Protein kinase C-δ (PKC-δ) has been demonstrated to be phosphorylated on tyrosine residue(s) in many different biological systems (Li, 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-δ has also been shown to occur in vitro when purified PKC-δ is coincubated with different tyrosine kinase sources. However, the tyrosine phosphorylation site(s) is currently unknown and the exact effect of this phosphorylation on its serine/threonine kinase activity and biological functions is still controversial. To directly investigate the potential role of PKC-δ tyrosine phosphorylation, tyrosine 187 was converted to phenylalanine (PKC-δY187F) by site-directed mutagenesis, and expression vectors containing PKC-δYI87F cDNAs were transfected into both 32D myeloid progenitor cells and NIH 3T3 fibroblasts. The results showed that tyrosine 187 of PKC-δ became phosphorylated in vivo in response to 12-O- tetradecanoylphorbol-13-acetate stimulation or platelet-derived growth factor receptor activation. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that one phosphopeptide was absent in PKC-δ187F when compared to wild type PKC-δ, further substantiating that tyrosine 187 of PKC-δ is phosphorylated in vivo. Although the phosphotyrosine content of PKC-δY187F was reduced compared with PKC-δWT, the kinase activity of PKC-δY187F toward a PKC-δ substrate was not altered. Moreover, 12-O-tetradecanoylphorbol-13-acetate-mediated monocytic differentiation of 32D cells was not affected by expression of the PKC- δY187F mutant. Taken together, these results suggest that tyrosine phosphorylation of PKC-δ on 187 may not influence PKC-δ activation and known functions.
Identification of tyrosine 187 as a protein kinase C-δ phosphorylation site / Li, W., Li, W., Chen, X.-H., Kelley, C.A., Alimandi, M., Zhang, J., Chen, Q., Bottaro, D.P., Pierce, J.H.. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 271:42(1996), pp. 26404-26409. [10.1074/jbc.271.42.26404]
Identification of tyrosine 187 as a protein kinase C-δ phosphorylation site
Alimandi M.Methodology
;
1996
Abstract
Protein kinase C-δ (PKC-δ) has been demonstrated to be phosphorylated on tyrosine residue(s) in many different biological systems (Li, 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-δ has also been shown to occur in vitro when purified PKC-δ is coincubated with different tyrosine kinase sources. However, the tyrosine phosphorylation site(s) is currently unknown and the exact effect of this phosphorylation on its serine/threonine kinase activity and biological functions is still controversial. To directly investigate the potential role of PKC-δ tyrosine phosphorylation, tyrosine 187 was converted to phenylalanine (PKC-δY187F) by site-directed mutagenesis, and expression vectors containing PKC-δYI87F cDNAs were transfected into both 32D myeloid progenitor cells and NIH 3T3 fibroblasts. The results showed that tyrosine 187 of PKC-δ became phosphorylated in vivo in response to 12-O- tetradecanoylphorbol-13-acetate stimulation or platelet-derived growth factor receptor activation. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that one phosphopeptide was absent in PKC-δ187F when compared to wild type PKC-δ, further substantiating that tyrosine 187 of PKC-δ is phosphorylated in vivo. Although the phosphotyrosine content of PKC-δY187F was reduced compared with PKC-δWT, the kinase activity of PKC-δY187F toward a PKC-δ substrate was not altered. Moreover, 12-O-tetradecanoylphorbol-13-acetate-mediated monocytic differentiation of 32D cells was not affected by expression of the PKC- δY187F mutant. Taken together, these results suggest that tyrosine phosphorylation of PKC-δ on 187 may not influence PKC-δ activation and known functions.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
