Comparison of baculovirus-expressed c-Abl and BCR/ABL protein tyrosine kinases.
Mouse c-Abl type IV and human BCR/ABL proteins have been expressed in insect cells using the baculovirus system. The proteins were expressed as full-length polypeptides as judged by electrophoresis in denaturing gels. They were identified by immunoprecipitation and immunoblotting with antibodies against ABL peptides and, for BCR/ABL, against a BCR peptide. In these immunoprecipitates both proteins gave autophosphorylation principally on tyrosine. Both proteins were active tyrosine kinases, phosphorylating a variety of tyrosine-containing substrates. In fresh extracts both proteins contained phosphotyrosine as shown by Western blots with antiphosphotyrosine antibodies. Partial purification could be achieved readily using ion exchange columns, and the BCR/ABL protein, p210BCR/ABL, could be further purified to near-homogeneity using an antiphosphotyrosine column. Both enzymes required a divalent metal ion for activity. At low concentrations of ATP (2 microM) and with angiotensin II as substrate both enzymes were activated by Mn2+ or by Mg2+. No major differences in catalytic properties were found between the two isolated enzymes in solution. The oncogenic properties of p210BCR/ABL may be due to its different subcellular location, or to the presence of an intracellular inhibitor of c-Abl that does not inhibit BCR/ABL, or to altered substrate-specificity such that it can phosphorylate a unique substrate which is not recognised by c-Abl.
Reynolds
C H
CH
Wellcome Research Laboratories, Beckenham, UK.
Willson
M G
MG
Groffen
J
J
Heisterkamp
N
N
Peakman
T C
TC
Page
M J
MJ
D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002413:Cations, Divalent; D016044:Fusion Proteins, bcr-abl / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016313:Genes, abl; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012996:Solutions; D013379:Substrate Specificity
D002413:Cations, Divalent; D012996:Solutions; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl