Comparison of baculovirus-expressed c-Abl and BCR/ABL protein tyrosine kinases

Biochim Biophys Acta. 1993 Apr 30;1181(2):122-30. doi: 10.1016/0925-4439(93)90100-f.

Abstract

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.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Baculoviridae / genetics*
  • Baculoviridae / metabolism
  • Base Sequence
  • Cations, Divalent
  • Fusion Proteins, bcr-abl / genetics*
  • Fusion Proteins, bcr-abl / isolation & purification
  • Fusion Proteins, bcr-abl / metabolism
  • Genes, abl*
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Protein-Tyrosine Kinases / genetics*
  • Protein-Tyrosine Kinases / isolation & purification
  • Protein-Tyrosine Kinases / metabolism
  • Solutions
  • Substrate Specificity

Substances

  • Cations, Divalent
  • Solutions
  • Protein-Tyrosine Kinases
  • Fusion Proteins, bcr-abl