Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction

1990 ◽  
Vol 10 (2) ◽  
pp. 801-809
Author(s):  
L Severinsson ◽  
B Ek ◽  
K Mellström ◽  
L Claesson-Welsh ◽  
C H Heldin
Keyword(s):  
Growth Factor ◽  
Protein Tyrosine Kinase ◽  
Catalytic Efficiency ◽  
Kinase Domain ◽  
Platelet Derived Growth Factor ◽  
Tyrosine Kinase Domain ◽  
Wild Type ◽  
Mutant Receptor ◽  
Beta Receptor ◽  
Type Receptor

A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor.

scholarly journals Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction.

1990 ◽  
Vol 10 (2) ◽  
pp. 801-809 ◽  
Author(s):  
L Severinsson ◽  
B Ek ◽  
K Mellström ◽  
L Claesson-Welsh ◽  
C H Heldin
Keyword(s):  
Growth Factor ◽  
Protein Tyrosine Kinase ◽  
Catalytic Efficiency ◽  
Kinase Domain ◽  
Platelet Derived Growth Factor ◽  
Tyrosine Kinase Domain ◽  
Wild Type ◽  
Mutant Receptor ◽  
Beta Receptor ◽  
Type Receptor

A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor.

scholarly journals Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach.

1993 ◽  
Vol 13 (6) ◽  
pp. 3567-3576 ◽  
Author(s):  
G Panayotou ◽  
G Gish ◽  
P End ◽  
O Truong ◽  
I Gout ◽  
...  
Keyword(s):  
Growth Factor ◽  
Kinetic Parameters ◽  
Protein Tyrosine Kinase ◽  
Platelet Derived Growth Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
High Association ◽  
Sh2 Domains ◽  
Beta Receptor ◽  
Growth Factor Beta ◽  
Phosphatidylinositol 3

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.

scholarly journals SH1 domain autophosphorylation of P210 BCR/ABL is required for transformation but not growth factor independence.

1993 ◽  
Vol 13 (3) ◽  
pp. 1728-1736 ◽  
Author(s):  
A M Pendergast ◽  
M L Gishizky ◽  
M H Havlik ◽  
O N Witte
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Specific Activity ◽  
Kinase Domain ◽  
Myelogenous Leukemia ◽  
Tyrosine Kinase Domain ◽  
Myc Gene ◽  
Efficient Induction ◽  
Transforming Activity

P210 BCR/ABL is a chimeric oncogene implicated in the pathogenesis of chronic myelogenous leukemia. BCR sequences have been shown to be required for activation of the tyrosine kinase and transforming functions of BCR/ABL. In this work, we show that two other structural requirements for full transforming activity of P210 BCR/ABL include a functional tyrosine kinase and the presence of tyrosine 1294, a site of autophosphorylation within the tyrosine kinase domain. Replacement of tyrosine 1294 with phenylalanine (1294F) greatly diminishes the transforming activity of BCR/ABL without affecting the specific activity of the protein tyrosine kinase. Expression of an exogenous myc gene in fibroblasts partially complements the transforming capacity of mutant P210 BCR/ABL (1294F). Surprisingly, tyrosine 1294 is not required for efficient induction of growth factor-independence in hematopoietic cell lines by P210 BCR/ABL. These results suggest that autophosphorylation at tyrosine 1294 may be important for recognition and phosphorylation of cellular substrates in the pathway of transformation, but it is not critical for mediating the events which lead to growth factor independence.

Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach

1993 ◽  
Vol 13 (6) ◽  
pp. 3567-3576 ◽  
Author(s):  
G Panayotou ◽  
G Gish ◽  
P End ◽  
O Truong ◽  
I Gout ◽  
...  
Keyword(s):  
Growth Factor ◽  
Kinetic Parameters ◽  
Protein Tyrosine Kinase ◽  
Platelet Derived Growth Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
High Association ◽  
Sh2 Domains ◽  
Beta Receptor ◽  
Growth Factor Beta ◽  
Phosphatidylinositol 3

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.

