scholarly journals Activated release of membrane-anchored TGF-alpha in the absence of cytosol

1993 ◽  
Vol 122 (1) ◽  
pp. 95-101 ◽  
Author(s):  
MW Bosenberg ◽  
A Pandiella ◽  
J Massagué
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Atp Hydrolysis ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Vesicular Traffic ◽  
Kinase C ◽  
Streptolysin O ◽  
Independent Protein

The ectodomain of proTGF-alpha, a membrane-anchored growth factor, is converted into soluble TGF-alpha by a regulated cellular proteolytic system that recognizes proTGF-alpha via the C-terminal valine of its cytoplasmic tail. In order to define the biochemical components involved in proTGF-alpha cleavage, we have used cells permeabilized with streptolysin O (SLO) that have been extensively washed to remove cytosol. PMA, acting through a Ca(2+)-independent protein kinase C, activates cleavage as efficiently in permeabilized cells as it does in intact cells. ProTGF-alpha cleavage is also stimulated by GTP gamma S through a mechanism whose pharmacological properties suggest the involvement of a heterotrimeric G protein acting upstream of the PMA-sensitive Ca(2+)-independent protein kinase C. Activated proTGF-alpha cleavage is dependent on ATP hydrolysis, appears not to require vesicular traffic, and acts specifically on proTGF-alpha that has reached the cell surface. These results indicate that proTGF-alpha is cleaved from the cell surface by a regulated system whose signaling, recognition, and proteolytic components are retained in cells devoid of cytosol.

Arachidonic acid-induced LH release is ATP-independent and insensitive to N-ethyl maleimide

1992 ◽  
Vol 132 (1) ◽  
pp. 77-82 ◽  
Author(s):  
P. V. Kaye ◽  
P. A. van der Merwe ◽  
R. P. Millar ◽  
J. S. Davidson
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Pituitary Cells ◽  
Cell Permeabilization ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Kinase C ◽  
Lh Release ◽  
Stimulation Of

ABSTRACT The mechanism of arachidonic acid (AA)-induced LH release was characterized using sheep pituitary cells in primary culture permeabilized with Staphylococcal α-toxin. In intact cells, exogenous AA evoked release of LH in a manner which was partially dependent on extracellular Ca2+. At similar concentrations, AA also caused cell permeabilization as monitored by efflux of [3H]2-deoxyglucose metabolites. In α-toxin-permeabilized cells where cytosolic Ca2+ was clamped at resting levels, AA retained its ability to cause LH release. Unlike the stimulation of exocytosis produced by Ca2+, phorbol ester or cyclic AMP, AA-evoked release was independent of ATP and was not inhibited by pretreatment with N-ethyl maleimide. These findings indicated that exogenous AA does not cause LH release by Ca2+ influx or mobilization or by activating protein kinase C. The results suggest that LH release induced by exogenous AA is probably due to its detergent-like properties, and does not represent true exocytosis. Journal of Endocrinology (1992) 132, 77–82

scholarly journals Activation of protein kinase C is not an absolute requirement for amylase release from permeabilized rat pancreatic acini

1992 ◽  
Vol 285 (2) ◽  
pp. 597-601 ◽  
Author(s):  
A J O'Sullivan ◽  
J D Jamieson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Peptide Inhibitor ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Permeabilized Cells ◽  
Kinase C ◽  
Streptolysin O ◽  
Absolute Requirement ◽  
Messenger System

The effect of protein kinase C (PKC) on amylase discharge from streptolysin-O-permeabilized rat pancreatic acini was investigated. Addition of phorbol 12-myristate 13-acetate (PMA) to permeabilized cells potentiated Ca(2+)-stimulated release, but had no effect on discharge at non-stimulatory Ca2+ concentrations. PMA markedly shifted the Ca(2+)-concentration-dependence of amylase discharge to the left, by enhancing the time over which the permeabilized cells release. This effect was inhibited by both staurosporine and PKC-19-31-amide peptide inhibitor, indicating that the effect of PMA was due to its action on PKC. Staurosporine also partially inhibited amylase release at the optimal concentration of Ca2+; this effect was not replicated by the more specific PKC-19-31-amide peptide inhibitor and may be due to an effect on another second-messenger system. PKC appears to be an important modulator of release in pancreatic acini, but its activation is not an absolute requirement for Ca(2+)-dependent amylase discharge.

