STIMULATION OF PROHORMONE THIOL PROTEASE (PTP) AND [MET]ENKEPHALIN BYFORSKOLIN - BLOCKADE OF ELEVATED [MET]ENKEPHALIN BY A CYSTEINE PROTEASE INHIBITOR OF PTP

Proenkephalin and other prohormones require proteolytic processing at paired basic and monobasic residues for the biosynthesis of active neu ropeptides. The novel ''prohormone thiol protease'' (PTP) has been pro posed as a candidate proenkephalin processing enzyme for the productio n of [Met]enkephalin in chromaffin granules (Krieger, T. J., and Hook, V. Y. H. (1991) J. Biol. Chem. 266, 88376-8383). In this study, PTP w as examined during elevation of cellular [Met]enkephalin by forskolin, a direct activator of adenylate cyclase that produces cAMP. Treatment of chromaffin cells with forskolin for 72 h increased enkephalin prec ursor cleaving activity (measured by following the conversion of the m odel substrate [S-35-Met]preproenkephalin to trichloroacetic acid-solu ble radioactivity) in isolated chromaffin granules by 170-180% over co ntrols (100%). The increased activity was associated with the membrane fraction, rather than the soluble fraction, of chromaffin granules. T he elevated activity was inhibited by E-64c, which is a potent inhibit or of PTP and cysteine proteases; however, the activity was not inhibi ted by serine or aspartic protease inhibitors. The elevated activity w as identified as PTP based on immunoprecipitation by anti-PTP immunogl obulins. Stimulation of PTP synthesis was involved in the forskolin-in duced increase in PTP activity, as demonstrated by a 10 fold increase in [S-35]PTP pulse labeling in forskolin-treated chromaffin cells. For skolin elevation of PTP protein levels within chromaffin granules was also detected in Western blots. Importantly, the forskolin-mediated ri se in cellular [Met]enkephalin levels was completely blocked when cell s were preincubated with the cysteine protease inhibitor Ep453, which is known to be converted by intracellular esterases to the more effect ive inhibitor E-64c (Buttle, D. J., Saklatvala, J., Tamai, M., and Bar rett, A. J. (1992) Biochem. J. 281, 175-177). Both E-64c and Ep453 inh ibit PTP, with E-64c being more potent (Azaryan, A. V., and Hook, V. Y . H. (1994b) Arch. Biochem. Biophys. 314, 171-177). These results demo nstrate a role for PTP in proenkephalin processing in chromaffin cells and indicate that [Met] enkephalin formation and PTP are both regulat ed by cAMP.