locomoting lymphocytes.

A selective protein kinase C inhibitor (CGP 41251) positively and negatively modulates melanoma cell MSH receptors.

Melanoma cells express receptors for melanocyte-stimulating hormone (MSH) in variable abundance. CGP 41251, a derivative of staurosporine with an increased selectivity for protein kinase C (PKC) inhibition, was found to modulate MSH receptors in human D10 and HBL cells and in the mouse B16 cell line. Up-regulation was observed in D10 and B16 cells at a concentration of 290 nM and 190 nM, respectively. In HBL cells, however, the PKC inhibitor induced a pronounced MSH receptor down-regulation with an EC50 of only 32 nM. In D10 and HBL cells, alpha-MSH and CGP 41251 synergistically regulated MSH receptors whereas these agents had an antagonistic effect in B16 cells. PKC stimulation by short-term treatment with phorbol ester had an opposite effect on MSH receptors as compared to CGP 41251. In B16 cells, CGP 41251 at a concentration of 100 nM increased the sensitivity to MSH-induced melanogenesis. The staurosporine derivative inhibited proliferation of HBL, B16, and D10 cells at EC50s of 180 nM, 190 nM, and 520 nM, respectively. Furthermore, CGP 41251 increased the dendricity of the cells. In a concentration range between 300 nM and 1 mu M, CGP 41251 induced a sharp increase of the mean cell diameter from 16 mu m to 19 mu m. Thus, the effects of the selective PKC inhibitor on MSH receptors are induced at lower concentrations than needed for the inhibition of proliferation or for the change in cell morphology. These results suggest that the number of MSH receptors expressed on the surface of cultured melanoma cells correlates with the level of constitutive PKC activity in individual cell lines.

Identification of Two Distinct Populations of Protein Kinase C in Rat Brain Membranes

The regulatory enzyme protein kinase C (PKC) is proposed to be activated on its translocation from the cytosol to the membrane. However, a portion of the native activity is always associated with the membrane fraction. Using a noninvasive procedure to extract this endogenous activity from rat brain membranes, it has been possible to characterize the activity in a partially purified reconstituted system bearing resemblance to the in vivo system. Two subpopulations of membrane-associated PKC were identified and characterized at the level of activation, inhibition, and isozyme immunologic characteristics and chromatographic properties. One peak had properties similar to those of cytosolic PKC, whereas the second population, extracted as protein-lipid complexes, had considerable constitutive activity that could be stimulated further on addition of PKC activators. This latter activity was relatively resistant to staurosporine inhibition and phorbol ester treatment, but it phosphorylated the exogenous PKC substrates, histone 1 and the epidermal growth factor receptor peptide KTRLRR. The constitutive activity was totally dependent on its endogenous associated lipids coextracted by the solubilization procedure. The ratio between these two populations was ontogenetically regulated and modulated by phorbol ester treatment, suggesting that different PKC populations may serve unique functions in the rat brain regulated by the lipid environment. Analyses of the phospholipids extracted in these protein-lipid complexes showed differences in the major classes correlating to age. However, apart from a markedly lower cholesterol content in these complexes, no direct relationship between a specific lipid composition and the amount of constitutive PKC activity was evident.

Differences in the effects of phorbol esters and diacylglycerols on protein kinase C.

The binding of protein kinase C (PKC) to membranes and appearance of kinase activity are separable events. Binding is a two-step process consisting of a reversible calcium-dependent interaction followed by an irreversible interaction that can only be dissociated by detergents. The irreversibly bound PKC is constitutively active, and the second step of binding may be a major mechanism of PKC activation [Bazzi & Nelsestuen (1988) Biochemistry 27, 7589]. This study examined the activity of other forms of membrane-bound PKC and compared the effects of phorbol esters and diacylglycerols. Like the membrane-binding event, activation of PKC was a two-stage process. Diacylglycerols (DAG) participated in forming an active PKC which was reversibly bound to the membrane. In this case, both activity and membrane binding were terminated by addition of calcium chelators. DAG functioned poorly in generating the constitutively active, irreversible PKC-membrane complex. These properties differed markedly from phorbol esters which activated PKC in a reversible complex but also promoted constitutive PKC activation by forming the irreversible PKC-membrane complex. The concentration of phorbol esters needed to generate the irreversible PKC-membrane complex was slightly higher than the concentration needed to activate PKC. In addition, high concentrations of phorbol esters (greater than or equal to 50 nM) activated PKC and induced irreversible PKC-membrane binding in the absence of calcium. Despite these striking differences, DAG prevented binding of phorbol esters to high-affinity sites on the PKC-membrane complex.(ABSTRACT TRUNCATED AT 250 WORDS)