Protein kinase C in mouse kidney: subcellular distribution and endogenous substrates

1988 ◽  
Vol 66 (4) ◽  
pp. 262-272 ◽  
Author(s):  
Avihu Boneh ◽  
Harriet S. Tenenhouse
Keyword(s):  
Protein Kinase C ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Brush Border ◽  
Subcellular Distribution ◽  
Brush Border Membrane ◽  
Mouse Kidney ◽  
Kidney Cortex ◽  
Kinase C ◽  
Endogenous Substrates

The subcellular distribution, kinetic properties, and endogenous substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) were examined in mouse kidney cortex. Protein kinase C associated with the particulate, mitochondrial, and brush border membrane fractions was assayed after solubilization in 0.2% Triton X-100 under conditions shown to be noninhibitory to catalytic activity. Of recovered activity, 52% was associated with the cytosolic fraction; mitochondrial and brush border membrane associated protein kinase C constituted 12 and 3%, respectively, of the activity recovered in the particulate fraction. Protein kinase C associated with brush border membranes exhibited a high affinity for ATP (apparent Km = 62 ± 10 μM) and the highest apparent maximal velocity (1146 ± 116 pmol P/(mg protein∙min)) of the renal fractions examined. Maximal stimulation of protein kinase C by diacylglycerol (in the presence of phosphatidylserine) was achieved at both 25 and 300 μM calcium in all renal fractions. These results are consistent with previous reports demonstrating that diacylglycerol increases the apparent affinity of protein kinase C for calcium. Phorbol 12-myristate 13-acetate, but not 4α-phorbol, was able to substitute for diacylglycerol and stimulate cytosolic and particulate renal protein kinase C. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a specific inhibitor of protein kinase C, led to significant inhibition of catalytic activity in all renal subcellular fractions. Endogenous substrates for protein kinase C were demonstrated in renal cytosolic (26, 45, 63, and 105 kilodaltons (kDa)), particulate (26, 33, 68, and 105 kDa), mitochondrial (43 kDa), and brush border membrane (26, 41, 52, 88, and 105 kDa) fractions. The possible physiological significance of protein kinase C in mammalian kidney is discussed.

Phorbol ester inhibition of chicken intestinal brush-border sodium-proton exchange

1991 ◽  
Vol 260 (6) ◽  
pp. C1264-C1272 ◽  
Author(s):  
E. B. Chang ◽  
M. W. Musch ◽  
D. Drabik-Arvans ◽  
M. C. Rao
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Brush Border ◽  
Time Course ◽  
Brush Border Membrane ◽  
Phorbol Esters ◽  
Kinase C ◽  
Na Uptake ◽  
Exchange Activity

Phorbol esters, specific activators of protein kinase C, inhibit amiloride-sensitive Na uptake from the mucosal medium in intact intestinal mucosa as well as in isolated chicken villus enterocytes. In isolated cells, maximal inhibition is observed at 60 s, and influx returns to control values within 15 min. This effect can be measured either as initial 22Na influx rates or by following changes in intracellular pH using the pH-sensitive fluorescent dye 5,6-carboxyfluorescein. The effects of amiloride and phorbol esters were not additive, suggesting inhibition of a common transport system, i.e., Na-H exchange. In brush-border membrane vesicles (BBMV) made from villus enterocytes, amiloride-sensitive Na-H exchange activity was significantly inhibited in phorbol ester-treated cells. The degree of inhibition of 22Na uptake by BBMV had the same time course and dose-effect relationship as phorbol ester-inhibited cellular Na uptake. Similarly, the time course of protein kinase C translocation from cytosol to particulate or brush-border membrane fractions correlated with Na uptake measurements made in whole cells and BBMV. These results suggest that protein kinase C activation in chicken villus enterocytes inhibits brush-border membrane Na-H exchange activity.

scholarly journals Stimulation of fructose transport across the intestinal brush-border membrane by PMA is mediated by GLUT2 and dynamically regulated by protein kinase C

2000 ◽  
Vol 350 (1) ◽  
pp. 149-154 ◽  
Author(s):  
Philip A. HELLIWELL ◽  
Michael RICHARDSON ◽  
Julie AFFLECK ◽  
George L. KELLETT
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Basolateral Membrane ◽  
Fold Increase ◽  
Sugar Absorption ◽  
Kinase C

