Transformation by pp60src or stimulation of cells with epidermal growth factor induces the stable association of tyrosine-phosphorylated cellular proteins with GTPase-activating protein

GTPase-activating protein (GAP) is a cytosolic protein that stimulates the rate of hydrolysis of GTP (GTP to GDP) bound to normal p21ras, but does not catalyze the hydrolysis of GTP bound to oncogenic, activated forms of the ras protein. Transformation of cells with v-src or activated transforming variants of c-src or stimulation of cells with epidermal growth factor resulted in the stable association of GAP with two tyrosine-phosphorylated cellular proteins of 64 kDa (p64) and 190 kDa (p190). Analysis of GAP immune complexes isolated from extracts of metabolically labeled src-transformed cells and epidermal growth factor-stimulated cells indicated that tyrosine phosphorylation of p64 and p190 appeared to be coincident with the stable association of these proteins with GAP. Quantitation of the amount of p64 associated with GAP in v-src-transformed cells, however, indicated that only 15 to 25% of tyrosine-phosphorylated p64 was found in complex with GAP. Mutations within the SH2 region of pp60src that render activated pp60src defective for transformation inhibited the efficient formation of complexes between GAP and the tyrosine-phosphorylated forms of p64 and p190. From these data, we suggest that tyrosine phosphorylation and stable association of p64 with GAP is an important step in mediating cellular signaling through the p21ras-GAP pathway.

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Tyrosine phosphorylation of ras GTPase-activating protein does not require association with the epidermal growth factor receptor.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)80641-3 ◽

1993 ◽

Vol 268 (29) ◽

pp. 22010-22019

Author(s):

C Soler ◽

L Beguinot ◽

A Sorkin ◽

G Carpenter

Keyword(s):

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Phosphorylation ◽

Growth Factor Receptor ◽

Gtpase Activating Protein ◽

Ras Gtpase ◽

Epidermal Growth

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Tyrosine phosphorylation of common and specific sets of cellular proteins rapidly induced by insulin, insulin-like growth factor I, and epidermal growth factor in an intact cell.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)48242-7 ◽

1987 ◽

Vol 262 (15) ◽

pp. 7342-7350 ◽

Cited By ~ 25

Author(s):

T Kadowaki ◽

S Koyasu ◽

E Nishida ◽

K Tobe ◽

T Izumi ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Phosphorylation ◽

Intact Cell ◽

Cellular Proteins ◽

Insulin Like Growth Factor ◽

Factor I ◽

Epidermal Growth ◽

Growth Factor I

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Signal transduction through epidermal growth factor receptor is altered in HeLa monolayer cells during mitosis

Biochemical Journal ◽

10.1042/bj3220937 ◽

1997 ◽

Vol 322 (3) ◽

pp. 937-946 ◽

Cited By ~ 10

Author(s):

Susanne KLEIN ◽

Marietta KASZKIN ◽

Holger BARTH ◽

Volker KINZEL

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Phosphorylation ◽

Egf Receptor ◽

Phosphoinositide Hydrolysis ◽

Phospholipase Cγ1 ◽

Epidermal Growth ◽

G2 Cells ◽

Stimulation Of ◽

Mitotic Cells

Epidermal growth factor (EGF)-induced signalling was studied separately in the mitosis and G2-phases of HeLa monolayer cells presynchronized (1) by amethopterin inhibition and thymidine release or (2) by nocodazole. For comparison, cells were treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA). In contrast with the observed responses effected by PMA, which seem to be independent of cell cycle and synchronization conditions, those induced by EGF are greatly influenced by both criteria. Synchronization with nocodazole abolished the EGF-induced stimulation of phosphoinositide hydrolysis in G2 as well as in mitotic cells although tyrosine phosphorylation of the EGF receptor and phospholipase Cγ1 could be shown to occur, especially in G2 cells. Synchronization with amethopterin/thymidine showed that, in contrast with G2 cells, mitotic cells were not able to react to EGF with an increase in phosphoinositide hydrolysis although a certain degree of EGF receptor dimerization and autophosphorylation as well as tyrosine phosphorylation of phospholipase Cγ1 could still be shown to occur in mitosis. The results seem to indicate that the EGF pathway leading to a stimulation of phosphoinositide hydrolysis is attenuated at different levels and requires a cytoskeletal condition that is not present either after treatment (24 h) with nocodazole or during normal mitosis of a monolayer cell.

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Rap2B-Dependent Stimulation of Phospholipase C-ε by Epidermal Growth Factor Receptor Mediated by c-Src Phosphorylation of RasGRP3

Molecular and Cellular Biology ◽

10.1128/mcb.24.11.4664-4676.2004 ◽

2004 ◽

Vol 24 (11) ◽

pp. 4664-4676 ◽

Cited By ~ 31

Author(s):

Matthias B. Stope ◽

Frank vom Dorp ◽

Daniel Szatkowski ◽

Anja Böhm ◽

Melanie Keiper ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Egf Receptor ◽

Dominant Negative ◽

Nucleotide Exchange ◽

Egf Receptors ◽

Epidermal Growth ◽

Stimulation Of

ABSTRACT Receptor tyrosine kinase regulation of phospholipase C-ε (PLC-ε), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca2+ signaling by the EGF receptor, which activated both PLC-γ1 and PLC-ε, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-ε, and Rap2B-dependent translocation of PLC-ε to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca2+ and expression of lipase-inactive PLC-γ1 but not of PLC-ε. Expression of RasGRP3, a Ca2+/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca2+ signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-γ1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-ε.

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The epidermal growth factor receptor phosphorylates GTPase-activating protein (GAP) at Tyr-460, adjacent to the GAP SH2 domains.

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2511 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2511-2516 ◽

Cited By ~ 41

Author(s):

X Q Liu ◽

T Pawson

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Egf Receptor ◽

Sh2 Domain ◽

Ras Signaling ◽

Gtpase Activating Protein ◽

Epidermal Growth

GTPase-activating protein (GAP) stimulates the ability of p21ras to hydrolyze GTP to GDP. Since GAP is phosphorylated by a variety of activated or oncogenic protein-tyrosine kinases, it may couple tyrosine kinases to the Ras signaling pathway. The epidermal growth factor (EGF) receptor cytoplasmic domain phosphorylated human GAP in vitro within a single tryptic phosphopeptide. The same GAP peptide was also apparently phosphorylated on tyrosine in EGF-stimulated rat fibroblasts. Circumstantial evidence suggested that residue 460 might be the site of GAP tyrosine phosphorylation. This possibility was confirmed by phosphorylation of a synthetic peptide corresponding to the predicted tryptic peptide containing Tyr-460. Alteration of Tyr-460 to phenylalanine by site-directed mutagenesis diminished the in vitro phosphorylation of a bacterial GAP polypeptide by the EGF receptor. We conclude that Tyr-460 is a site of GAP tyrosine phosphorylation by the EGF receptor in vitro and likely in vivo. GAP Tyr-460 is located immediately C terminal to the second GAP SH2 domain, suggesting that its phosphorylation might have a role in regulating protein-protein interactions.

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Tyrosine phosphorylation of a 120-kilodalton pp60src substrate upon epidermal growth factor and platelet-derived growth factor receptor stimulation and in polyomavirus middle-T-antigen-transformed cells.

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.713 ◽

1991 ◽

Vol 11 (2) ◽

pp. 713-720 ◽

Cited By ~ 83

Author(s):

S B Kanner ◽

A B Reynolds ◽

J T Parsons

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

T Antigen ◽

Platelet Derived Growth Factor ◽

Transformed Cells ◽

Epidermal Growth ◽

Middle T Antigen ◽

In Cells

The monoclonal antibody 2B12 is directed toward p120, a 120-kDa cellular protein originally identified as a protein tyrosine kinase substrate in cells expressing membrane-associated oncogenic variants of pp60src. In this report, we show that p120 was tyrosine phosphorylated in avian cells expressing membrane-associated, enzymatically activated variants of c-src, including variants having structural alterations in the src homology regions 2 and 3. In contrast, p120 was not tyrosine phosphorylated in cells expressing enzymatically activated, nonmyristylated pp60src. Furthermore, p120 was tyrosine phosphorylated in avian cells expressing middle T antigen, the transforming protein of polyomavirus, as well as in rodent cells stimulated with either epidermal growth factor (EGF) or platelet-derived growth factor. Analysis of the time course of p120 tyrosine phosphorylation in EGF-stimulated cells revealed a rapid onset of tyrosine phosphorylation. In addition, both the extent and duration of p120 phosphorylation increased when cells overexpressing the EGF receptor were stimulated with EGF. Biochemical analysis showed that p120 (in both normal and src-transformed cells) was membrane associated, was myristylated, and was phosphorylated on serine and threonine residues. Hence, p120 appears to be a substrate of both nonreceptor- and ligand-activated transmembrane receptor tyrosine kinases and of serine/threonine kinases and is perhaps a component of both mitogen-stimulated and tyrosine kinase oncogene-induced signaling pathways.

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The epidermal growth factor receptor phosphorylates GTPase-activating protein (GAP) at Tyr-460, adjacent to the GAP SH2 domains

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2511-2516.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2511-2516 ◽

Cited By ~ 1

Author(s):

X Q Liu ◽

T Pawson

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Egf Receptor ◽

Sh2 Domain ◽

Ras Signaling ◽

Gtpase Activating Protein ◽

Epidermal Growth

GTPase-activating protein (GAP) stimulates the ability of p21ras to hydrolyze GTP to GDP. Since GAP is phosphorylated by a variety of activated or oncogenic protein-tyrosine kinases, it may couple tyrosine kinases to the Ras signaling pathway. The epidermal growth factor (EGF) receptor cytoplasmic domain phosphorylated human GAP in vitro within a single tryptic phosphopeptide. The same GAP peptide was also apparently phosphorylated on tyrosine in EGF-stimulated rat fibroblasts. Circumstantial evidence suggested that residue 460 might be the site of GAP tyrosine phosphorylation. This possibility was confirmed by phosphorylation of a synthetic peptide corresponding to the predicted tryptic peptide containing Tyr-460. Alteration of Tyr-460 to phenylalanine by site-directed mutagenesis diminished the in vitro phosphorylation of a bacterial GAP polypeptide by the EGF receptor. We conclude that Tyr-460 is a site of GAP tyrosine phosphorylation by the EGF receptor in vitro and likely in vivo. GAP Tyr-460 is located immediately C terminal to the second GAP SH2 domain, suggesting that its phosphorylation might have a role in regulating protein-protein interactions.

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Tyrosine phosphorylation of a 120-kilodalton pp60src substrate upon epidermal growth factor and platelet-derived growth factor receptor stimulation and in polyomavirus middle-T-antigen-transformed cells

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.713-720.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 713-720

Author(s):

S B Kanner ◽

A B Reynolds ◽

J T Parsons

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

T Antigen ◽

Platelet Derived Growth Factor ◽

Transformed Cells ◽

Epidermal Growth ◽

Middle T Antigen ◽

In Cells

The monoclonal antibody 2B12 is directed toward p120, a 120-kDa cellular protein originally identified as a protein tyrosine kinase substrate in cells expressing membrane-associated oncogenic variants of pp60src. In this report, we show that p120 was tyrosine phosphorylated in avian cells expressing membrane-associated, enzymatically activated variants of c-src, including variants having structural alterations in the src homology regions 2 and 3. In contrast, p120 was not tyrosine phosphorylated in cells expressing enzymatically activated, nonmyristylated pp60src. Furthermore, p120 was tyrosine phosphorylated in avian cells expressing middle T antigen, the transforming protein of polyomavirus, as well as in rodent cells stimulated with either epidermal growth factor (EGF) or platelet-derived growth factor. Analysis of the time course of p120 tyrosine phosphorylation in EGF-stimulated cells revealed a rapid onset of tyrosine phosphorylation. In addition, both the extent and duration of p120 phosphorylation increased when cells overexpressing the EGF receptor were stimulated with EGF. Biochemical analysis showed that p120 (in both normal and src-transformed cells) was membrane associated, was myristylated, and was phosphorylated on serine and threonine residues. Hence, p120 appears to be a substrate of both nonreceptor- and ligand-activated transmembrane receptor tyrosine kinases and of serine/threonine kinases and is perhaps a component of both mitogen-stimulated and tyrosine kinase oncogene-induced signaling pathways.

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