scholarly journals A protein kinase C pseudosubstrate peptide inhibits phosphorylation of the CD3 antigen in streptolysin-O-permeabilized human T lymphocytes

1989 ◽  
Vol 260 (3) ◽  
pp. 893-901 ◽  
Author(s):  
D R Alexander ◽  
J M Hexham ◽  
S C Lucas ◽  
J D Graves ◽  
D A Cantrell ◽  
...  
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
T Lymphocytes ◽  
Gamma Chain ◽  
Permeabilized Cells ◽  
Human T Lymphocytes ◽  
Kinase C ◽  
Streptolysin O ◽  
Subsequent Addition

Activation of human T lymphocytes leads to the phosphorylation of the CD3-antigen gamma polypeptide. We have investigated a possible role for protein kinase C (PKC) in mediating this phosphorylation event by using T cells permeabilized with streptolysin-O in the presence of 120 mM-K+ buffers containing Ca2+-EGTA. The gamma-chain was phosphorylated by [gamma-32P]ATP in permeabilized T lymphoblasts in the presence of phorbol 12,13-dibutyrate (Pdbu) or phytohaemagglutinin (PHA). Ca2+ alone in the range 0.5-1.0 microM also induced gamma-chain phosphorylation in some T-lymphoblast preparations; that in Jurkat-6 cells occurred at lower concentrations (50-500 nM). Two experimental approaches were used to investigate the possible involvement of PKC. Firstly, when permeabilization was carried out in buffer lacking free Ca2+, PKC was lost from the cells, and gamma-chain phosphorylation could then no longer be induced on subsequent addition of Pdbu or PHA in 400 nM-Ca2+, or 800 nM-Ca2+ alone, to permeabilized cells. However, when permeabilization was carried out in the presence of these three agents, PKC was translocated to intracellular membranes, and subsequent addition of [gamma-32P]ATP to these cells then resulted in gamma-chain phosphorylation. In the second approach, induction of gamma-chain phosphorylation by Pdbu, 1-oleoyl-2-acetylglycerol, 1,2-diolein, PHA or Ca2+ alone was effectively blocked by permeabilizing T cells in the presence of a PKC pseudosubstrate peptide (50 microM). Pseudosubstrate concentrations in the range 7-20 microM inhibited gamma-chain phosphorylation by 50%. In contrast, addition of four other ‘irrelevant’ basic peptides (50 microM) did not result in detectable inhibition, and 50 microM-pseudosubstrate did not inhibit the phosphorylation of 17 other polypeptides isolated from permeabilized T cells. These data suggest that Pdbu-, 1,2-diacylglycerol-, PHA- and Ca2+-induced phosphorylation of the CD3-antigen gamma chain in permeabilized T cells is mediated by PKC.

Regulation of protein kinase C activity in neuronal differentiation induced by the N-ras oncogene in PC-12 cells

1990 ◽  
Vol 10 (6) ◽  
pp. 2983-2990
Author(s):  
J C Lacal ◽  
A Cuadrado ◽  
J E Jones ◽  
R Trotta ◽  
D E Burstein ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Neuronal Differentiation ◽  
Phorbol Esters ◽  
Ras Oncogene ◽  
Intact Cells ◽  
Pc 12 Cells ◽  
Kinase C ◽  
Ras P21

Expression of the N-ras oncogene under the control of the glucocorticoid-responsive promoter in the pheochromocytoma cell line UR61, a subline of PC-12 cells, has been used to investigate the differentiation process to neuronal cells triggered by ras oncogenes (I. Guerrero, A. Pellicer, and D. E. Burstein, Biochem. Biophys. Res. Commun. 150:1185-1192, 1988). Using ras-inducible cell lines, we observed that expression of the oncogenic N-ras p21 protein interferes with the ability of phorbol esters to induce downregulation of protein kinase C. This effect was associated with the appearance of immunologically detectable protein kinase C as well as the activity of the enzyme as analyzed either by binding of [3H]phorbol-12,13-dibutyrate in intact cells or by in vitro kinase activity. These results indicate a relationship between ras p21 and protein kinase C in neuronal differentiation in this model system. Comparison to the murine fibroblast system suggests that this relationship may be functional.

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

1988 ◽  
Vol 106 (4) ◽  
pp. 1061-1066 ◽  
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.

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

2016 ◽  
Vol 310 (9) ◽  
pp. F821-F831 ◽  
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.

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

2000 ◽  
Vol 11 (7) ◽  
pp. 2497-2511 ◽  
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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