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

1990 ◽  
Vol 265 (15) ◽  
pp. 8369-8372
Author(s):  
C Godson ◽  
B A Weiss ◽  
P A Insel
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Differential Activation ◽  
Kinase C ◽  
Protein Kinase C Alpha ◽  
Arachidonate Release ◽  
Darby Canine Kidney ◽  
Canine Kidney

scholarly journals Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells

2003 ◽  
Vol 373 (3) ◽  
pp. 917-924 ◽  
Author(s):  
Jorge CERBÓN ◽  
Rosa del Carmen LÓPEZ-SÁNCHEZ
Keyword(s):  
Cell Proliferation ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Fold Increase ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Kinase C ◽  
Pkc Activation ◽  
Darby Canine Kidney ◽  
Canine Kidney

We have investigated the effects of inhibiting sphingomyelin (SM) biosynthesis on cellular diacylglycerol (DAG) content and protein kinase C (PKC) activation during growth initiation in Madin–Darby canine kidney cells. We utilized β-chloroalanine (BCA) to inactivate serine C-palmitoyltransferase, the first enzyme in the sphingolipid biosynthesis pathway. This inactivation prevented growth, but did not affect viability. When the inhibitor was replaced with fresh culture medium, the cells continued their proliferation in a normal way. BCA (2 mM) inhibited [32P]Pi, [3H]palmitic acid and [methyl-3H]choline incorporation into SM, but did not influence the synthesis of other major phospholipids. SM synthesis and DAG generation were decreased by 51% and 47.6% respectively. Particulate PKC activity was not observed in cells incubated with BCA, in contrast with a 5-fold increase in control cells. BCA inhibited 75% of the [3H]thymidine incorporation, and the cells were arrested before the S phase of the cell cycle. Moreover, exogenous d-erythrosphingosine restored SM synthesis, DAG generation and cell proliferation. These data indicate that the contribution of DAG generated during SM synthesis plays an important role in PKC activation and cell proliferation.

Regulation of eicosanoid biosynthesis by phorbol ester in Madin Darby canine kidney cells

1990 ◽  
Vol 259 (4) ◽  
pp. F698-F703 ◽  
Author(s):  
D. W. Coyne ◽  
M. Mordhorst ◽  
A. R. Morrison
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Mdck Cells ◽  
Pge2 Production ◽  
Madin Darby Canine Kidney ◽  
Kinase C ◽  
Protein Kinase C Inhibitor ◽  
Peptide Agonist ◽  
Darby Canine Kidney ◽  
Canine Kidney

We assessed the effects of the peptide agonist, bradykinin (BK), and phorbol myristate acetate (PMA) on prostaglandin E2 (PGE2) production, cyclooxygenase (COX) activity and mass, and arachidonic acid (AA) release in Madin Darby canine kidney (MDCK) cells. PMA stimulated PGE2 production by increasing both AA release and the activity of COX. Using [35S]methionine labeling and immunoprecipitation, we demonstrated that the increased COX activity is due to new COX synthesis. Actinomycin D and cycloheximide blocked the PMA-stimulated COX activity but not AA release. Both PMA-stimulated AA release and COX activity were reduced by the protein kinase C inhibitor staurosporine (STP). Glucocorticoids failed to alter PMA- or BK-stimulated PGE2 production was reduced by STP, indicating BK acts in part through protein kinase C activation. BK increased PGE2 production in PMA-treated cells, suggesting a protein kinase C-independent mechanism of action as well. BK did not stimulate any change in COX activity. We conclude that in MDCK cells PMA, but not BK, can stimulate both AA release and COX synthesis. Stimulation of COX synthesis requires either prolonged activation of protein kinase C and/or an additional nonprotein kinase C-mediated effect of PMA.

scholarly journals Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover

2017 ◽  
Vol 28 (25) ◽  
pp. 3595-3608 ◽  
Author(s):  
Anastasia F. Thévenin ◽  
Rachel A. Margraf ◽  
Charles G. Fisher ◽  
Rachael M. Kells-Andrews ◽  
Matthias M. Falk
Keyword(s):  
Protein Kinase ◽  
Gap Junction ◽  
Wild Type ◽  
Madin Darby Canine Kidney ◽  
Kinase C ◽  
Zonula Occludens ◽  
Zonula Occludens 1 ◽  
Darby Canine Kidney ◽  
Canine Kidney

To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 located in the C-terminal domain of connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All connexin mutants were translated into stable, full-length proteins and assembled into GJs when expressed in HeLa or Madin–Darby canine kidney epithelial cells. However, mutants with S residues exchanged at positions 365, 368, and 373 exhibited a significantly altered ZO-1 interaction profile, while mutants with S residues exchanged at 255 and 279/282 did not. Unlike wild-type Cx43, in which ZO-1 binding is restricted to the periphery of GJ plaques, S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. Inability to disengage from ZO-1 correlated with increased GJ plaque size and increased connexin protein half-life, while maintaining GJ channels in an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations in clathrin-binding efficiency, suggesting that the inability to disengage from ZO-1 prevented maturation of functional into nonfunctional/endocytic channels, rather than ZO-1 interfering with GJ endocytosis directly. Collectively, our results indicate that ZO-1 binding regulates channel accrual, while disengagement from ZO-1 is critical for GJ channel closure and transitioning GJ channels for endocytosis. Intriguingly, these transitional ZO-1 binding/release and channel-aging steps are mediated by a series of hierarchical phosphorylation/dephosphorylation events at S373, S365, and S368, well-known Cx43 Akt, protein kinase A, and protein kinase C phosphorylation sites located in the vicinity of the ZO-1 binding site.

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