Perfusion of rat jejunum in vitro with PMA increased fructose transport by 70% compared with control values and was blocked by the protein kinase C (PKC) inhibitor chelerythrine. The brush-border membrane contained both the fructose transporters GLUT5 and GLUT2; the presence of the latter was confirmed by luminal biotinylation. PMA increased the GLUT2 level 4-fold within minutes, so that the level was comparable with that of the basolateral membrane, but had no effect on GLUT5 level. GLUT2 was functional, accessible to luminal fructose and could be inhibited selectively by phloretin to permit determination of GLUT2- and GLUT5-mediated transport components. The 4-fold increase in GLUT2 level induced by PMA was matched by a 4-fold increase in GLUT2-mediated transport: there was a compensatory fall in the GLUT5-mediated rate. The pattern of dynamic trafficking was seen only for GLUT2, not GLUT5 or SGLT1, implying that GLUT2 trafficks to the brush-border membrane by a different pathway. Trafficking of GLUT2 to the brush-border membrane correlated with activation of PKC βII, implying that this isoenzyme is likely to control trafficking. Since PKC is activated by endogenous hormones, GLUT2 levels in vivo are 3–4-fold those in vitro; moreover, because PKC is inactivated as soon as intestine is excised, GLUT2 is lost from the brush-border within minutes in vitro. It is therefore difficult to detect GLUT2 in most in vitro preparations and its role in intestinal sugar absorption across the brush-border membrane has accordingly been overlooked.

scholarly journals Protein Kinase C Mediates the Mitogenic Action of Thrombopoietin in c-Mpl–Expressing UT-7 Cells

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 813-822 ◽  
Author(s):  
Ying Hong ◽  
Dominique Dumènil ◽  
Bernd van der Loo ◽  
Frédérique Goncalves ◽  
William Vainchenker ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Subcellular Distribution ◽  
Membrane Fraction ◽  
Induced Expression ◽  
Gm Csf ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Mitogenic Action ◽  
Pkc Activation

Protein kinase C (PKC) has been implicated in signal transduction events elicited by several hematopoietic growth factors. Thrombopoietin (TPO) is the major regulator of megakaryocytic lineage development, and its receptor, c-Mpl, transduces signals for the proliferation and differentiation of hematopoietic progenitors. In this study we have examined the effect of TPO on the subcellular distribution of PKC (a measure of enzyme activation) in a growth factor-dependent pluripotent hematopoietic cell line that was engineered to express the c-Mpl receptor (UT-7/mpl). In addition, we have assessed the significance of this activation for the induction of both mitogenesis and differentiation. Using a PKC translocation assay, TPO was found to stimulate a time- and dose-dependent increase in the total content of PKC activity present in the membrane fraction of UT-7/mpl cells (maximum increase = 2.3-fold above basal level after 15 minutes with 40 ng/mL TPO, EC50 = 7 ng/mL). Accordingly, a decrease of PKC content in the cytosolic fraction was observed. Immunoblot analysis using PKC isotype-specific antibodies showed that TPO treatment led to a marked increase of the Ca2+/diacylglycerol-sensitive PKC isoforms α and β found in the membrane fraction. In contrast, the subcellular distribution of these isoforms did not change after treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF). Exposure of UT-7/mpl cells to the selective PKC inhibitor GF109203X completely inhibited the PKC activity associated to the membrane fraction after TPO treatment, and blocked the mitogenic effect of TPO. In contrast, GF109203X had no effect on the TPO-induced expression of GpIIb, a megakaryocytic differentiation antigen. Downregulation of PKC isoforms α and β to less than 25% of their initial level by treatment with phorbol 12,13-dibutyrate also abolished the TPO-induced mitogenic response, but had no significant effect when this response was induced by GM-CSF. Taken together, these findings suggest that (1) TPO stimulates the activation of PKC, (2) PKC activation mediates the mitogenic action of TPO, and (3) PKC activation is not required for TPO-induced expression of megakaryocytic surface markers.

scholarly journals Protein kinase C in rod outer segments: effects of phosphorylation of the phosphodiesterase inhibitory subunit

1996 ◽  
Vol 317 (1) ◽  
pp. 291-295 ◽  
Author(s):  
Igor P. UDOVICHENKO ◽  
Jess CUNNICK ◽  
Karen GONZALEZ ◽  
Alexander YAKHNIN ◽  
Dolores J. TAKEMOTO
Keyword(s):  
Protein Kinase C ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Direct Interaction ◽  
Inhibitory Effect ◽  
Rod Outer Segments ◽  
Visual Transduction ◽  
Gtp Hydrolysis ◽  
Outer Segments ◽  
Kinase C

The inhibitory subunit (PDEγ) of the cGMP phosphodiesterase (PDEαβγ2) in rod outer segments (ROS) realizes its regulatory role in phototransduction by inhibition of PDEαβ catalytic activity. The photoreceptor G-protein, transducin, serves as a transducer from the receptor (rhodopsin) to the effector (PDE) and eliminates the inhibitory effect of PDEγ by direct interaction with PDEγ. Our previous study [Udovichenko, Cunnick, Gonzalez and Takemoto (1994) J. Biol. Chem. 269, 9850–9856] has shown that PDEγ is a substrate for protein kinase C (PKC) from ROS and that phosphorylation by PKC increases the ability of PDEγ to inhibit PDEαβ catalytic activity. Here we report that transducin is less effective in activation of PDEαβ(γp)2 (a complex of PDEαβ with phosphorylated PDEγ, PDEγp) than PDEαβγ2. PDEγp also increases the rate constant of GTP hydrolysis of transducin (from 0.16 s-1 for non-phosphorylated PDEγ to 0.21 s-1 for PDEγp). These data suggest that phosphorylation of the inhibitory subunit of PDE by PKC may regulate the visual transduction cascade by decreasing the photoresponse.

