The relationship between lysosomal enzyme release and protein phosphorylation in human monocytes stimulated by...

Since it was established that phorbol esters bind to and activate protein kinase C, a proposed mechanism of action for these compounds has been the phosphorylation of specific protein substrates (Niedel, J. E., Kuhn, L. J., and Vandenbark, G. R. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 36-40; Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851). To better understand this proposed relationship, we investigated the ability of a series of phorbol esters to elicit lysosomal enzyme release (LER) and specific substrate phosphorylation in human monocytes. In this system, phorbol 12-myristate 13-acetate stimulated the secretion of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase in both a time- and concentration-dependent manner. Furthermore, the ability of a series of phorbol esters to stimulate LER was characterized and found to be in good agreement with the relative order of these compounds to stimulate the phosphorylation of four endogenous protein substrates. Phorbol ester-stimulated protein phosphorylation was examined in intact cell preparations and found to be concentration and structure-dependent. The phosphoproteins (pp) were designated pp28, pp55, pp61, and pp66 corresponding to their molecular masses in kilodaltons. These findings are consistent with the hypothesis that phorbol ester-mediated effects are the result of protein kinase C activation and subsequent protein phosphorylation. Finally, opsonized zymosan was found to elicit a concentration-dependent stimulation of N-acetyl-beta-D-glucosaminidase release similar in magnitude and time course to phorbol ester-stimulated LER. Opsonized zymosan also stimulated the phosphorylation of two phosphoproteins (pp61 and pp66) in a concentration-dependent manner. Specific phosphorylation of pp61 and pp66 by both secretagogues, phorbol 12-myristate 13-acetate and opsonized zymosan, suggests these two proteins may be key to the functional response of LER in human monocytes.