A phorbol ester binding domain of protein kinase C gamma. Deletion analysis of the Cys2 domain defines a minimal 43-amino acid peptide.
Cysteine-rich regions of protein kinase C (PKC) are critical for the lipid-dependent regulation of activity and are implicated in the coordination of zinc. A glutathione S-transferase fusion protein containing the second cysteine-rich region, Cys2, of PKC gamma with bound zinc with a stoichiometry of 1.8 +/- 0.1 mol of zinc/mol of protein. Deletion analysis within this cysteine-rich region defined amino acids essential for zinc coordination. An NH2-terminal histidine (His102) and a COOH-terminal cysteine (Cys151) were both critical for the coordination of distinct zinc atoms. Both represent the ultimate residues of a 50-amino acid consensus motif with six conserved cysteines and two conserved histidines present in the cysteine-rich regions of all PKC isoforms. Removal of histidine His102 abolished phorbol ester binding, while deletion of cysteine Cys151 did not. Deletion of valine (Val147) greatly diminished phorbol ester binding, which was completely lost only when valine (Val144) was also deleted. No significant further reduction in zinc stoichiometry below one resulted even when three COOH-terminal conserved cysteines (Cys151, Cys143, and Cys135) and a conserved histidine (His140) were deleted. These results are consistent with a model in which two zinc atoms are tetracoordinated per cysteine-rich region in two independent coordination spheres that are not functionally equivalent. These analyses determine a minimal peptide (residues 102-144) of 43 amino acids capable of [3H]PDBu binding.
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Section of Cell Growth, Regulation and Oncogenesis, Duke University Medical Center, Durham, North Carolina 27710.
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Bell
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D017124:Conserved Sequence; D003545:Cysteine; D017931:DNA Primers; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000378:metabolism; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009036:Moths; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D011487:Protein Conformation; D011493:Protein Kinase C / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D015032:Zinc / Q000032:analysis / Q000378:metabolism
D017931:DNA Primers; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D015240:Phorbol 12,13-Dibutyrate; D005982:Glutathione Transferase; D011493:Protein Kinase C; D015032:Zinc; D003545:Cysteine