Regulation of α1-adrenoceptor-mediated contractions of uterine arteries by PKC: effect of pregnancy

2006 ◽  
Vol 291 (5) ◽  
pp. H2282-H2289 ◽  
Author(s):  
Hongying Zhang ◽  
DaLiao Xiao ◽  
Lawrence D. Longo ◽  
Lubo Zhang
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Western Blot ◽  
Subcellular Distribution ◽  
Response Curve ◽  
Uterine Artery ◽  
Uterine Arteries ◽  
Kinase C ◽  
Pkc Isozymes ◽  
Near Term

Protein kinase C (PKC) plays an important role in the regulation of uterine artery contractility and its adaptation to pregnancy. The present study tested the hypothesis that PKC differentially regulates α1-adrenoceptor-mediated contractions of uterine arteries isolated from nonpregnant (NPUA) and near-term pregnant (PUA) sheep. Phenylephrine-induced contractions of NPUA and PUA sheep were determined in the absence or presence of the PKC activator phorbol 12,13-dibutyrate (PDBu). In NPUA sheep, PDBu produced a concentration-dependent potentiation of phenylephrine-induced contractions and shifted the dose-response curve to the left. In contrast, in PUA sheep, PDBu significantly inhibited phenylephrine-induced contractions and decreased their maximum response. Simultaneous measurement of contractions and intracellular free Ca2+ concentrations ([Ca2+]i) in the same tissues revealed that PDBu inhibited phenylephrine-induced [Ca2+]i and contractions in PUA sheep. In NPUA sheep, PDBu increased phenylephrine-induced contractions without changing [Ca2+]i. Western blot analysis showed six PKC isozymes, α, βI, βII, δ, ε, and ζ, in uterine arteries, among which βI, βII, and ζ isozymes were significantly increased in PUA sheep. In contrast, PKC-α was decreased in PUA sheep. In addition, analysis of subcellular distribution revealed a significant decrease in the particulate-to-cytosolic ratio of PKC-ε in PUA compared with that in NPUA sheep. The results suggest that pregnancy induces a reversal of PKC regulatory role on α1-adrenoceptor-mediated contractions from a potentiation in NPUA sheep to an inhibition in PUA sheep. The differential expression of PKC isozymes and their subcellular distribution in uterine arteries appears to play an important role in the regulation of Ca2+ mobilization and Ca2+ sensitivity in α1-adrenoceptor-mediated contractions and their adaptation to pregnancy.

Regulation of Jak2 tyrosine kinase by protein kinase C during macrophage differentiation of IL-3–dependent myeloid progenitor cells

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1626-1632 ◽  
Author(s):  
Panu E. Kovanen ◽  
Ilkka Junttila ◽  
Kati Takaluoma ◽  
Pipsa Saharinen ◽  
Leena Valmu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Growth Arrest ◽  
Macrophage Differentiation ◽  
Myeloid Progenitor ◽  
Kinase C ◽  
Myeloid Progenitor Cells

Differentiation of macrophages from myeloid progenitor cells depends on a discrete balance between cell growth, survival, and differentiation signals. Interleukin-3 (IL-3) supports the growth and survival of myeloid progenitor cells through the activation of Jak2 tyrosine kinase, and macrophage differentiation has been shown to be regulated by protein kinase C (PKC). During terminal differentiation of macrophages, the cells lose their mitogenic response to IL-3 and undergo growth arrest, but the underlying signaling mechanisms have remained elusive. Here we show that in IL-3–dependent 32D myeloid progenitor cells, the differentiation-inducing PKC isoforms PKC- and PKC-δ specifically caused rapid inhibition of IL-3–induced tyrosine phosphorylation. The target for this inhibition was Jak2, and the activation of PKC by 12-O-tetradecanoyl-phorbol-13-acetate treatment also abrogated IL-3–induced tyrosine phosphorylation of Jak2 in Ba/F3 cells. The mechanism of this regulation was investigated in 32D and COS7 cells, and the inhibition of Jak2 required catalytic activity of PKC-δ and involved the phosphorylation of Jak2 on serine and threonine residues by the associated PKC-δ. Furthermore, PKC-δ inhibited the in vitro catalytic activity of Jak2, indicating that Jak2 was a direct target for PKC-δ. In 32D cells, the inhibition of Jak2 either by PKC-δ, tyrosine kinase inhibitor AG490, or IL-3 deprivation caused a similar growth arrest. Reversal of PKC-δ–mediated inhibition by the overexpression of Jak2 promoted apoptosis in differentiating 32D cells. These results demonstrate a PKC-mediated negative regulatory mechanism of cytokine signaling and Jak2, and they suggest that it serves to integrate growth-promoting and differentiation signals during macrophage differentiation.

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