Cytosolic pH sensitivity of an expressed human NHE-1 Na(+)-H+ exchanger

1993 ◽  
Vol 264 (4) ◽  
pp. C944-C950 ◽  
Author(s):  
K. Takaichi ◽  
D. F. Balkovetz ◽  
E. Van Meir ◽  
D. G. Warnock
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Wild Type Mouse ◽  
Host Cells ◽  
Wild Type ◽  
Cytosolic Ph ◽  
Kinase C ◽  
In The Wild ◽  
Mouse Cells ◽  
The Hill

These studies examined the effects of protein kinase C activation and calmodulin inhibition on the amiloride-sensitive NHE-1 isoform of the Na(+)-H+ exchanger in defined host cells. Our objective was to define differences in the cellular regulatory responses using a specified isoform of the Na(+)-H+ exchanger. Suspended cells were loaded with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and preacidified to a cytosolic pH of 6.2. Wild-type mouse Ltk- cells, human A-431 cells, and mutant mouse fibroblasts stably transfected with the human NHE-1 isoform (LAP+ cells) were examined to define the maximal rate of transport (Vmax) in response to 140 mM external Na+, the Hill stoichiometric coefficient, and the cytosolic pH at which the NHE-1 isoform was half-maximally stimulated (pH50). The mouse NHE-1 isoform had a greater affinity for cytosolic H+ than the human NHE-1 isoforms. Calmodulin antagonism with N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide reduced the Vmax and shifted the pH50 in the acidic direction, especially in the A-431 cells. Protein kinase C stimulation had a similar effect in A-431 cells and little effect in the wild-type (Ltk-) and transfected (LAP+) mouse cells. While the NHE-1 isoform contains several potential phosphorylation sites, the cellular milieu in which the isoform is expressed has an important effect on the modulation of NHE-1 activity.

Regulation of rat Na+-K+-ATPase activity by PKC is modulated by state of phosphorylation of Ser-943 by PKA

1997 ◽  
Vol 273 (6) ◽  
pp. C1981-C1986 ◽  
Author(s):  
Xian-Jun Cheng ◽  
Jan-Olov Höög ◽  
Angus C. Nairn ◽  
Paul Greengard ◽  
Anita Aperia
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Response To Treatment ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Present Evidence ◽  
Cos Cells ◽  
Kinase C ◽  
Kinase A

We have previously shown that the rat Na+-K+-ATPase α1-isoform is phosphorylated at Ser-943 by protein kinase A (PKA) and at Ser-23 by protein kinase C (PKC), which in both cases results in inhibition of enzyme activity. We now present evidence that suggests that the phosphorylation of Ser-943 by PKA modulates the response of Na+-K+-ATPase to PKC. Rat Na+-K+-ATPase α1 or a mutant in which Ser-943 was changed to Ala-943 was stably expressed in COS cells. The inhibition of enzyme activity measured in response to treatment with the phorbol ester, phorbol 12,13-dibutyrate (PDBu; 10−6 M), was significantly reduced in the cells expressing the Ala-943 mutant compared with that observed in cells expressing wild-type enzyme. In contrast, for cells expressing Na+-K+-ATPase α1 in which Ser-943 was mutated to Asp-943, the effect of PDBu was slightly enhanced. The PDBu-induced inhibition was not mediated by activation of the adenosine 3′,5′-cyclic monophosphate/PKA system and was not achieved via direct phosphorylation of Ser-943. Sp-5,6-DCl-cBIMPS, a specific PKA activator, increased the phosphorylation of Ser-943, and this was associated with an enhanced response to PDBu. Thus the effect of PKC on rat Na+-K+-ATPase α1 is determined not only by the activity of PKC but also by the state of phosphorylation of Ser-943.

scholarly journals Targeting of Protein Kinase C-ϵ during Fcγ Receptor-dependent Phagocytosis Requires the ϵC1B Domain and Phospholipase C-γ1

2006 ◽  
Vol 17 (2) ◽  
pp. 799-813 ◽  
Author(s):  
Keylon L. Cheeseman ◽  
Takehiko Ueyama ◽  
Tanya M. Michaud ◽  
Kaori Kashiwagi ◽  
Demin Wang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Fcγ Receptor ◽  
Kinase C ◽  
Green Fluorescent

Protein kinase C-ϵ (PKC-ϵ) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-ϵ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ϵC1 and ϵC1B domains, or the ϵC1B point mutant ϵC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ϵC259G, ϵC1, and ϵC1B accumulation at phagosomes was significantly less than that of intact PKC-ϵ. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-ϵ translocation. Thus, DAG binding to ϵC1B is necessary for PKC-ϵ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-γ1, and PI-PLC-γ2 in PKC-ϵ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-ϵ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-ϵ accumulation. Although expression of PI-PLC-γ2 is higher than that of PI-PLC-γ1, PI-PLC-γ1 but not PI-PLC-γ2 consistently concentrated at phagosomes. Macrophages from PI-PLC-γ2-/-mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-ϵ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-γ1 as the enzyme that supports PKC-ϵ localization and phagocytosis. That PI-PLC-γ1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-γ1 provides DAG that binds to ϵC1B, facilitating PKC-ϵ localization to phagosomes for efficient IgG-mediated phagocytosis.

scholarly journals Cytosolic pH regulation in osteoblasts. Regulation of anion exchange by intracellular pH and Ca2+ ions.

1990 ◽  
Vol 95 (1) ◽  
pp. 121-145 ◽  
Author(s):  
J Green ◽  
D T Yamaguchi ◽  
C R Kleeman ◽  
S Muallem
Keyword(s):  
Protein Kinase C ◽  
Membrane Potential ◽  
Protein Kinase ◽  
Anion Exchange ◽  
Cytosolic Ph ◽  
Subsequent Recovery ◽  
Kinase C ◽  
Recovery From Acidification ◽  
Umr 106 ◽  
Cytosolic Acidification

Measurements of cytosolic pH (pHi) 36Cl fluxes and free cytosolic Ca2+ concentration ([Ca2+]i) were performed in the clonal osteosarcoma cell line UMR-106 to characterize the kinetic properties of Cl-/HCO3- (OH-) exchange and its regulation by pHi and [Ca2+]i. Suspending cells in Cl(-)-free medium resulted in rapid cytosolic alkalinization from pHi 7.05 to approximately 7.42. Subsequently, the cytosol acidified to pHi 7.31. Extracellular HCO3- increased the rate and extent of cytosolic alkalinization and prevented the secondary acidification. Suspending alkalinized and Cl(-)-depleted cells in Cl(-)-containing solutions resulted in cytosolic acidification. All these pHi changes were inhibited by 4',4',-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and H2DIDS, and were not affected by manipulation of the membrane potential. The pattern of extracellular Cl- dependency of the exchange process suggests that Cl- ions interact with a single saturable external site and HCO3- (OH-) complete with Cl- for binding to this site. The dependencies of both net anion exchange and Cl- self-exchange fluxes on pHi did not follow simple saturation kinetics. These findings suggest that the anion exchanger is regulated by intracellular HCO3- (OH-). A rise in [Ca2+]i, whether induced by stimulation of protein kinase C-activated Ca2+ channels, Ca2+ ionophore, or depolarization of the plasma membrane, resulted in cytosolic acidification with subsequent recovery from acidification. The Ca2+-activated acidification required the presence of Cl- in the medium, could be blocked by DIDS, and H2DIDS and was independent of the membrane potential. The subsequent recovery from acidification was absolutely dependent on the initial acidification, required the presence of Na+ in the medium, and was blocked by amiloride. Activation of protein kinase C without a change in [Ca2+]i did not alter pHi. Likewise, in H2DIDS-treated cells and in the absence of Cl-, an increase in [Ca2+]i did not activate the Na+/H+ exchanger in UMR-106 cells. These findings indicate that an increase in [Ca2+]i was sufficient to activate the Cl-/HCO3- exchanger, which results in the acidification of the cytosol. The accumulated H+ in the cytosol activated the Na+/H+ exchanger. Kinetic analysis of the anion exchange showed that at saturating intracellular OH-, a [Ca2+]i increase did not modify the properties of the extracellular site. A rise in [Ca2+]i increased the apparent affinity for intracellular OH- (or HCO3-) of both net anion and Cl- self exchange. These results indicate that [Ca2+]i modifies the interaction of intracellular OH- (or HCO3-) with the proposed regulatory site of the anion exchanger in UMR-106 cells.

scholarly journals Inhibition of myogenesis by the H-ras oncogene: implication of a role for protein kinase C.

