scholarly journals Characterization of the tyrosine phosphorylation of calpactin I (annexin II) induced by platelet-derived growth factor

1991 ◽  
Vol 278 (2) ◽  
pp. 447-452 ◽  
Author(s):  
R Brambilla ◽  
R Zippel ◽  
E Sturani ◽  
L Morello ◽  
A Peres ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Platelet Derived Growth Factor ◽  
Annexin Ii ◽  
Necessary Condition ◽  
Pdgf Receptor ◽  
Permeabilized Cells ◽  
Phosphorylated Proteins ◽  
3T3 Fibroblasts

Stimulation in vivo of Swiss 3T3 fibroblasts with platelet-derived growth factor (PDGF) in the presence of orthovanadate induces the tyrosine phosphorylation of a 39 kDa protein, identified as the phosphorylated slow-migrating form of calpactin I (annexin II) heavy chain, p36. In fact, in PDGF-stimulated cells, anti-(calpactin I) antibodies recognize a doublet of bands, p36 and p39, and the latter disappears upon treatment with phosphatase. In many regards phosphorylation of p39 differs from the rapid and transient phosphorylation of the PDGF receptor and of other substrates: (a) it has slower kinetics but is then stable for longer periods of time; (b) it occurs at 37 degrees C but not at 4 degrees C; and (c) whereas most of the tyrosine-phosphorylated proteins are associated with membrane-enriched preparations, membrane association of p39 only occurs in the presence of Ca2+. Moreover, calpactin I leaks out of permeabilized cells at 0.1 microM free Ca2+, whereas it remains associated with the cells at concentrations of Ca2+ greater than or equal to 1 microM. PDGF does not stimulate phosphoinositide turnover (and thus Ca2+ mobilization) at 4 degrees C; thus it can be suggested that the Ca(2+)-dependent translocation of the protein to membrane/cytoskeletal structures is a necessary condition for its phosphorylation. In addition, calpactin I may not be a direct substrate for the PDGF receptor kinase, but rather the substrate of another tyrosine kinase activated by the receptor.

Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex

1990 ◽  
Vol 10 (5) ◽  
pp. 2359-2366
Author(s):  
D K Morrison ◽  
D R Kaplan ◽  
S G Rhee ◽  
L T Williams
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Serine Phosphorylation ◽  
Platelet Derived Growth Factor ◽  
Pdgf Receptor ◽  
Wild Type ◽  
Receptor Proteins ◽  
Signaling Complex

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.

scholarly journals Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex.

1990 ◽  
Vol 10 (5) ◽  
pp. 2359-2366 ◽  
Author(s):  
D K Morrison ◽  
D R Kaplan ◽  
S G Rhee ◽  
L T Williams
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Serine Phosphorylation ◽  
Platelet Derived Growth Factor ◽  
Pdgf Receptor ◽  
Wild Type ◽  
Receptor Proteins ◽  
Signaling Complex

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.

The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency

1991 ◽  
Vol 11 (4) ◽  
pp. 2040-2048
Author(s):  
F Fazioli ◽  
U H Kim ◽  
S G Rhee ◽  
C J Molloy ◽  
O Segatto ◽  
...  
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Phospholipase C ◽  
Signaling Pathway ◽  
Tyrosine Phosphorylation ◽  
Platelet Derived Growth Factor ◽  
Mitogenic Signaling ◽  
Gtpase Activating Protein ◽  
3T3 Fibroblasts ◽  
Nih 3T3

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.

Kinetics and Regulation of Tyrosine Phosphorylation of the Platelet Derived Growth Factor Receptor

1989 ◽  
Vol 75 (4) ◽  
pp. 362-366
Author(s):  
Lilia Alberghina ◽  
Renata Zippel ◽  
Enzo Martegani ◽  
Emmapaola Sturani
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Tyrosine Phosphorylation ◽  
Growth Factor Receptor ◽  
Growth Conditions ◽  
Platelet Derived Growth Factor ◽  
Dependent Manner ◽  
Pdgf Receptor ◽  
Receptor Complexes ◽  
Phosphorylated Form

Platelet derived growth factor (PDGF) interaction with the cells induces rapid tyrosine phosphorylation of the PDGF receptor in a dose dependent manner. At 37 °C phosphorylation of the receptor is followed by its dephosphorylation and internalization. It is observed that the higher the ligand concentration, the more transient is the response, and the observed kinetics are explained by a simple kinetic model. At 4 °C the phosphorylated form of the receptor is more stable; however, if PDGF is dissociated from the cell surface-associated ligand-receptor complexes, the receptors are rapidly dephosphorylated, indicating that phosphatases specific for phosphotyrosine groups are very active within the cells. In fact, addition of orthovanadate stabilizes the phosphorylated form of the receptor and helps in recognizing possible physiological substrates of the PDGF receptor kinase. The expression of PDGF receptors on the cell surface has been investigated under different growth conditions: a positive correlation exists between the amount of PDGF receptors and the duplication times of exponentially growing cultures. Moreover, during exponential growth the PDGF receptors are scarcely expressed, and their number increases reaching a maximal value when the population enters the stationary phase.

scholarly journals The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency.

