scholarly journals A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine.

1991 ◽  
Vol 11 (2) ◽  
pp. 1125-1132 ◽  
Author(s):  
J A Escobedo ◽  
D R Kaplan ◽  
W M Kavanaugh ◽  
C W Turck ◽  
L T Williams
Keyword(s):  
Growth Factor ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Platelet Derived Growth Factor ◽  
Pdgf Receptor ◽  
Phosphatidylinositol 3 Kinase ◽  
Cell Lysates ◽  
Phosphorylated Peptides ◽  
Insert Region ◽  
Phosphatidylinositol 3

Platelet-derived growth factor (PDGF) stimulates autophosphorylation of the PDGF receptor and association of the receptor with several cytoplasmic molecules, including phosphatidylinositol-3 kinase (PI3 kinase). In this study we examined the association of PI3 kinase with immunoprecipitated autophosphorylated PDGF receptor in vitro. The PI3 kinase from cell lysates bound to the wild-type receptor but not to a mutant receptor that had a deletion of the kinase insert region. A protein of an apparent size of 85 kDa bound to the receptor, consistent with previous observations that a protein of this size is associated with PI3 kinase activity. In addition, 110- and 74-kDa proteins bound to the phosphorylated receptor. Dephosphorylated receptors lost the ability to bind PI3 kinase activity as well as the 85-kDa protein. A 20-amino-acid peptide composed of a sequence in the kinase insert region that included one of the autophosphorylation sites of the receptor (tyrosine 719) as well as a nearby tyrosine (Y708) blocked the binding of PI3 kinase to the receptor, but only when the peptide was phosphorylated on tyrosine residues. A scrambled version of the peptide did not block PI3 kinase binding to the receptor even when it was phosphorylated on tyrosine. These tyrosine-phosphorylated peptides did not block binding of phospholipase C-gamma or GTPase-activating protein to the receptor. In separate experiments (receptor blots), soluble radiolabeled receptor bound specifically to an 85-kDa protein present in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated 3T3 cell lysates that were transferred to nitrocellulose paper. The binding was blocked by the same tyrosine-phosphorylated peptides that prevented binding of PI3 kinase activity to immobilized receptors. These findings show that the PDGF receptor binds directly to an 85-kDa protein and to a PI3 kinase activity through specific sequences in the kinase insert region. The association of a 110-kDa protein with the receptor also involve these sequences, suggesting that this protein may be a subunit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor.

scholarly journals A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine

1991 ◽  
Vol 11 (2) ◽  
pp. 1125-1132
Author(s):  
J A Escobedo ◽  
D R Kaplan ◽  
W M Kavanaugh ◽  
C W Turck ◽  
L T Williams
Keyword(s):  
Growth Factor ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Platelet Derived Growth Factor ◽  
Pdgf Receptor ◽  
Phosphatidylinositol 3 Kinase ◽  
Cell Lysates ◽  
Phosphorylated Peptides ◽  
Insert Region ◽  
Phosphatidylinositol 3

Platelet-derived growth factor (PDGF) stimulates autophosphorylation of the PDGF receptor and association of the receptor with several cytoplasmic molecules, including phosphatidylinositol-3 kinase (PI3 kinase). In this study we examined the association of PI3 kinase with immunoprecipitated autophosphorylated PDGF receptor in vitro. The PI3 kinase from cell lysates bound to the wild-type receptor but not to a mutant receptor that had a deletion of the kinase insert region. A protein of an apparent size of 85 kDa bound to the receptor, consistent with previous observations that a protein of this size is associated with PI3 kinase activity. In addition, 110- and 74-kDa proteins bound to the phosphorylated receptor. Dephosphorylated receptors lost the ability to bind PI3 kinase activity as well as the 85-kDa protein. A 20-amino-acid peptide composed of a sequence in the kinase insert region that included one of the autophosphorylation sites of the receptor (tyrosine 719) as well as a nearby tyrosine (Y708) blocked the binding of PI3 kinase to the receptor, but only when the peptide was phosphorylated on tyrosine residues. A scrambled version of the peptide did not block PI3 kinase binding to the receptor even when it was phosphorylated on tyrosine. These tyrosine-phosphorylated peptides did not block binding of phospholipase C-gamma or GTPase-activating protein to the receptor. In separate experiments (receptor blots), soluble radiolabeled receptor bound specifically to an 85-kDa protein present in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated 3T3 cell lysates that were transferred to nitrocellulose paper. The binding was blocked by the same tyrosine-phosphorylated peptides that prevented binding of PI3 kinase activity to immobilized receptors. These findings show that the PDGF receptor binds directly to an 85-kDa protein and to a PI3 kinase activity through specific sequences in the kinase insert region. The association of a 110-kDa protein with the receptor also involve these sequences, suggesting that this protein may be a subunit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor.

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.

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.

TEL/platelet-derived growth factor receptor β activates phosphatidylinositol 3 (PI3) kinase and requires PI3 kinase to regulate the cell cycle

Blood ◽  
2002 ◽  
Vol 99 (5) ◽  
pp. 1758-1765 ◽  
Author(s):  
Jamil Dierov ◽  
Qing Xu ◽  
Raia Dierova ◽  
Martin Carroll
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Kinase Activity ◽  
Growth Factor Receptor ◽  
Protein Product ◽  
Pi3 Kinase ◽  
Platelet Derived Growth Factor ◽  
Myeloproliferative Disease ◽  
Kinase Activation ◽  
Phosphatidylinositol 3

TEL/platelet-derived growth factor receptor β (PDGFβR) is the protein product of the t(5;12) translocation in chronic myelomonocytic leukemia. TEL/PDGFβR transforms interleukin-3 (IL-3)–dependent Ba/F3 and 32D cells to IL-3 independence and induces a murine myeloproliferative disease in a bone marrow transplantation model of leukemogenesis. The fusion protein encodes a constitutively activated, cytoplasmic tyrosine kinase that activates multiple signal transduction pathways. To identify the signaling pathways that are necessary for transformation by TEL/PDGFβR, transformed Ba/F3 and 32D cells were studied. TEL/PDGFβR activates the kinase activity of phosphatidylinositol-3 (PI3) kinase and stimulates phosphorylation of its downstream substrates, including Akt and p70S6 kinase. Activation of this pathway requires the kinase activity of TEL/PDGFβR and is inhibited by the PDGFβR inhibitor, STI571. Furthermore, inhibition of PI3 kinase with the pharmacologic inhibitor, LY294002, inhibits growth of the transformed cells. Treated cells arrest in the G1 phase of the cell cycle within 16 hours but do not undergo apoptosis. To study the mechanism of cell cycle arrest by LY294002, the activity of the cdk4 complex, which regulates the transit of cells from the G1 to S phase in hematopoietic cells, was examined. Both STI571 and LY294002 lead to a decrease in the activity of cdk4 kinase activity and a decrease in expression of both Cyclin D2 and Cyclin E within several hours. These studies demonstrate the presence of a signaling pathway from TEL/PDGFβR to PI3 kinase and subsequently to regulation of the cdk4 kinase complex. Activation of this pathway is necessary for transformation by TEL/PDGFβR.

scholarly journals Involvement of a phosphotyrosine protein phosphatase in the suppression of platelet-derived growth factor receptor autophosphorylation in ras-transformed cells

1993 ◽  
Vol 293 (1) ◽  
pp. 215-221 ◽  
Author(s):  
L Tomáska ◽  
R J Resnick
Keyword(s):  
Growth Factor ◽  
Phosphatase Activity ◽  
Receptor Tyrosine Kinase ◽  
Protein Phosphatase ◽  
Kinase Activity ◽  
Platelet Derived Growth Factor ◽  
Transformed Cells ◽  
Pdgf Receptor ◽  
Tyrosine Kinase Activity ◽  
Phosphotyrosine Protein Phosphatase

The nature of the suppression of platelet-derived growth factor (PDGF) receptor autophosphorylation in ras-transformed NIH 3T3 fibroblasts was investigated. The PDGF receptor from ras-transformed cells that had been purified by wheatgerm-lectin affinity chromatography displayed normal PDGF-induced autophosphorylation, indicating that the receptor is not irreversibly modified. Various phosphotyrosine-protein-phosphatase inhibitors did not reverse the inhibition of PDGF-receptor kinase in crude membrane preparations from ras-transformed cells. However, treatment of intact ras-transformed cells both with 2 mM sodium orthovanadate and with 20 microM phenylarsine oxide restored PDGF-receptor tyrosine-kinase activity to a level similar to that observed in normal cells. Direct measurement of the phosphatase activities in crude cellular fractions revealed a 2.5-fold higher membrane-associated phosphotyrosine-protein-phosphatase activity in ras-transformed cells, whereas phosphoserine-protein-phosphatase activity remained unchanged between the cell lines. These data suggest that the suppression of the PDGF-receptor tyrosine-kinase activity in ras-transformed cells is mediated via an inhibitory component, distinct from the receptor, that may be positively regulated by the dephosphorylation of tyrosine residue(s).

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