1991 ◽  
Vol 114 (4) ◽  
pp. 809-820 ◽  
Author(s):  
T B Vaidya ◽  
C M Weyman ◽  
D Teegarden ◽  
C L Ashendel ◽  
E J Taparowsky
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Transcriptional Activation ◽  
Signal Transduction Pathway ◽  
Phospholipid Metabolism ◽  
Skeletal Myogenesis ◽  
Wild Type ◽  
Myogenic Regulatory Factor ◽  
Kinase C ◽  
Ras P21

Expression of the oncogenic form of H-ras p21 in the mouse myogenic cell line, 23A2, blocks myogenesis and inhibits expression of the myogenic regulatory factor gene, MyoD1. Previous studies from a number of laboratories have demonstrated that the activation of ras p21 is associated with changes in phospholipid metabolism that directly, or indirectly, lead to elevated levels of intracellular diacylglycerol and the subsequent activation of protein kinase C (PKC). To assess the importance of PKC activity to the ras-induced inhibition of skeletal myogenesis, we examined the levels of PKC activity associated with the terminal differentiation of wild-type myoblasts and with the differentiation-defective phenotype of 23A2 ras cells. We demonstrate that there is a 50% reduction in PKC activity during normal myogenesis and that PKC activity is required for myoblast fusion, but not for the transcriptional activation of muscle-specific genes. In contrast, we found that the differentiation-defective 23A2 ras cells possess two- to threefold more PKC activity than wild-type myofibers and that reducing the PKC activity in these cultures does not reverse their non-myogenic phenotype. On the other hand, if PKC activity is downregulated in 23A2 cells before the expression of activated ras p21, myogenesis is not inhibited. These results suggest that activated ras p21 relies on a PKC-dependent signal transduction pathway to initiate, but not to sustain, its negative effects on 23A2 skeletal myogenesis and underscore the potential importance of PKC activity to the proper control of skeletal muscle differentiation.

scholarly journals Phosphorylation of human pleckstrin on Ser-113 and Ser-117 by protein kinase C

1996 ◽  
Vol 314 (3) ◽  
pp. 937-942 ◽  
Author(s):  
Karen L. CRAIG ◽  
Calvin B. HARLEY
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Cos Cells ◽  
Phospholipid Hydrolysis ◽  
Kinase C

During platelet activation, receptor-coupled phospholipid hydrolysis stimulates protein kinase C (PKC) and results in the phosphorylation of several proteins, the most prominent being pleckstrin. Pleckstrin is composed of two repeated domains, now called pleckstrin homology (PH) domains, separated by a spacer region that contains several consensus PKC phosphorylation sites. To determine the role of PKC-dependent phosphorylation in pleckstrin function, we mapped the phosphorylation sites in vivo of wild-type and site-directed mutants of pleckstrin expressed in COS cells. Phosphorylation was found to occur almost exclusively on Ser-113 and Ser-117 within the sequence 108-KFARKS*TRRS*IRL-120. Phosphorylation of these sites was confirmed by phosphorylation of the corresponding wild-type and mutant synthetic peptides in vitro.

scholarly journals CalDAG-GEFI and protein kinase C represent alternative pathways leading to activation of integrin αIIbβ3 in platelets

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 1696-1703 ◽  
Author(s):  
Stephen M. Cifuni ◽  
Denisa D. Wagner ◽  
Wolfgang Bergmeier
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Small Gtpase ◽  
Wild Type ◽  
Pkc Inhibitor ◽  
Integrin Αiibβ3 ◽  
Alternative Pathways ◽  
Kinase C ◽  
Induced Aggregation ◽  
Inside Out

