Active recombinant human tyrosine kinase c-Yes: expression in baculovirus system, purification, comparison to c-Src, and inhibition by a c-Src inhibitor.
C-Yes is a non-receptor-type tyrosine kinase of the Src family that is most closely related to c-Src. C-Yes has been implicated in development of some human cancers. Here we report on the expression, purification, and characterization of the active human recombinant c-Yes. A full-length human c-Yes clone has been generated and the protein was expressed in insect Sf9 cells. Active c-Yes was purified by liquid chromatography to yield a preparation with a high specific activity (160 nmol/min/mg using an optimal Src substrate peptide). In a comparison between human c-Yes and c-Src enzymes, relative phosphorylation efficiencies on nine protein and four peptide substrates were different. However, the recently described Src inhibitor CGP77675 inhibited human c-Yes with a potency similar to that of c-Src (IC(50) value of 6.5 nM). The purified preparation of active c-Yes provides a good basis for further enzymatic characterization and for the development of c-Yes-specific inhibitors.
Susa
M
M
Research Bone Metabolism, Novartis Pharma AG, Basel, CH-4002, Switzerland. mira.susa_spring@pharma.novartis.com
Luong-Nguyen
N H
NH
Crespo
J
J
Maier
R
R
Missbach
M
M
McMaster
G
G
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000737:chemistry; D006801:Humans; D007313:Insecta / Q000166:cytology; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051575:Proto-Oncogene Proteins c-yes; D011743:Pyrimidines / Q000737:chemistry; D011758:Pyrroles / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019061:src-Family Kinases
D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D011743:Pyrimidines; D011758:Pyrroles; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D051575:Proto-Oncogene Proteins c-yes; D019061:src-Family Kinases; C000631346:CSK protein, human; C119957:CGP 77675