SH1 domain autophosphorylation of P210 BCR/ABL is required for transformation but not growth factor independence

1993 ◽  
Vol 13 (3) ◽  
pp. 1728-1736
Author(s):  
A M Pendergast ◽  
M L Gishizky ◽  
M H Havlik ◽  
O N Witte
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Specific Activity ◽  
Kinase Domain ◽  
Myelogenous Leukemia ◽  
Tyrosine Kinase Domain ◽  
Myc Gene ◽  
Efficient Induction ◽  
Transforming Activity

P210 BCR/ABL is a chimeric oncogene implicated in the pathogenesis of chronic myelogenous leukemia. BCR sequences have been shown to be required for activation of the tyrosine kinase and transforming functions of BCR/ABL. In this work, we show that two other structural requirements for full transforming activity of P210 BCR/ABL include a functional tyrosine kinase and the presence of tyrosine 1294, a site of autophosphorylation within the tyrosine kinase domain. Replacement of tyrosine 1294 with phenylalanine (1294F) greatly diminishes the transforming activity of BCR/ABL without affecting the specific activity of the protein tyrosine kinase. Expression of an exogenous myc gene in fibroblasts partially complements the transforming capacity of mutant P210 BCR/ABL (1294F). Surprisingly, tyrosine 1294 is not required for efficient induction of growth factor-independence in hematopoietic cell lines by P210 BCR/ABL. These results suggest that autophosphorylation at tyrosine 1294 may be important for recognition and phosphorylation of cellular substrates in the pathway of transformation, but it is not critical for mediating the events which lead to growth factor independence.

scholarly journals Ligand-independent activation of the platelet-derived growth factor beta receptor: requirements for bovine papillomavirus E5-induced mitogenic signaling.

1995 ◽  
Vol 15 (5) ◽  
pp. 2570-2581 ◽  
Author(s):  
D A Drummond-Barbosa ◽  
R R Vaillancourt ◽  
A Kazlauskas ◽  
D DiMaio
Keyword(s):  
Growth Factor ◽  
Complex Formation ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Platelet Derived Growth Factor ◽  
Bovine Papillomavirus ◽  
Wild Type ◽  
Beta Receptor ◽  
E5 Protein ◽  
Mitogenic Signal

The E5 protein of bovine papillomavirus type 1 binds to and activates the endogenous platelet-derived growth factor (PDGF) beta receptor in fibroblasts, resulting in cell transformation. We have developed a functional assay to test the ability of PDGF beta receptor mutants to mediate a mitogenic signal initiated by the E5 protein. Lymphoid Ba/F3 cells are strictly dependent on interleukin-3 for growth, but coexpression of the wild-type PDGF beta receptor and the E5 or v-sis-encoded protein generated a mitogenic signal which allowed Ba/F3-derived cells to proliferate in the absence of interleukin-3. In these cells, the E5 protein bound to and caused increased tyrosine phosphorylation of both the mature and the precursor forms of the wild-type PDGF beta receptor. The tyrosine kinase activity of the receptor was necessary for E5-induced receptor tyrosine phosphorylation and mitogenic activity but not for complex formation with the E5 protein. In contrast, the PDGF-binding domain of the receptor was not required for complex formation with the E5 protein, E5-induced tyrosine phosphorylation or mitogenic activity, demonstrating that E5-mediated receptor activation is ligand independent. Analysis of receptor mutants lacking various combinations of tyrosine phosphorylation sites revealed that the E5 and v-sis-encoded proteins display similar requirements for signaling and suggested that the wild-type PDGF beta receptor can generate multiple independent mitogenic signals. Importantly, these mutants dissociated two activities of the PDGF beta receptor tyrosine kinase, both of which are required for sustained mitogenic signaling: (i) receptor autophosphorylation and creation of binding sites for SH2 domain-containing proteins and (ii) phosphorylation of substrates other than the receptor itself.

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