IDENTIFICATION OF TYROSINE-187 AS A PROTEIN-KINASE C-DELTA PHOSPHORYLATION SITE

Protein kinase C-delta (PKC-delta) has been demonstrated to be phospho rylated on tyrosine residue(s) in many different biological systems (L i, W., Yu, J.-C., Michieli, P., Beeler, J. F., Ellmore, N., Heidaran, M. A., and Pierce, J. H. (1994) Mol. Cell. Biol. 14, 6727-6735; Li, W. , Mischak, H., Yu, J.-C., Wang, L.-M., Mushinski, J. F., Heidaran, M. A., and Pierce, J. H. (1994) J. Biol. Chem. 269, 2349-2352; Denning, M . F., Dlugosz, A. A., Howett, M. A., and Yuspa, S. H. (1993) J. Biol. Chem. 268, 26079-26081). Tyrosine phosphorylation of PKC-delta has als o been shown to occur in vitro when purified PKC-delta is coincubated with different tyrosine kinase sources. However, the tyrosine phosphor ylation site(s) is currently unknown and the exact effect of this phos phorylation on its serine/threonine kinase activity and biological fun ctions is still controversial. To directly investigate the potential r ole of PKC-delta tyrosine phosphorylation, tyrosine 187 was converted to phenylalanine (PKC-delta Y187F) by site-directed mutagenesis, and e xpression vectors containing PKC-delta Y187F cDNAs were transfected in to both 32D myeloid progenitor cells and NIH 3T3 fibroblasts. The resu lts showed that tyrosine 187 of PKC-delta became phosphorylated in viv o in response to 12-O-tetradecanoylphorbol-13-acetate stimulation or p latelet-derived growth factor receptor activation. In vivo labeling an d subsequent two-dimensional phosphopeptide analysis demonstrated that one phosphopeptide was absent in PKC-delta Y187F when compared to wil d type PKC-delta, further substantiating that tyrosine 187 of PKC-delt a is phosphorylated in vivo. Although the phosphotyrosine content of P KC-delta Y187F was reduced compared with PKC-delta WT, the kinase acti vity of PKC-delta Y187F toward a PKC-delta substrate was not altered. Moreover, 12-O-tetradecanoylphorbol-13-acetate-mediated monocytic diff erentiation of 32D cells was not affected by expression of the PKC-del ta Y187F mutant. Taken together, these results suggest that tyrosine p hosphorylation of PKC-delta on 187 may not influence PKC-delta activat ion and known functions.