Journal: Molecular and Cellular Biology

Article Title: Inhibition of Bcr serine kinase by tyrosine phosphorylation

doi: 10.1128/mcb.16.3.998

Figure Lengend Snippet: FIG. 1. 2-D tryptic mapping of in vitro-autophosphorylated wild-type and mutant Bcr-Abl proteins. Mutant and wild-type Bcr-Abl proteins were expressed in COS-1 cells as described in Materials and Methods. Bcr-Abl proteins were autophosphorylated in vitro with anti-Abl (51-64) immune complexes, digested with trypsin, and separated on thin-layer plates. Dashed circles identify the positions of missing peptide spots. (A) Wild-type P210BCR-ABL; (B) Y276F P210BCR-ABL; (C) Y283F P210BCR-ABL; (D) Y360F P185BCR-ABL. Radioactive patterns were detected with a PhosphorImager (Molecular Dynamics).

Article Snippet: Casein bands were quantitated with a PhosphorImager (Molecular Dynamics). on M ay 18, 2016 by guest http://m cb.asm .org/ D ow nloaded from sion of a tyrosine kinase-inactive form of P210BCR-ABL (K1172R) (22) should have little effect on Bcr’s transkinase activity.

Techniques: In Vitro, Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Bcr serine kinase by tyrosine phosphorylation

doi: 10.1128/mcb.16.3.998

Figure Lengend Snippet: FIG. 3. Tyrosine 360 of Bcr is required for Bcr transkinase activity. (A) Mutant Bcr (Y360F) is defective in transkinase activity. Wild-type and mutant Bcr (Y360F) proteins were expressed in COS-1 cells, lysates were made, and immune complexes were harvested with anti-Bcr(1256-1271). Kinase assays were conducted in the presence and absence of 10 mg of casein. Lane 1, wild-type Bcr; lane 2, wild-type Bcr plus casein; lane 3, Y360F mutant Bcr; lane 4, Y360F mutant Bcr plus casein. Radioactive bands were detected with a Phosphor- Imager. (B) Western blotting of wild-type and mutant Bcr proteins expressed in COS-1 cells. Proteins were detected with anti-Bcr(1256-1271) by ECL in analyses of 5 3 106 cells. Lane 1, wild-type P160BCR; lane 2, Y283F Bcr; lane 3, Y360F Bcr. The SDS–12% polyacrylamide gel was exposed for 30 s. (C) Bcr-Abl weakly phosphorylated casein compared with P160BCR. COS-1 cells expressing either P210BCR-ABL or P160BCR were lysed and processed as described for panel A. Lane 1, untransfected COS-1 cells immunoprecipitated with anti-Bcr(1256- 1271); lane 2, COS-1 cells transfected with P210BCR-ABL cDNA and immuno- precipitated with anti-Bcr(898-911); lane 3, COS-1 cells transfected with wild- type P160BCR cDNA processed as described for lane 1. Ten micrograms of casein was added to each assay. Bands were detected with a PhosphorImager.

Article Snippet: Casein bands were quantitated with a PhosphorImager (Molecular Dynamics). on M ay 18, 2016 by guest http://m cb.asm .org/ D ow nloaded from sion of a tyrosine kinase-inactive form of P210BCR-ABL (K1172R) (22) should have little effect on Bcr’s transkinase activity.

Techniques: Activity Assay, Mutagenesis, Western Blot, Expressing, Immunoprecipitation, Transfection

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Bcr serine kinase by tyrosine phosphorylation

doi: 10.1128/mcb.16.3.998

Figure Lengend Snippet: FIG. 5. In vitro tyrosine phosphorylation of Bcr by Bcr-Abl inhibits Bcr serine/threonine protein kinase activity. (A) SDS gel patterns of kinase reactions. Bcr was expressed in COS-1 cells in the presence and absence of P210BCR-ABL. Lysates were immunoprecipitated with anti-Bcr(1256-1271) or anti-Abl(51-64). Immunocomplexes were assayed for kinase activity with and without added casein. Lanes 1 and 2, anti-Bcr immune complexes from Bcr-expressing COS-1 cells without (2) and with (1) casein, respectively; lane 3, anti-Bcr immune complexes on protein A-Sepharose beads from Bcr-expressing cells mixed with anti-Abl immune complexes on beads from P210BCR-ABL-expressing cells; as indicated, kinase assays were performed with 10 mg of casein. Bands were detected with a PhosphorImager. (B) Phosphoamino acid analysis of casein

Article Snippet: Casein bands were quantitated with a PhosphorImager (Molecular Dynamics). on M ay 18, 2016 by guest http://m cb.asm .org/ D ow nloaded from sion of a tyrosine kinase-inactive form of P210BCR-ABL (K1172R) (22) should have little effect on Bcr’s transkinase activity.

Techniques: In Vitro, Phospho-proteomics, Activity Assay, SDS-Gel, Immunoprecipitation, Expressing, Phosphoamino Acid Analysis