The regulatory domain of protein kinase C coordinates four atoms of zinc.
Protein kinase C (PKC) was found to be a zinc metallo-enzyme. Atomic absorption measurements on the intact enzyme indicated that four zinc atoms (4.2 +/- 0.5) were bound per PKC alpha molecule. Similar stoichiometric ratios were determined for PKC beta II and PKC gamma, other PKC isoforms individually expressed in the baculovirus-insect cell expression system, as well as for purified rat brain PKC. By trypsin treatment of PKC alpha, a 32-kDa lipid binding regulatory and a 50-kDa catalytic domain were generated that were subsequently completely separated by gel filtration in the presence of Triton X-100/phosphatidylserine mixed micelles. Zinc was present at levels significantly above background in fractions that contained the 32-kDa fragment and displayed phorbol ester binding activity. Lipid association and phorbol ester binding did not lead to displacement of zinc from the protein. The stoichiometry determined for this fragment (4.7 +/- 0.9) suggested that zinc was bound exclusively within the lipid binding regulatory domain of intact PKC. Furthermore, this stoichiometry is consistent with zinc being coordinated between 6 cysteine residues in a structural motif related to the Zn(II)2Cys6 binuclear cluster identified in the GAL4 transcriptional factor (Pan, T., and Coleman, J.E. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2077-2081).
Quest
A F
AF
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Bloomenthal
J
J
Bardes
E S
ES
Bell
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007313:Insecta; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011493:Protein Kinase C / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D014162:Transfection; D015032:Zinc / Q000032:analysis*
D000906:Antibodies; D010446:Peptide Fragments; D010455:Peptides; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D015032:Zinc