Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1

Increased expression of endothelin converting enzyme-1 (ECE-1) is associated with diabetic nephropathy. The molecular mechanisms underlying this association, as yet unknown, possibly involve protein kinase C (PKC) pathways. In the present study, we examined the effects of high glucose and PKC activation on ECE-1 expression in primary human umbilical vein endothelial cells (HUVECs) and in HUVEC line (EA.hy926). Increasing glucose concentration, but not mannitol, from 5.5–22.2 mmol/liter for 3 d, enhanced prepro endothelin-1 (ET-1) mRNA expression, ET-1 levels, ECE-1 protein, and mRNA expressions by 7, 4, 20, and 2.6-fold, respectively. High glucose increased ECE-1 protein expression dose and time dependently. By Western blot analysis, PKC-β1, -β2, and -δ isoform levels were significantly increased relative to other isoforms when glucose level was increased. Treatment with Rottlerin, a PKC-δ isoform inhibitor, reduced significantly the glucose-induced ET-1 secretion, and ECE-1 protein expression, but (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16,21-dimetheno 1H,13H-dibenzo[e,k]pyrrolo[3,4-h] (1, 4, 3) oxadiaza-cyclohexadecene-1,3(2H)-dione or Gö6976, specific PKC-β and -α inhibitors, respectively, did not. Overexpression of PKC-δ but not PKC-α or -β1 isoforms by adenovirus vector containing the respective cDNA in HUVECs incubated with 5.5 mmol/liter glucose, increased in parallel PKC proteins, and glucose-induced endothein-1 and ECE-1 protein expression by 4- to 6-fold. These results show that enhanced ECE-1 expression induced by hyperglycemia is partly due to activation of the PKC-δ isoform. Thus, inhibition of this PKC isoform may prevent diabetes-related increase in ET-1. Hyperglycemia-induced enhanced endothelin converting enzyme-1 expression is mediated by PKC-δ. Inhibition of this PKC isoform may prevent diabetes-related increase in endothelin-1.

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Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2010.06.045 ◽

2010 ◽

Vol 398 (2) ◽

pp. 173-177 ◽

Cited By ~ 12

Author(s):

Sanjaya Kuruppu ◽

Natalie Tochon-Danguy ◽

A. Ian Smith

Keyword(s):

Endothelial Cells ◽

Protein Kinase C ◽

Protein Kinase ◽

Converting Enzyme ◽

Kinase C ◽

Endothelin Converting Enzyme

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Protein Kinase C recognition sites in the cytoplasmic domain of Endothelin Converting Enzyme-1c

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2012.09.105 ◽

2012 ◽

Vol 427 (3) ◽

pp. 606-610 ◽

Cited By ~ 7

Author(s):

Sanjaya Kuruppu ◽

Nathalie Tochon-Danguy ◽

A. Ian Smith

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cytoplasmic Domain ◽

Converting Enzyme ◽

Kinase C ◽

Recognition Sites ◽

Endothelin Converting Enzyme

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Transcriptional Mechanism of Protein Kinase C-Induced Isoform-Specific Expression of the Gene for Endothelin-Converting Enzyme-1in Human Endothelial Cells

Molecular Pharmacology ◽

10.1124/mol.60.6.1332 ◽

2001 ◽

Vol 60 (6) ◽

pp. 1332-1342 ◽

Cited By ~ 16

Author(s):

Hans-Dieter Orzechowski ◽

Astrid Günther ◽

Stefan Menzel ◽

Andreas Zimmermann ◽

Heiko Funke-Kaiser ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Kinase C ◽

Protein Kinase ◽

Converting Enzyme ◽

Human Endothelial Cells ◽

Specific Expression ◽

Kinase C ◽

Endothelin Converting Enzyme

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Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation.

The Journal of Cell Biology ◽

10.1083/jcb.106.4.1061 ◽

1988 ◽

Vol 106 (4) ◽

pp. 1061-1066 ◽

Cited By ~ 41

Author(s):

T E McGraw ◽

K W Dunn ◽

F R Maxfield

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cell Surface ◽

Phorbol Ester ◽

Cho Cells ◽

Transferrin Receptor ◽

Phosphorylation Site ◽

Major Protein ◽

Kinase C ◽

Recycling Pathway

In Chinese hamster ovary (CHO) fibroblast cells the protein kinase C activating phorbol ester, phorbol myristate acetate (PMA), stimulates an increase in cell surface transferrin receptor (TR) expression by increasing the exocytic rate of the recycling pathway. The human TR expressed in CHO cells is similarly affected by PMA treatment. A mutant human TR in which the major protein kinase C phosphorylation site, serine 24, has been replaced with the non-phosphorylatable amino acid glycine has been constructed to investigate the role of receptor phosphorylation in the PMA induced up-regulation. The Gly-24-substituted receptor binds, internalizes, and recycles Tf. Furthermore, the altered receptor mediates cellular Fe accumulation from diferric-Tf, thereby fulfilling the receptor's major biological role. The Gly-24 TR behaves identically to the wild-type TR when cells are treated with PMA. Therefore, Ser-24 phosphorylation is not required for the PMA-induced redistribution of the human TR expressed in CHO cells. The increased TR expression on the cell surface after PMA treatment results from an increase in the rate of exocytosis of the recycling receptors. No change in the endocytic rate or the size of the recycling receptor pool was observed. These results indicate that the PMA effect on the TR surface expression may result from a more general perturbation of membrane trafficking rather than a specific modulation of the TR.

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Nedd4-2 but not Nedd4-1 is critical for protein kinase C-regulated ubiquitination, expression, and transport activity of human organic anion transporter 1

AJP Renal Physiology ◽

10.1152/ajprenal.00522.2015 ◽

2016 ◽

Vol 310 (9) ◽

pp. F821-F831 ◽

Cited By ~ 11

Author(s):

Da Xu ◽

Haoxun Wang ◽

Qiang Zhang ◽

Guofeng You

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cell Surface ◽

Organic Anion ◽

The Body ◽

Organic Anion Transporter ◽

Transport Activity ◽

Kinase C ◽

Anion Transporter ◽

In Cells

Human organic anion transporter 1 (hOAT1) expressed at the membrane of the kidney proximal tubule cells mediates the body disposition of a diverse array of clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, and antiinflammatories. Therefore, understanding the regulation of hOAT1 will provide significant insights into kidney function and dysfunction. We previously established that hOAT1 transport activity is inhibited by activation of protein kinase C (PKC) through accelerating hOAT1 internalization from cell surface into intracellular endosomes and subsequent degradation. We further established that PKC-induced hOAT1 ubiquitination is an important step preceding hOAT1 internalization. In the current study, we identified two closely related E3 ubiquitin ligases, neural precursor cell expressed, developmentally downregulated 4-1 and 4-2 (Nedd4-1 and Nedd4-2), as important regulators for hOAT1: overexpression of Nedd4-1 or Nedd4-2 enhanced hOAT1 ubiquitination, reduced the hOAT1 amount at the cell surface, and suppressed hOAT1 transport activity. In further exploring the relationship among PKC, Nedd4-1, and Nedd4-2, we discovered that PKC-dependent changes in hOAT1 ubiquitination, expression, and transport activity were significantly blocked in cells transfected with the ligase-dead mutant of Nedd4-2 (Nedd4-2/C821A) or with Nedd4-2-specific siRNA to knockdown endogenous Nedd4-2 but not in cells transfected with the ligase-dead mutant of Nedd4-1 (Nedd4-1/C867S) or with Nedd4-1-specific siRNA to knockdown endogenous Nedd4-1. In conclusion, this is the first demonstration that both Nedd4-1 and Nedd4-2 are important regulators for hOAT1 ubiquitination, expression, and function. Yet they play distinct roles, as Nedd4-2 but not Nedd4-1 is a critical mediator for PKC-regulated hOAT1 ubiquitination, expression, and transport activity.

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Entry of the Two Infectious Forms of Vaccinia Virus at the Plasma Membane Is Signaling-Dependent for the IMV but Not the EEV

Molecular Biology of the Cell ◽

10.1091/mbc.11.7.2497 ◽

2000 ◽

Vol 11 (7) ◽

pp. 2497-2511 ◽

Cited By ~ 127

Author(s):

Jacomine Krijnse Locker ◽

Annett Kuehn ◽

Sibylle Schleich ◽

Gaby Rutter ◽

Heinrich Hohenberg ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Cell Surface ◽

Vaccinia Virus ◽

Hela Cells ◽

Signaling Cascade ◽

Enveloped Virus ◽

Kinase C ◽

Mature Virus

The simpler of the two infectious forms of vaccinia virus, the intracellular mature virus (IMV) is known to infect cells less efficiently than the extracellular enveloped virus (EEV), which is surrounded by an additional, TGN-derived membrane. We show here that when the IMV binds HeLa cells, it activates a signaling cascade that is regulated by the GTPase rac1 and rhoA, ezrin, and both tyrosine and protein kinase C phosphorylation. These cascades are linked to the formation of actin and ezrin containing protrusions at the plasma membrane that seem to be essential for the entry of IMV cores. The identical cores of the EEV also appear to enter at the cell surface, but surprisingly, without the need for signaling and actin/membrane rearrangements. Thus, in addition to its known role in wrapping the IMV and the formation of intracellular actin comets, the membrane of the EEV seems to have evolved the capacity to enter cells silently, without a need for signaling.

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Protein Kinase C-ε Induces Caveolin-Dependent Internalization of Vascular Adenosine 5′-Triphosphate–Sensitive K + Channels

Hypertension ◽

10.1161/hypertensionaha.108.110817 ◽

2008 ◽

Vol 52 (3) ◽

pp. 499-506 ◽

Cited By ~ 32

Author(s):

Jundong Jiao ◽

Vivek Garg ◽

Baofeng Yang ◽

Terry S. Elton ◽

Keli Hu

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Angiotensin Ii ◽

Protein Kinase ◽

Cell Surface ◽

Small Interfering Rna ◽

Caveolin 1 ◽

Kinase C ◽

Interfering Rna ◽

Pkc Activation

Vascular ATP-sensitive K + (K ATP ) channels are critical regulators of arterial tone and, thus, blood flow in response to local metabolic needs. They are important targets for clinically used drugs to treat hypertensive emergency and angina. It is known that protein kinase C (PKC) activation inhibits K ATP channels in vascular smooth muscles. However, the mechanism by which PKC inhibits the channel remains unknown. Here we report that caveolin-dependent internalization is involved in PKC-ε–mediated inhibition of vascular K ATP channels (Kir6.1 and SUR2B) by phorbol 12-myristate 13-acetate or angiotensin II in human embryonic kidney 293 cells and immortalized human saphenous vein vascular smooth muscle cells. We showed that Kir6.1 substantially overlapped with caveolin-1 at the cell surface. Cholesterol depletion with methyl-β-cyclodextrin significantly reduced, whereas overexpression of caveolin-1 largely enhanced, PKC-induced inhibition of Kir6.1/SUR2B currents. Importantly, we demonstrated that activation of PKC-ε caused internalization of K ATP channels, the effect that was blocked by depletion of cholesterol with methyl-β-cyclodextrin, expression of dominant-negative dynamin mutant K44E, or knockdown of caveolin-1 with small interfering RNA. Moreover, patch-clamp studies revealed that PKC-ε–mediated inhibition of the K ATP current induced by PMA or angiotensin II was reduced by a dynamin mutant, as well as small interfering RNA targeting caveolin-1. The reduction in the number of plasma membrane K ATP channels by PKC activation was further confirmed by cell surface biotinylation. These studies identify a novel mechanism by which the levels of vascular K ATP channels could be rapidly downregulated by internalization. This finding provides a novel mechanistic insight into how K ATP channels are regulated in vascular smooth muscle cells.

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