scholarly journals Thrombin Ca2+-dependently stimulates protein tyrosine phosphorylation in BC3H1 muscle cells

1993 ◽  
Vol 290 (1) ◽  
pp. 27-32 ◽  
Author(s):  
S Offermanns ◽  
E Bombien ◽  
G Schultz
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Cellular Response ◽  
Cell Types ◽  
Ionophore A23187 ◽  
Mouse Muscle ◽  
Kinase C ◽  
Epidermal Growth ◽  
Different Cell Types ◽  
G Protein Coupled

The proteinase thrombin, known to act via heptahelical G-protein-coupled receptors, is a mitogenic agent for different cell types, including the mouse muscle cell line BC3H1. In this study, the effect of thrombin on tyrosine phosphorylation was examined using anti-phosphotyrosine antibodies. Thrombin was found to induce phosphorylation of 65-70 and 110-120 kDa proteins in BC3H1 cells. The effect of thrombin was concentration-dependent, being half-maximal and maximal at concentrations of 0.03 and 1 unit/ml respectively. The thrombin-induced increase in phosphorylation was rapid (< or = 10 s) and transient, with a peak response after about 1-2 min. The effect of thrombin could be mimicked by the thrombin receptor agonist peptide SFLLRN-NH2. Preincubation of cells with pertussis toxin (PT) had no effect on thrombin-induced tyrosine phosphorylation. Epidermal growth factor, platelet-derived growth factor and insulin stimulated tyrosine phosphorylation of different proteins, among which were 65-70 and 110-120 kDa proteins. The phorbol ester 12-myristate 13-acetate (PMA) as well as the Ca2+ ionophore A23187 both stimulated tyrosine phosphorylation of proteins identical to those phosphorylated by thrombin, suggesting that activation of protein kinase C (PKC) and elevation of the cytosolic Ca2+ concentration alone are sufficient to induce tyrosine phosphorylation. However, calphostin C and other PKC inhibitors, which completely inhibited tyrosine phosphorylation induced by PMA, had no influence on the effect of thrombin, whereas loading of cells with the intracellular Ca2+ chelator bis-(O-aminophenoxy)ethane-NNN'N'-tetra-acetic acid totally blocked thrombin-stimulated tyrosine phosphorylation. Thus tyrosine phosphorylation stimulated by thrombin is an early PT-insensitive cellular response which is either directly mediated by elevation of cytosolic Ca2+ concentration or by a presently unknown mechanism that requires an elevated cytosolic Ca2+ concentration.

scholarly journals Gαs protein binds ubiquitin to regulate epidermal growth factor receptor endosomal sorting

2017 ◽  
Vol 114 (51) ◽  
pp. 13477-13482 ◽  
Author(s):  
Xuezhi Li ◽  
Danny Létourneau ◽  
Brian Holleran ◽  
Richard Leduc ◽  
Pierre Lavigne ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cellular Response ◽  
Growth Factor Receptor ◽  
Fine Tuning ◽  
Lysosomal Degradation ◽  
Endosomal Sorting ◽  
Epidermal Growth ◽  
G Protein Coupled

The Gαs subunit is classically involved in the signal transduction of G protein-coupled receptors (GPCRs) at the plasma membrane. Recent evidence has revealed noncanonical roles for Gαs in endosomal sorting of receptors to lysosomes. However, the mechanism of action of Gαs in this sorting step is still poorly characterized. Here, we report that Gαs interacts with ubiquitin to regulate the endosomal sorting of receptors for lysosomal degradation. We reveal that the N-terminal extremity of Gαs contains a ubiquitin-interacting motif (UIM), a sorting element usually found in the endosomal sorting complex required for transport (ESCRT) machinery responsible for sorting ubiquitinated receptors into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). Mutation of the UIM in Gαs confirmed the importance of ubiquitin interaction for the sorting of epidermal growth factor receptor (EGFR) into ILVs for lysosomal degradation. These findings demonstrate a role for Gαs as an integral component of the ubiquitin-dependent endosomal sorting machinery and highlight the dual role of Gαs in receptor trafficking and signaling for the fine-tuning of the cellular response.

scholarly journals Differential induction of phosphatidylcholine hydrolysis, diacylglycerol formation and protein kinase C activation by epidermal growth factor and transforming growth factor-α in normal human skin fibroblasts and keratinocytes

