scholarly journals Expression and characterization of the p85 subunit of the phosphatidylinositol 3-kinase complex and a related p85β protein by using the baculovirus expression system

1992 ◽  
Vol 288 (2) ◽  
pp. 395-405 ◽  
Author(s):  
I Gout ◽  
R Dhand ◽  
G Panayotou ◽  
M J Fry ◽  
I Hiles ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinases ◽  
Growth Regulation ◽  
Kinase Activity ◽  
Insect Cells ◽  
Expression System ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
T Complex

PtdIns 3-kinase associates with certain activated protein-tyrosine kinase receptors and with the pp60c-src/polyoma middle-T complex, suggesting that the enzyme is involved in growth regulation. The purified PtdIns 3-kinase appears to have two subunits, of 85 kDa and 110 kDa. Structural analysis at protein and cDNA levels revealed two forms of the 85 kDa subunit, one which associates with PtdIns 3-kinase activity termed p85 alpha, and a protein of unknown function, p85 beta. Both 85 kDa proteins contain src-homology regions 2 and 3 (SH2 and SH3), but lack enzymic activity, suggesting that they may be regulatory subunits of PtdIns 3-kinase. To probe their structure and function further, p85 alpha and p85 beta have been expressed and purified in large amounts from insect cells by using baculovirus vectors. Specific antisera detect p85 alpha, but not p85 beta, associated with PtdIns 3-kinase activity in various cell types. Co-expression studies in insect cells have shown that p85 alpha and p85 beta are substrates for the protein-tyrosine kinases of epidermal growth factor, colony-stimulating factor 1 and c-erbB2 receptors and the src family kinase p59c-fyn. Both p85 alpha and p85 beta form tight complexes with these protein-tyrosine kinases as measured by immunoprecipitation and kinase assays in vitro. The specificity of binding of free p85 is less restricted than that of p85 in the active PtdIns 3-kinase complex with the 110 kDa protein. The relevance of these results to growth-factor-induced PtdIns 3-kinase activation is discussed.

scholarly journals CGP 57148, a Tyrosine Kinase Inhibitor, Inhibits the Growth of Cells Expressing BCR-ABL, TEL-ABL, and TEL-PDGFR Fusion Proteins

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4947-4952 ◽  
Author(s):  
Martin Carroll ◽  
Sayuri Ohno-Jones ◽  
Shu Tamura ◽  
Elisabeth Buchdunger ◽  
Jürg Zimmermann ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Philadelphia Chromosome ◽  
Lymphocytic Leukemia ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine

Abstract CGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL–expressing cells. To extend these observations, we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases, including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation, inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound, the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL–positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome–positive ALL patient. As CGP 57148 inhibits the PDGFR kinase, we also showed that cells expressing an activated PDGFR tyrosine kinase, TEL-PDGFR, are sensitive to this compound. Thus, this compound may be useful for the treatment of a variety of BCR-ABL–positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein.

scholarly journals CGP 57148, a Tyrosine Kinase Inhibitor, Inhibits the Growth of Cells Expressing BCR-ABL, TEL-ABL, and TEL-PDGFR Fusion Proteins

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4947-4952 ◽  
Author(s):  
Martin Carroll ◽  
Sayuri Ohno-Jones ◽  
Shu Tamura ◽  
Elisabeth Buchdunger ◽  
Jürg Zimmermann ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Philadelphia Chromosome ◽  
Lymphocytic Leukemia ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine

CGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL–expressing cells. To extend these observations, we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases, including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation, inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound, the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL–positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome–positive ALL patient. As CGP 57148 inhibits the PDGFR kinase, we also showed that cells expressing an activated PDGFR tyrosine kinase, TEL-PDGFR, are sensitive to this compound. Thus, this compound may be useful for the treatment of a variety of BCR-ABL–positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein.

scholarly journals Capture by chemical crosslinkers provides evidence that integrin α IIb β 3 forms a complex with protein tyrosine kinases in intact platelets

1995 ◽  
Vol 309 (2) ◽  
pp. 481-490 ◽  
Author(s):  
D J Dorahy ◽  
M C Berndt ◽  
G F Burns
Keyword(s):  
Tyrosine Kinase ◽  
Platelet Activation ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Spatial Association ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Kinase Reaction

