Non-equivalent roles for the first and second zinc fingers of protein kinase Cdelta. Effect of their mutation on phorbol ester-induced translocation in NIH 3T3 cells.
Classical and novel protein kinase C (PKC) isozymes contain two, so-called cysteine-rich zinc finger domains that represent the binding sites for phorbol esters and the diacylglycerols. X-ray crystallographic, mutational, and modeling studies are providing detailed understanding of the interactions between the phorbol esters and individual PKC zinc fingers. In the present study, we explore the roles of the individual zinc fingers in the context of the intact enzyme. Our approach was to mutate either the first, the second, or both zinc fingers of PKCdelta, to express the mutated enzyme in NIH 3T3 cells, and to monitor the effect of the mutations on the dose-response curve for translocation induced by phorbol 12-myristate 13-acetate. The introduced mutations change into glycine the consensus proline in the phorbol ester binding loop of the zinc finger; in the isolated zinc finger, this mutation causes a 125-fold decrease in phorbol ester binding affinity. We observed that mutation in the first zinc finger caused almost no shift in the dose-response curve for translocation; mutation in the second zinc finger caused a 21-fold shift, whereas mutation in both zinc fingers caused a 138-fold shift. We conclude that the zinc fingers in the intact PKC are not equivalent and that the second zinc finger plays the predominant role in translocation of protein kinase Cdelta in response to phorbol 12-myristate 13-acetate. Our findings have important implications for the understanding and design of PKC inhibitors targeted to the zinc finger domains.
Szallasi
Z
Z
Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Bogi
K
K
Gohari
S
S
Biro
T
T
Acs
P
P
Blumberg
P M
PM
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active / Q000187:drug effects; D017931:DNA Primers / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017354:Point Mutation; D011493:Protein Kinase C / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D016335:Zinc Fingers / Q000235:genetics* / Q000502:physiology*
D017931:DNA Primers; D007527:Isoenzymes; C497259:Prkcd protein, mouse; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate