A truncated v-abl-derived tyrosine-specific tyrosine kinase expressed in Escherichia coli.
Several biochemical properties of a 43 kDa v-abl-encoded tyrosine-specific protein kinase (p43v-abl) expressed in Escherichia coli were examined. p43v-abl is a fragment of a 60 kDa v-abl-encoded precursor, p60v-abl, and could be generated by limited proteolysis of a purified p60v-abl with trypsin. Tryptic cleavage of p60v-abl was prevented in the presence of ATP. These results suggest that the catalytic kinase domain of v-abl-derived protein can be separated from other (regulatory) domains by limited proteolysis. p43v-abl readily phosphorylated tyrosine residues on several different protein and peptide substrates, including peptides containing only two amino acid residues. However, the local sequence of the tyrosine-containing peptide substrate significantly affected its rate of phosphorylation. Thus the primary structure and local conformation at the tyrosine acceptor site can play an important role in determining the substrate specificity of v-abl-derived kinase. Phosphorylation by p43v-abl requires Mn2+, Co2+ or Mg2+ and exhibits a strong preference for ATP as phosphate donor. Analogues of ATP and the thiol-reactive reagent N-ethylmaleimide inhibited p43v-abl kinase activity. Purified p43v-abl is intrinsically thermolabile (t1/2 = 5 min at 40 degrees C) and phosphorylates glycerol inefficiently (Km = 1.4 M).
Pritchard
M L
ML
Department of Cell Biology, Smith Kline and French Laboratory, Philadelphia, PA.
Rieman
D
D
Feild
J
J
Kruse
C
C
Rosenberg
M
M
Poste
G
G
Greig
R G
RG
Ferguson
B Q
BQ
D000011:Abelson murine leukemia virus / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005814:Genes, Viral; D006358:Hot Temperature; D009052:Leukemia Virus, Murine / Q000201:enzymology*; D008670:Metals / Q000378:metabolism; D009857:Oncogenes; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D008670:Metals; D010455:Peptides; D011505:Protein-Tyrosine Kinases