Platelet activation is accompanied by a cascade of kinase reactions in which numerous specific proteins are phosphorylated on tyrosine. These events are strictly dependent upon functional activation of an integrin receptor, generally alpha IIb beta 3 (also known as glycoprotein IIb-IIIa). It is not known how alpha IIb beta 3 regulates protein tyrosine kinase activation and, in particular, neither this nor any other integrin has been shown to associate with a protein tyrosine kinase. We employed chemical crosslinking of intact platelets with the bifunctional reagents dithiobis(succinimidyl propionate) (DSP) (lipid-soluble) and dithiobis(sulphosuccinimidyl propionate) (DTSSP) (lipid-insoluble) followed by in vitro kinase assays of immunoprecipitated proteins to identify kinase activity associated with alpha IIb beta 3 in intact platelets. It was found that DSP but not DTSSP crosslinked kinase activity to alpha IIb beta 3, suggesting an internal association. In these immunoprecipitates from DSP-crosslinked platelets, the in vitro kinase reaction revealed a complex of several phosphoproteins in association with alpha IIb beta 3. This association was not seen when the resting platelets were lysed before crosslinking, indicating the specificity of the reaction in crosslinking only molecules in preformed spatial association. The beta 3 subunit of alpha IIb beta 3 was identified as one of the phosphoproteins in the complex obtained after subjecting anti-beta 3 immunoprecipitates from lysates of DSP-treated platelets to an in vitro kinase reaction and SDS/PAGE analysis. Phosphorylation of this subunit is shown to be predominantly on tyrosine. Affinity purification of the crosslinked phosphoprotein complex with anti-beta 3 followed by elution and re-precipitation identified pp60c-src and pp54/58c-lyn as two protein tyrosine kinases associating with the integrin. These results suggest that, upon platelet activation, alpha IIb beta 3 may provide a transmembrane focus for proteins involved in signal transduction.

Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK

1994 ◽  
Vol 14 (1) ◽  
pp. 147-155
Author(s):  
B S Cobb ◽  
M D Schaller ◽  
T H Leu ◽  
J T Parsons
Keyword(s):  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Src Family Kinases ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Sh2 Domains ◽  
Embryo Cells ◽  
Stable Association

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.

Association between B-lymphocyte membrane immunoglobulin and multiple members of the Src family of protein tyrosine kinases

1992 ◽  
Vol 12 (5) ◽  
pp. 2315-2321
Author(s):  
M A Campbell ◽  
B M Sefton
Keyword(s):  
B Cells ◽  
B Cell ◽  
Tyrosine Kinases ◽  
B Lymphocyte ◽  
Protein Tyrosine Kinases ◽  
Cell Type ◽  
Protein Tyrosine ◽  
Membrane Immunoglobulin ◽  
Family Protein

Treatment of B lymphocytes with antibodies to membrane immunoglobulin (Ig) stimulates protein tyrosine phosphorylation. We have examined the phosphorylation in vitro of proteins associated with membrane Ig. The Src family protein tyrosine kinases p53/56lyn, p59fyn, and p56lck are associated with membrane Ig in spleen B cells and B-cell lines and undergo phosphorylation in vitro. The pattern of expression of Src family protein tyrosine kinases in B cells varied. Our studies suggest that multiple kinases can potentially interact with membrane Ig and that within any one B-cell type, all of the Src family kinases expressed can be found in association with membrane Ig. We also observed that the Ig-associated Ig alpha protein, multiple forms of Ig beta, and proteins of 100 and 25 kDa were tyrosine phosphorylated in vitro. The 100- and 25-kDa proteins remain unidentified.

scholarly journals The ras-related gene rhoB is an immediate-early gene inducible by v-Fps, epidermal growth factor, and platelet-derived growth factor in rat fibroblasts.

1991 ◽  
Vol 11 (7) ◽  
pp. 3682-3690 ◽  
Author(s):  
D Jähner ◽  
T Hunter
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinases ◽  
Platelet Derived Growth Factor ◽  
Immediate Early ◽  
Ras Gene ◽  
Protein Tyrosine ◽  
Sarcoma Virus ◽  
Epidermal Growth

A set of genes is rapidly inducible when quiescent fibroblasts are stimulated by growth factors or by the activation of temperature-sensitive retroviral protein-tyrosine kinases. Most of these so-called immediate-early genes were cloned by differential cDNA hybridization. DNA sequence analysis identified many of them as putative members of the growth factor or of the transcription factor gene family, suggesting a role in signal transmission during the G0-to-G1 transition. In this study, we identified one of the genes that are rapidly inducible by the retroviral protein-tyrosine kinases v-Src and v-Fps of Rous sarcoma virus and Fujinami sarcoma virus, respectively, as the rhoB gene, a member of the ras gene superfamily whose products are GTP-binding proteins, rhoB is transiently activated at the transcriptional level by v-Fps and by epidermal growth factor. Its labile RNA is inducible in the presence of cycloheximide but not of actinomycin D. rhoB is strongly induced by epidermal growth factor and by platelet-derived growth factor both in subconfluent, serum-starved and in density-arrested Rat-2 fibroblasts. Fetal calf serum is a poor inducer, particularly in density-arrested cells, and phorbol esters do not increase rhoB expression at all. These data suggest that rhoB is inducible by protein-tyrosine kinases through a pathway not involving the activation of protein kinase C. Neither the closely related rhoC and rhoA genes nor the distantly related c-H-ras gene is rapidly inducible by mitogens. Thus, rhoB is the first known member of the small GTP-binding proteins among the immediate-early genes.

Specific inhibition of cytoplasmic protein tyrosine kinases by herbimycin A in vitro

1991 ◽  
Vol 42 (9) ◽  
pp. 1661-1671 ◽  
Author(s):  
Fukazawa Hidesuke ◽  
Li Pei-Mlng ◽  
Yamamoto Chii ◽  
Murakami Yuko ◽  
Mizuno Satoshi ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Protein Tyrosine Kinases ◽  
Specific Inhibition ◽  
Cytoplasmic Protein ◽  
Protein Tyrosine ◽  
Herbimycin A
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