Journal zyxwvutsrqponmlk of Neurochemisrry zyxwvutsrqpon Raven Press, Ltd., New York zyxwvutsrqpon 0 1992 International Society for Neurochernistry 6-Methylmercaptopurine Riboside Is a Potent and Selective Inhibitor of Nerve Growth Factor-Activated Protein Kinase N Cinzia Volontk and Lloyd A. Greene Department of Pathology and Centerfor Neurobiology and Behavior, College of Physicians and Surgeons zyx of Columbia University, New York, New York, U.S.A. Abstract: Protein kinase N (PKN) is a soluble, apparently novel serine protein kinase that is activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma cells as well as in several nonneuronal cell lines. Purine an- alogs, such as 6-thioguanine and 2-aminopurine, have been found to inhibit PKN in vitro. When applied to intact cells, these compounds suppress certain biological responses to NGF, but not others, a finding suggesting the presence of multiple pathways in the NGF mechanism. We report here that 6-methylmercaptopurine riboside (6-MMPR) inhibits NGF-stimulated PKN activity in vitro with an apparent K, of zyxwvutsrqp - 5 nM. This is - 1,000-fold lower than the zyxwvut Ki of the most potent purine inhibitor of PKN. Compounds similar to 6- MMPR, but lacking the methyl or riboside groups, were much less potent as PKN inhibitors. zyxwvuts A survey of six additional pu- rified protein kinases shows no inhibitory effect of 6-MMPR, thus indicating a good degree of specificity of this compound Nerve growth factor (NGF) (Levi-Montalcini and Angeletti, 1968; Levi-Montalcini, 1987)exerts trophic and differentiative influences on neurons by means of mechanisms that are only incompletely understood (Levi et al., 1988). Nevertheless, several investigations have indicated that a crucial parameter likely to be involved in many of the events controlled by NGF is the regulation of protein phosphorylation (Halegoua and Patrick, 1980; Blenis and Erikson, 1986; Cremins et al., 1986; Hama et al., 1986; Romano et al., 1987; Rowland et al., 1987; Aletta et al., 1988; Mutoh et al., 1988; Vulliet et al., 1989; Hashimoto and Hagino, 1990; Landreth et al., 1990; Sano et al., 1990; Tsao et al., 1990). Among the protein b a s e s known to be ~~ ~~~~ Received February 15, 199 1 ; revised manuscript received June 6, 199 1 ; accepted July 8, 199 1. Address correspondence and reprint requests to Dr. C. Volontt at Department of Pathology, College of Physicians and Surgeons of Columbia University, 630 West 168th Street, New York, NY 10032, U.S.A. Abbreviations zyxwvutsrqp used: 2-AP, 2-aminopurine; CaMKI, CaMKII, and for PKN. In contrast to NGF-stimulated PKN, a PKN-like activity stimulated in PC12 cells in response to activation of cyclic AMP-dependent protein kinase was nearly insensitive to 6-MMPR. Application of 6-MMPR to intact PC12 cells resulted in blockade of several responses to NGF (neurite regeneration and ornithine decarboxylase induction) but not of several others (rapid enhancement of tyrosine hydroxylase phosphorylation and PKN activation). These findings suggest that 6-MMPR is a potent and selective agent for character- izing PKN in vitro and for assessing its potential role in the multiple pathways of the NGF mechanism of action. Key Words: Nerve growth factor-Protein kinases-Protein ki- nase N-PC 1 2 cells-6-Methylmercaptopurine riboside. Volontb C. and Greene L. A. 6-Methylmercaptopurine ri- boside is a potent and selective inhibitor of nerve growth factor-activated protein kinase N. J. Neurochem. 58, 700- 708 (1 992). regulated by NGF is one designated protein kinase N (PKN) (Rowland et al., 1987). This kinase is rapidly activated by NGF and other agents in cultured rat PC 12 pheochromocytoma cells (Blenis and Erikson, 1986; Rowland-GagnC and Greene, 1990) and in several nonneuronal cell lines that possess functional NGF re- ceptors (VolontC and Greene, 1990~). PKN is a serine kinase and uses histone HF1, tyrosine hydroxylase, and ribosomal S6 protein as in vitro substrates. Further- more, PKN is soluble, is not inhibited by Mn2+, does not require cofactors, and appears to be distinct from other well-characterized protein kinases (Blenis and Erikson, 1986; Rowland et al., 1987; Rowland-GagnC and Greene, 1990). CaMKIII, Ca2+/calmodulin-dependent kinase I, 11, and 111, respec- tively; CAMP, cyclic AMP CasKI1, casein kinase 11; CPT-CAMP, 8-(4chlorophenylthio)-cyclic AMP; 6-MMPR, 6-methylmercapto- purine riboside; NGF, nerve growth factor; ODC, ornithine decar- boxylase; PAGE, polyacrylamide gel electrophoresis; PKA, cyclic AMPdependent protein kinase; PKN, protein kinase N; SDS, zy sodium dodecyl sulfate; 6-TG, 6-thioguanosine. 700