1993 ◽  
Vol 294 (2) ◽  
pp. 535-544 ◽  
Author(s):  
N J Reynolds ◽  
H S Talwar ◽  
J J Baldassare ◽  
P A Henderson ◽  
J T Elder ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Skin Fibroblasts ◽  
Kinase C ◽  
Epidermal Growth

We have investigated coupling between the epidermal growth factor (EGF) receptor and the phospholipase C (PLC)/protein kinase C (PKC) signal-transduction system in normal skin fibroblasts and keratinocytes, for which EGF and transforming growth factor alpha (TGF-alpha) are mitogenic. EGF and TGF-alpha induced a rapid increase in tyrosine phosphorylation of the EGF receptor, in both fibroblasts and keratinocytes, but failed to induce tyrosine phosphorylation of PLC-gamma 1 or detectable phosphoinositide hydrolysis, as measured by two sensitive assays. In fibroblasts, EGF induced phosphatidylcholine (PC) hydrolysis, resulting in increased diacylglycerol (DAG). In contrast, in keratinocytes, there was no detectable PC hydrolysis or elevation of DAG in response to EGF or TGF-alpha. EGF and TGF-alpha activated PKC in fibroblasts, as evidenced by increased phosphorylation of a specific cellular PKC substrate (myristoylated alanine-rich C-kinase substrate, ‘MARCKS’). In keratinocytes, TGF-alpha and EGF induced only a modest increase in MARCKS protein phosphorylation. This apparent modest activation of PKC, in the absence of detectable DAG formation, may have been mediated by arachidonic acid, which was released from keratinocytes in response to TGF-alpha, and has been shown to stimulate PKC activity in vitro. These data demonstrate that (1) in dermal fibroblasts and keratinocytes, which express normal levels of EGF receptors, EGF receptor activation is not coupled to tyrosine phosphorylation of PLC-gamma 1 or PtdIns hydrolysis, suggesting that these events are not required for the mitogenic activity of EGF or TGF-alpha in these cells, (2) coupling of EGF receptor to PC hydrolysis is cell-type specific, and (3) in skin fibroblasts, DAG, formed through EGF-induced PC hydrolysis, is capable of activating PKC.

scholarly journals Intracellular Ca2+ and phorbol esters synergistically inhibit internalization of epidermal growth factor in pancreatic acini

1984 ◽  
Vol 223 (3) ◽  
pp. 893-900 ◽  
Author(s):  
C D Logsdon ◽  
J A Williams
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Phorbol Esters ◽  
Acinar Cells ◽  
Ionophore A23187 ◽  
Intact Cells ◽  
Kinase C ◽  
Epidermal Growth

The association of 125I-labelled epidermal growth factor (125I-EGF) with mouse pancreatic acinar cells was inhibited by secretagogues which increase intracellular free Ca2+ concentrations. These agents included cholecystokinin-octapeptide (CCK8) and the Ca2+ ionophore A23187. Inhibition by CCK8 was blocked by lowering the incubation temperature from 37 degrees C to 15 degrees C. Moreover, in contrast with studies of intact acini, the binding of 125I-EGF to isolated acinar membrane particles was not affected either by CCK8, or by varying the level of Ca2+ in the incubation medium. These results indicated, therefore, that the inhibition of 125I-EGF association with acinar cells required intact cells that are metabolically active. Since intact cells at 37 degrees C are known to internalize bound EGF rapidly, acid washing was used to distinguish membrane-associated hormone from internalized hormone. Under steady-state conditions 86% of the 125I-EGF associated with the acini was found to be internalized by this technique. When agents that increased intracellular Ca2+ were tested they all markedly reduced the amount of internalized hormone, whereas surface binding was only minimally affected. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), which is known to activate protein kinase C, a Ca2+-regulated enzyme, also inhibited the association of EGF with acini. This inhibition was similar to that induced by elevated intracellular Ca2+. To test whether these two inhibitory phenomena were related, the effects of TPA in combination with the Ca2+ ionophore A23187 were examined. At low concentrations the effects were synergistic, whereas at high concentrations the maximal level of inhibition was not changed. We suggest therefore that elevated intracellular Ca2+ and phorbol esters may inhibit EGF internalization by a mechanism involving activation of protein kinase C.

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