Tyrosine phosphorylation of Ras GTPase-activating protein stabilizes its association with p62 at membranes of v-Src transformed cells.

Flk2/Flt3 is a recently identified receptor tyrosine kinase expressed in brain, placenta, testis, and primitive hematopoietic cells. The mitogenic signalling potential and biochemical properties of Flk2/Flt3 have been analyzed by using a chimeric receptor composed of the extracellular domain of the human colony-stimulating factor 1 receptor and the transmembrane and cytoplasmic domains of murine Flk2/Flt3. We demonstrate that colony-stimulating factor 1 stimulation of the Flk2/Flt3 kinase in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a full proliferative response in the absence of other growth factors. In transfected interleukin 3 (IL-3)-dependent Ba/F3 lymphoid cells, activation of the chimeric receptor can abrogate IL-3 requirement and sustain long-term proliferation. We show that phospholipase C-gamma 1, Ras GTPase-activating protein, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, Vav, Fyn, and Src are components of the Flk2/Flt3 signal transduction pathway. In addition, we demonstrate that phospholipase C-gamma 1, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, and Src family tyrosine kinases, but not Ras GTPase-activating protein, Vav, or Nck, physically associate with the Flk2/Flt3 cytoplasmic domain. Cell-type-specific differences in tyrosine phosphorylation of p85 and Shc are observed. A comparative analysis of the Flk2/Flt3 signal cascade with those of the endogenous platelet-derived growth factor and IL-3 receptors indicates that Flk2/Flt3 displays specific substrate preferences. Furthermore, tyrosine phosphorylation of p85 and Shc is similarly affected by totally different growth factors in the same cellular background.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.945 ◽

1991 ◽

Vol 11 (2) ◽

pp. 945-953 ◽

Cited By ~ 48

Author(s):

A H Bouton ◽

S B Kanner ◽

R R Vines ◽

H C Wang ◽

J B Gibbs ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Phosphorylation ◽

Cellular Proteins ◽

Transformed Cells ◽

Gtpase Activating Protein ◽

Epidermal Growth ◽

Stable Association ◽

Hydrolysis Of ◽

Stimulation Of

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.945-953.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 945-953

Author(s):

A H Bouton ◽

S B Kanner ◽

R R Vines ◽

H C Wang ◽

J B Gibbs ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Phosphorylation ◽

Cellular Proteins ◽

Transformed Cells ◽

Gtpase Activating Protein ◽

Epidermal Growth ◽

Stable Association ◽

Hydrolysis Of ◽

Stimulation Of

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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Transient Cerebral Ischemia Increases Tyrosine Phosphorylation of the Synaptic RAS-GTPase Activating Protein, SynGAP

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-200108000-00008 ◽

2001 ◽

Vol 21 (8) ◽

pp. 955-963 ◽

Cited By ~ 20

Author(s):

Lin Pei ◽

R. Lucy Teves ◽

M. Christopher Wallace ◽

James W. Gurd

Keyword(s):

Cerebral Ischemia ◽

Tyrosine Phosphorylation ◽

Mitogen Activated Protein Kinase ◽

Pdz Domains ◽

Gtpase Activating Protein ◽

Vessel Occlusion ◽

Sh2 Domains ◽

Ras Gtpase ◽

Mitogen Activated Protein ◽

The Brain

Cerebral ischemia results in activation of the mitogen-activated protein kinase pathway and increased tyrosine phosphorylation of proteins associated with postsynaptic densities (PSDs). The authors investigated the possible relation between these events by determining the effect of ischemia on tyrosine phosphorylation of the brain-specific, PSD-enriched, Ras-GTPase activating protein, SynGAP. Transient (15 minutes) global ischemia was produced in rats by 4-vessel occlusion and PSDs prepared from forebrains immediately after ischemia or at 20 minutes, 1 hour, or 24 hours of reperfusion. Tyrosine phosphorylation of SynGAP was elevated relative to sham-operated controls by 20 minutes of reperfusion and remained elevated for at least 24 hours. Tyrosine phosphorylation of SynGAP also increased in CA1 and CA3/DG subfields of the hippocampus. Enhanced tyrosine phosphorylation of SynGAP was not accompanied by a change in PSD RasGAP activity. SynGAP bound to the SH2 domains of Src and Fyn in a tyrosine phosphorylation-dependent fashion, and this interaction increased after ischemia. SynGAP binds to the PDZ domains of PSD-95/SAP90 and coimmunoprecipitated with PSD-95. The coimmunoprecipitation of SynGAP with PSD-95 decreased after ischemia. The results indicate that changes in the properties and interactions of SynGAP may be involved in the neuropathology of ischemia.

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Mitogenic signalling and substrate specificity of the Flk2/Flt3 receptor tyrosine kinase in fibroblasts and interleukin 3-dependent hematopoietic cells.

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6572 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6572-6585 ◽

Cited By ~ 113

Author(s):

M Dosil ◽

S Wang ◽

I R Lemischka

Keyword(s):

Growth Factors ◽

Tyrosine Phosphorylation ◽

Receptor Tyrosine Kinase ◽

Chimeric Receptor ◽

Colony Stimulating Factor ◽

Phosphatidylinositol 3 Kinase ◽

Gtpase Activating Protein ◽

Ras Gtpase ◽

Mitogenic Signalling ◽

Stimulating Factor

Flk2/Flt3 is a recently identified receptor tyrosine kinase expressed in brain, placenta, testis, and primitive hematopoietic cells. The mitogenic signalling potential and biochemical properties of Flk2/Flt3 have been analyzed by using a chimeric receptor composed of the extracellular domain of the human colony-stimulating factor 1 receptor and the transmembrane and cytoplasmic domains of murine Flk2/Flt3. We demonstrate that colony-stimulating factor 1 stimulation of the Flk2/Flt3 kinase in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a full proliferative response in the absence of other growth factors. In transfected interleukin 3 (IL-3)-dependent Ba/F3 lymphoid cells, activation of the chimeric receptor can abrogate IL-3 requirement and sustain long-term proliferation. We show that phospholipase C-gamma 1, Ras GTPase-activating protein, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, Vav, Fyn, and Src are components of the Flk2/Flt3 signal transduction pathway. In addition, we demonstrate that phospholipase C-gamma 1, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, and Src family tyrosine kinases, but not Ras GTPase-activating protein, Vav, or Nck, physically associate with the Flk2/Flt3 cytoplasmic domain. Cell-type-specific differences in tyrosine phosphorylation of p85 and Shc are observed. A comparative analysis of the Flk2/Flt3 signal cascade with those of the endogenous platelet-derived growth factor and IL-3 receptors indicates that Flk2/Flt3 displays specific substrate preferences. Furthermore, tyrosine phosphorylation of p85 and Shc is similarly affected by totally different growth factors in the same cellular background.

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