1991 ◽  
Vol 11 (4) ◽  
pp. 2040-2048 ◽  
Author(s):  
F Fazioli ◽  
U H Kim ◽  
S G Rhee ◽  
C J Molloy ◽  
O Segatto ◽  
...  
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Phospholipase C ◽  
Signaling Pathway ◽  
Tyrosine Phosphorylation ◽  
Platelet Derived Growth Factor ◽  
Mitogenic Signaling ◽  
Gtpase Activating Protein ◽  
3T3 Fibroblasts ◽  
Nih 3T3

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.

Platelet-derived growth factor increases the in vivo activity of phospholipase C-gamma 1 and phospholipase C-gamma 2

1991 ◽  
Vol 11 (4) ◽  
pp. 2018-2025
Author(s):  
L Sultzman ◽  
C Ellis ◽  
L L Lin ◽  
T Pawson ◽  
J Knopf
Keyword(s):  
Growth Factor ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Cell Surface Receptor ◽  
Platelet Derived Growth Factor ◽  
Surface Receptor ◽  
Phospholipase C Gamma

Upon binding to its cell surface receptor, platelet-derived growth factor (PDGF) causes the tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) and stimulates the production of diacylglycerol and inositol 1,4,5-triphosphate. We showed that following stimulation by PDGF, rat-2 cells overexpressing PLC-gamma 1 display an increase in the levels of both tyrosine-phosphorylated PLC-gamma 1 and inositol phosphates compared with the parental rat-2 cells. This increased responsiveness to PDGF is a direct effect of PLC-gamma 1 overexpression, as a cell line expressing similar levels of an enzymatically inactive point mutant of PLC-gamma 1, PLC-gamma 1 335Q, did not show elevated inositol phosphate production in response to PDGF. Hematopoietic cells express PLC-gamma 2, a PLC isoform that is closely related to PLC-gamma 1. When rat-2 cells overexpressing PLC-gamma 2 were treated with PDGF, an increase in both the tyrosine phosphorylation and the in vivo activity of PLC-gamma 2 was observed. Aluminum fluoride (AIF4-), a universal activator of PLC linked to G-proteins, did not produce an increase in the levels of inositol phosphates in either of the overexpressing cell lines compared with parental rat-2 cells, demonstrating that PLC-gamma isoforms respond specifically to a receptor with tyrosine kinase activity.

scholarly journals Tyrosine mutations within the alpha platelet-derived growth factor receptor kinase insert domain abrogate receptor-associated phosphatidylinositol-3 kinase activity without affecting mitogenic or chemotactic signal transduction.

1991 ◽  
Vol 11 (7) ◽  
pp. 3780-3785 ◽  
Author(s):  
J C Yu ◽  
M A Heidaran ◽  
J H Pierce ◽  
J S Gutkind ◽  
D Lombardi ◽  
...  
Keyword(s):  
Growth Factor ◽  
Kinase Activity ◽  
Growth Factor Receptor ◽  
Platelet Derived Growth Factor ◽  
Pdgf Receptor ◽  
Tyrosine Residue ◽  
Phosphatidylinositol 3 Kinase ◽  
Ligand Stimulation ◽  
Phosphatidylinositol 3

A phosphatidylinositol-3 (PI-3) kinase activity of unknown biological function associates with tyrosine kinase-containing proteins, including a number of growth factor receptors after ligand stimulation. In the beta platelet-derived growth factor (beta PDGF) receptor, phosphorylation of a specific tyrosine residue within the kinase insert domain was required for its interaction with this enzyme. We show that substitutions of phenylalanine for tyrosine residue 731 or 742 within the kinase insert domain of the alpha PDGF receptor do not impair PDGF-induced tyrosine phosphorylation of the receptor or of an in vivo substrate, phospholipase C-gamma. Moreover, phosphatidylinositol turnover in response to ligand stimulation is unaffected. However, both lesions markedly impair receptor association with PI-3 kinase. Antiphosphotyrosine antibody-recoverable PI-3 kinase was also dramatically reduced in PDGF-stimulated cells expressing either mutant receptor. Since neither mutation abolished PDGF-induced mitogenesis or chemotaxis, we conclude that alpha PDGF receptor-associated PI-3 kinase activity is not required for either of these major PDGF signalling functions.

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