Expression, purification, and characterization of Bacneu. A soluble protein tyrosine kinase domain encoded by the neu-oncogene.
To further characterize the structure and regulation of the tyrosine kinase encoded by the rodent neu oncogene, its cytoplasmic tyrosine kinase domain has been expressed as a soluble protein, called Bacneu, in Sf9 insect cells, using the baculovirus expression system. Expression of Bacneu was detected by immunoblotting with anti p185neu antisera and in vitro autophosphorylation analysis as early as 24 h postinfection. Maximal expression was observed at 48 h postinfection. The soluble kinase was purified to near homogeneity by sequential chromatography on DEAE-Sepharose, phosphocellulose, poly-L-lysine, and Sephacryl 300, yielding 0.55 mg Bacneu per L of Sf9 cells (4% yield). The kinase is more active in the presence of Mn2+ compared to Mg2+ ions. The specific activity of the kinase using poly(Glu4Tyr1) as a substrate is 179 nmol/min/mg. Maximal incorporation of 1.4 mol of phosphate per mol of enzyme by autophosphorylation was found to increase the activity of the enzyme 1.5- to twofold. These results indicate that the Bacneu kinase is activated by phosphorylation. Therefore, it will be a useful reagent for characterizing the effects that phosphorylation by other cellular kinases and dephosphorylation by phosphatases have on its activity.
Department of Pathology, University of Pennsylvania, Philadelphia.
J N
JN
Myers
C M
CM
LeVea
J E
JE
Smith
R G
RG
Kallen
L
L
Tung
M I
MI
Greene
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003593:Cytoplasm / Q000201:enzymology; D005815:Genetic Code / Q000235:genetics*; D015151:Immunoblotting; D007313:Insecta / Q000166:cytology / Q000378:metabolism; D009857:Oncogenes / Q000235:genetics*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification* / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases; D012995:Solubility
D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases