scholarly journals Autoinhibition of the Platelet-derived Growth Factor β-Receptor Tyrosine Kinase by Its C-terminal Tail

2004 ◽  
Vol 279 (19) ◽  
pp. 19732-19738 ◽  
Author(s):  
Federica Chiara ◽  
Subal Bishayee ◽  
Carl-Henrik Heldin ◽  
Jean-Baptiste Demoulin
Keyword(s):  
Growth Factor ◽  
Kinase Activity ◽  
Platelet Derived Growth Factor ◽  
Receptor Kinase ◽  
Wild Type ◽  
Focus Formation ◽  
Soluble Peptide ◽  
Β Receptor

In this report, we investigated the role of the C-terminal tail of the platelet-derived growth factor (PDGF) β-receptor in the control of the receptor kinase activity. Using a panel of PDGF β-receptor mutants with progressive C-terminal truncations, we observed that deletion of the last 46 residues, which contain a proline- and glutamic acid-rich motif, increased the autoactivation velocityin vitroand theVmaxof the phosphotransfer reaction, in the absence of ligand, as compared with wild-type receptors. By contrast, the kinase activity of mutant and wild-type receptors that were pre-activated by treatment with PDGF was comparable. Using a conformation-sensitive antibody, we found that truncated receptors presented an active conformation even in the absence of PDGF. A soluble peptide containing the Pro/Glu-rich motif specifically inhibited the PDGF β-receptor kinase activity. Whereas deletion of this motif was not enough to confer ligand-independent transforming ability to the receptor, it dramatically enhanced the effect of the weakly activating D850N mutation in a focus formation assay. These findings indicate that allosteric inhibition of the PDGF β-receptor by its C-terminal tail is one of the mechanisms involved in keeping the receptor inactive in the absence of ligand.

Basis of hematopoietic defects in platelet-derived growth factor (PDGF)-B and PDGF β-receptor null mice

Blood ◽  
2001 ◽  
Vol 97 (7) ◽  
pp. 1990-1998 ◽  
Author(s):  
Wolfgang E. Kaminski ◽  
Per Lindahl ◽  
Nancy L. Lin ◽  
Virginia C. Broudy ◽  
Jeffrey R. Crosby ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fetal Liver ◽  
Liver Cells ◽  
Platelet Derived Growth Factor ◽  
Wild Type ◽  
Liver Growth ◽  
Hematologic Disorders ◽  
Fetal Liver Cells ◽  
Β Receptor

Abstract Platelet-derived growth factor (PDGF)-B and PDGF β-receptor (PDGFRβ) deficiency in mice is embryonic lethal and results in cardiovascular, renal, placental, and hematologic disorders. The hematologic disorders are described, and a correlation with hepatic hypocellularity is demonstrated. To explore possible causes, the colony-forming activity of fetal liver cells in vitro was assessed, and hematopoietic chimeras were demonstrated by the transplantation of mutant fetal liver cells into lethally irradiated recipients. It was found that mutant colony formation is equivalent to that of wild-type controls. Hematopoietic chimeras reconstituted with PDGF-B−/−, PDGFRβ−/−, or wild-type fetal liver cells show complete engraftment (greater than 98%) with donor granulocytes, monocytes, B cells, and T cells and display none of the cardiovascular or hematologic abnormalities seen in mutants. In mouse embryos, PDGF-B is expressed by vascular endothelial cells and megakaryocytes. After birth, expression is seen in macrophages and neurons. This study demonstrates that hematopoietic PDGF-B or PDGFRβ expression is not required for hematopoiesis or integrity of the cardiovascular system. It is argued that metabolic stress arising from mutant defects in the placenta, heart, or blood vessels may lead to impaired liver growth and decreased production of blood cells. The chimera models in this study will serve as valuable tools to test the role of PDGF in inflammatory and immune responses.

