title: Kinetics of trkA tyrosine kinase activity and inhibition by K-252a.
abstract: The kinetic mechanism of the trk receptor-linked tyrosine kinase was determined using a baculovirus expressed trk kinase domain and a bacterially expressed phospholipase C-gamma/glutathione S-transferase (PLC-gamma/ GST) fusion protein as substrate. Product and dead-end inhibition studies indicate an ordered association of substrates to trkA kinase with the nucleotide ATP binding prior to the exogenous substrate PLC-gamma/GST, followed by release of the phosphorylated PLC-gamma/GST product prior to release of ADP (sequential ordered bi-bi mechanism). This is in contrast to the reported kinetic mechanisms of closely related EGF receptor and insulin receptor kinases which appear to proceed via a rapid equilibrium random mechanism. The indolocarbazole K-252a, which was previously shown to be a potent and relatively selective inhibitor of trk kinase activity, acts as a competitive inhibitor with respect to ATP. The data suggest that potent and selective kinase inhibitors can be rationally designed by exploring subtle variations surrounding the nucleotide binding sites of receptor tyrosine kinases.
authors.affiliation: Cephalon, Inc., West Chester, Pennsylvania 19380, USA. TAngeles@cephalon.com
authors.forename: T S
authors.initials: TS
authors.lastname: Angeles
authors.forename: S X
authors.initials: SX
authors.lastname: Yang
authors.forename: C
authors.initials: C
authors.lastname: Steffler
authors.forename: C A
authors.initials: CA
authors.lastname: Dionne
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology*; D002460:Cell Line; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D026121:Indole Alkaloids; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D002227:Carbazoles; D026121:Indole Alkaloids; D007527:Isoenzymes; D011518:Proto-Oncogene Proteins; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C049985:staurosporine aglycone; D005982:Glutathione Transferase; D020794:Receptor Protein-Tyrosine Kinases; D020917:Receptor, trkA; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; C002630:alpha,beta-methyleneadenosine 5'-triphosphate