The use of the fluorescence properties of indolocarbazole analogs of adenosine triphosphate as probes of the activating conformational change in protein kinase C

The fluorescence properties of the protein kinase C (PKC) inhibitors stauro sporine, a naturally occurring indolocarbazole, and the synthetic compound 12-(2-cyanoethyl)-6,7,12,13-teu-ahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrr olo[3,4-c]carbazole (GO 6976), which share the indolo[2,3-a]pyrrolo[3,4-c]c arbazole core, were used to probe the adenosine triphosphate (ATP)-binding site of PKC alpha. Upon binding to PKC alpha, the fluorescence emission of staurosporine and GO 6976 increased in intensity and the maxima shifted sli ghtly to shorter wavelengths. For staurosporine, while the fluorescence lif etime was relatively insensitive to the PKC activation state, a long rotati onal correlation time appeared, consistent with a more restricted motion wi thin the ATP-binding site. This effect was obtained only in the presence of the activators of the enzyme diacylglycerol or phorbol ester along with ca lcium and was obtained in spite of the inhibition of phosphorylation of a p eptide substrate by the effective blocking of the ATP-binding site by the p robe. This shows that the catalytic domain of PKC, within which the ATP-bin ding site resides, changes conformation upon enzyme activation. The results demonstrate the usefulness of indolocarbazole-analogs in probing ATP-bindi ng site conformational changes and show that the ATP-binding site within th e PKC catalytic domain participates in the activating conformational change in the enzyme. (C) 2001 Elsevier Science B.V. All rights reserved.