title: Characterization of specific [3H]dimethylstaurosporine binding to protein kinase C.
abstract: The microbial alkaloid staurosporine is a member of a recently described family of protein kinase inhibitors. [N,N-dimethyl-3H]N-dimethylstaurosporine ([3H]DMS) was prepared from staurosporine by methylation with [3H]methyl iodide. Since staurosporine inhibits protein kinase C (PKC) most potently, the binding of [3H]DMS to this enzyme was examined. Unlike [20-3H(N)]phorbol-12,13-dibutyrate ([3H]PDBu) binding to PKC, [3H]DMS binding was not calcium or phosphatidylserine (PS) dependent. Binding was reversible, with a T1/2 of 69 min and a Koff of 0.01/min. Non-specific binding was defined by a 500-fold molar excess of staurosporine and was less than 10% of total [3H]DMS binding. Specific binding of [3H]DMS was consistent with a single class of binding sites with a Kd of 3.8 +/- 0.6 nM and a Bmax of 675 +/- 30 pmol/g tissue. In competition experiments, staurosporine inhibited [3H]DMS binding with a Ki of 4.7 +/- 0.6 nM, indicating that the two alkaloids had a similar potency for PKC. Also, unlabeled DMS and staurosporine inhibited [3H]DMS binding and PKC catalysis with equivalent potencies. Highly purified rat brain PKC bound equimolar amounts of [3H]PDBu and [3H]DMS. In contrast, crude rat brain PKC, which had been proteolysed to generate a PS and Ca2+ independent enzyme (PK-M) retained the ability to bind [3H]DMS, but not [3H]PDBu. In addition, the kinase inhibitors K-252a and H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine] inhibited [3H]DMS binding, whereas PDBu did not. These results indicate that [3H]DMS is a useful ligand to identify catalytic inhibitors of kinase activity and to explore their mechanisms of action.
authors.affiliation: Medical Products Department, E. I. du Pont de Nemours & Co., Inc., Wilmington, DE 19898.
authors.forename: J L
authors.initials: JL
authors.lastname: Gross
authors.forename: W F
authors.initials: WF
authors.lastname: Herblin
authors.forename: U H
authors.initials: UH
authors.lastname: Do
authors.forename: J S
authors.initials: JS
authors.lastname: Pounds
authors.forename: L J
authors.initials: LJ
authors.lastname: Buenaga
authors.forename: L E
authors.initials: LE
authors.lastname: Stephens
mesh_terms: D000470:Alkaloids / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D051379:Mice; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D019311:Staurosporine / Q000031:analogs & derivatives; D014316:Tritium
chemical_list: D000470:Alkaloids; D014316:Tritium; C065079:N,N-dimethylstaurosporine; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D019311:Staurosporine