scholarly journals Platelet-derived growth factor (PDGF)-induced activation of signal transducer and activator of transcription (Stat) 5 is mediated by PDGF β-receptor and is not dependent on c-Src, Fyn, Jak1 or Jak2 kinases

2000 ◽  
Vol 345 (3) ◽  
pp. 759-766 ◽  
Author(s):  
Kirsi PAUKKU ◽  
Sigrídur VALGEIRSDÓTTIR ◽  
Pipsa SAHARINEN ◽  
Mathias BERGMAN ◽  
Carl-Henrik HELDIN ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinases ◽  
Mammalian Cells ◽  
Platelet Derived Growth Factor ◽  
Wild Type ◽  
Signal Transducers ◽  
Tyrosine Residues ◽  
Type C ◽  
Β Receptor

Several growth factors activate signal transducers and activators of transcription (Stats) but the mechanism of Stat activation in receptor tyrosine kinase signalling has remained elusive. In the present study we have analysed the roles of different platelet-derived growth factor (PDGF)-induced tyrosine kinases in the activation of Stat5. Co-expression experiments in insect and mammalian cells demonstrated that both PDGF β-receptor (PDGF β-R) and Jak1, but not c-Src, induced the activation of Stat5. Furthermore, immune-complex-purified PDGF β-R was able to phosphorylate Stat5 directly. The role of the cytoplasmic tyrosine kinases in the PDGF-induced activation of Stat5 was further investigated by overexpressing kinase-negative (KN) and wild-type Jak and c-Src kinases. Jak1-KN or Jak2-KN had no effect but both Src-KN and wild-type c-Src similarly decreased the PDGF-β-R-induced activation of Stat5. The activation of both Src and Stat5 is dependent on the same tyrosine residues Tyr579 and Tyr581 in PDGF β-R; thus the observed inhibition by Src might result from competition for binding of Stat5 to the receptor. Finally, fibroblasts derived from Src-/- and Fyn-/- mice showed normal pattern of PDGF-induced tyrosine phosphorylation of Stat5. Taken together, these results indicate that Stat5 is a direct substrate for PDGF β-R and that the activation does not require Jak1, Jak2, c-Src or Fyn tyrosine kinases.

Deletion or substitution within the alpha platelet-derived growth factor receptor kinase insert domain: effects on functional coupling with intracellular signaling pathways

1991 ◽  
Vol 11 (1) ◽  
pp. 134-142
Author(s):  
M A Heidaran ◽  
J H Pierce ◽  
D Lombardi ◽  
M Ruggiero ◽  
J S Gutkind ◽  
...  
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Intracellular Signaling ◽  
Kinase Activity ◽  
Platelet Derived Growth Factor ◽  
Functional Coupling ◽  
Wild Type ◽  
Pi Turnover

The tyrosine kinase domains of the platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1)/c-fms receptors are interrupted by kinase inserts (ki) which vary in length and amino acid sequence. To define the role of the ki in the human alpha PDGF receptor (alpha PDGFR), we generated deletion mutants, designated alpha R delta ki-1 and alpha R delta ki-2, which lacked 80 (710 to 789) and 95 (695 to 789) amino acids of the 104-amino-acid ki region, respectively. Their functional characteristics were compared with those of the wild-type alpha PDGFR following introduction into a naive hematopoietic cell line, 32D. Biochemical responses, including PDGF-stimulated PDGFR tyrosine phosphorylation, phosphatidylinositol (PI) turnover, and receptor-associated PI-3 kinase activity, were differentially impaired by the deletions. Despite a lack of any detectable receptor-associated PI-3 kinase activity, 32D cells expressing alpha R delta ki-1 showed only partially impaired chemotactic and mitogenic responses and were capable of sustained proliferation in vitro and in vivo under conditions of autocrine stimulation by the c-sis product. 32D transfectants expressing the larger ki deletion (alpha R delta ki-2) showed markedly decreased or abolished biochemical and biological responses. However, insertion of the highly unrelated smaller c-fms (685 to 750) ki domain into alpha R delta ki-2 restored each of these activities to wild-type alpha PDGFR levels. Since the CSF-1R does not normally induce PI turnover, the ability of the c-fms ki domain to reconstitute PI turnover in the alpha R delta ki-2 transfectant provides evidence that the ki domain of the alpha PDGFR does not directly couple with this pathway. Taken together, all od these bindings imply that their ki domains have evolved to play very similar roles in the known signaling functions PDGF and CSF-1 receptors.