AbstractSecond messenger-mediated inside-out activation of integrin αIIbβ3 is a key step in platelet aggregation. We recently showed strongly impaired but not absent αIIbβ3-mediated aggregation of CalDAG-GEFI–deficient platelets activated with various agonists. Here we further evaluated the roles of CalDAG-GEFI and protein kinase C (PKC) for αIIbβ3 activation in platelets activated with a PAR4 receptor–specific agonist, GYPGKF (PAR4p). Compared with wild-type controls, platelets treated with the PKC inhibitor Ro31-8220 or CalDAG-GEFI–deficient platelets showed a marked defect in aggregation at low (< 1mM PAR4p) but not high PAR4p concentrations. Blocking of PKC function in CalDAG-GEFI–deficient platelets, how-ever, strongly decreased aggregation at all PAR4p concentrations, demonstrating that CalDAG-GEFI and PKC represent separate, but synergizing, pathways important for αIIbβ3 activation. PAR4p-induced aggregation in the absence of CalDAG-GEFI required cosignaling through the Gαi-coupled receptor for ADP, P2Y12. Independent roles for CalDAG-GEFI and PKC/Gαi signaling were also observed for PAR4p-induced activation of the small GTPase Rap1, with CalDAG-GEFI mediating the rapid but reversible activation of this small GTPase. In summary, our study identifies CalDAG-GEFI and PKC as independent pathways leading to Rap1 and αIIbβ3 activation in mouse platelets activated through the PAR4 receptor.

Identification of protein kinase C subspecies in wild-type and multidrug-resistant cell lines

1993 ◽  
Vol 21 (4) ◽  
pp. 378S-378S
Author(s):  
James A. L. Fenton ◽  
Alex Paton ◽  
Nigel Groome ◽  
J. Roger Warr ◽  
Martin G. Rumsby
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Lines ◽  
Multidrug Resistant ◽  
Resistant Cell ◽  
Wild Type ◽  
Kinase C

High Density Lipoproteins Enhance the Na+/H+ Antiport in Human Platelets

1996 ◽  
Vol 75 (04) ◽  
pp. 635-641 ◽  
Author(s):  
Jerzy-Roch Nofer ◽  
Martin Tepel ◽  
Beate Kehrel ◽  
Michael Walter ◽  
Udo Seedorf ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Human Platelets ◽  
Covalent Modification ◽  
High Density ◽  
High Density Lipoproteins ◽  
Free Calcium ◽  
Sodium Propionate ◽  
Cytosolic Ph ◽  
Kinase C

SummaryIn the present study, we investigated the effect of high density lipoproteins 3 (HDL3) on Na+/H+ exchanger activity and cytosolic pH (pHi) in human platelets. HDL3 alone failed to affect pHi? but preincubation with HDL3 significantly enhanced the Na+/H+ antiport activation brought about by acidification with 100 mM sodium propionate or stimulation with 0.05 U/ml thrombin. The stimulatory effect of HDL3 was unaffected by indomethacin excluding a role for cyclooxygenase products. The HDL3 effect was not mediated by Ca2+/calmodulin-dependent protein kinase as HDL3 failed to increase cytosolic free calcium concentration. However, the potentiating effect of HDL3 was completely blocked in the presence of the protein kinase C inhibitor, bisindoylmaleimide and the phosphatidylcholine-specific phospholi-pase C inhibitor, D609. Furthermore, the effect of HDL3 was abolished after covalent modification of HDL3 with dimethylsuberimidate and was not observed in platelets from Glanzmann thrombasthenia type 1 which do not express GP IIb/IIIa, as well as in platelets preincubated with anti-GP Ilb/IIIa polyclonal antibodies. We conclude that HDL3 enhances the sodium propionate- and thrombin-induced Na+/H+ antiport activity in human platelets via binding to GP Ilb/IIIa and activation of protein kinase C and phosphatidylcholine-specific phospholipase C.

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