scholarly journals Deletion or substitution within the alpha platelet-derived growth factor receptor kinase insert domain: effects on functional coupling with intracellular signaling pathways.

1991 ◽  
Vol 11 (1) ◽  
pp. 134-142 ◽  
Author(s):  
M A Heidaran ◽  
J H Pierce ◽  
D Lombardi ◽  
M Ruggiero ◽  
J S Gutkind ◽  
...  
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Intracellular Signaling ◽  
Kinase Activity ◽  
Platelet Derived Growth Factor ◽  
Functional Coupling ◽  
Wild Type ◽  
Pi Turnover

The tyrosine kinase domains of the platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1)/c-fms receptors are interrupted by kinase inserts (ki) which vary in length and amino acid sequence. To define the role of the ki in the human alpha PDGF receptor (alpha PDGFR), we generated deletion mutants, designated alpha R delta ki-1 and alpha R delta ki-2, which lacked 80 (710 to 789) and 95 (695 to 789) amino acids of the 104-amino-acid ki region, respectively. Their functional characteristics were compared with those of the wild-type alpha PDGFR following introduction into a naive hematopoietic cell line, 32D. Biochemical responses, including PDGF-stimulated PDGFR tyrosine phosphorylation, phosphatidylinositol (PI) turnover, and receptor-associated PI-3 kinase activity, were differentially impaired by the deletions. Despite a lack of any detectable receptor-associated PI-3 kinase activity, 32D cells expressing alpha R delta ki-1 showed only partially impaired chemotactic and mitogenic responses and were capable of sustained proliferation in vitro and in vivo under conditions of autocrine stimulation by the c-sis product. 32D transfectants expressing the larger ki deletion (alpha R delta ki-2) showed markedly decreased or abolished biochemical and biological responses. However, insertion of the highly unrelated smaller c-fms (685 to 750) ki domain into alpha R delta ki-2 restored each of these activities to wild-type alpha PDGFR levels. Since the CSF-1R does not normally induce PI turnover, the ability of the c-fms ki domain to reconstitute PI turnover in the alpha R delta ki-2 transfectant provides evidence that the ki domain of the alpha PDGFR does not directly couple with this pathway. Taken together, all od these bindings imply that their ki domains have evolved to play very similar roles in the known signaling functions PDGF and CSF-1 receptors.

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 role of platelet-derived growth factor on human pluripotent progenitor (CFU-GEMM) growth in vitro

1985 ◽  
Vol 9 (3) ◽  
pp. 399-405 ◽  
Author(s):  
Rita Michalevicz ◽  
Gillian E. Francis ◽  
Gillian M. Price ◽  
A.Victor Hoffbrand
Keyword(s):  
Growth Factor ◽  
Platelet Derived Growth Factor

scholarly journals Tyr-716 in the platelet-derived growth factor beta-receptor kinase insert is involved in GRB2 binding and Ras activation.

1994 ◽  
Vol 14 (10) ◽  
pp. 6715-6726 ◽  
Author(s):  
A K Arvidsson ◽  
E Rupp ◽  
E Nånberg ◽  
J Downward ◽  
L Rönnstrand ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Growth Factor ◽  
Platelet Derived Growth Factor ◽  
Receptor Kinase ◽  
Aortic Endothelial Cells ◽  
Beta Receptor ◽  
Beta Receptors ◽  
Pdgfr Beta

Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.

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