title: The cysteine-rich domain of human proteins, neuronal chimaerin, protein kinase C and diacylglycerol kinase binds zinc. Evidence for the involvement of a zinc-dependent structure in phorbol ester binding.
abstract: Diacylglycerol (DG) and its analogue phorbol 12-myristate 13-acetate (PMA) activate the ubiquitous phospholipid/Ca2(+)-dependent protein kinase, protein kinase C (PKC), and cause it to become tightly associated with membranes. DG is produced transiently as it is rapidly metabolized by DG kinase (DGK) to phosphatidic acid. Phorbol esters such as PMA are not metabolized and induced a prolonged membrane association of PKC. Until recently, PKC was the only known phorbol ester receptor. We have shown that a novel brain-specific cDNA, neuronal chimaerin (NC), expressed in Escherichia coli, binds phorbol ester with high affinity, stereospecificity and a phospholipid requirement [Ahmed, Kozma, Monfries, Hall, Lim, Smith & Lim (1990) Biochem. J. 272, 767-773]. The proteins NC, PKC and DGK possess a cysteine-rich domain with the motif HX11/12CX2CXnCX2CX4HX2CX6/7C (where n varies between 12 and 14). The partial motif, CX2CX13CX2C, is present in a number of transcription factors including the steroid hormone receptors and the yeast protein, GAL4, in which zinc plays a structural role of co-ordinating cysteine residues and is essential for DNA binding (protein-nucleic acid interactions). The cysteine-rich domain of NC and PKC is required for phospholipid-dependent phorbol is required for phospholipid-dependent phorbol ester binding, suggesting an involvement of this domain in protein-lipid interactions. We have expressed recombinant NC, PKC and DGK glutathione S-transferase and TrpE fusion proteins in E. coli to investigate the relationship between the cysteine-rich motif, HX11/12CX2CX10-14CX2CX4HX2CX6/7C, zinc and phorbol ester binding. The cysteine-rich domain of NC, PKC and DGK bound 65Zn2+ but only NC and PKC bound [3H]phorbol 12,13-dibutyrate. When NC and PKC were subjected to treatments known to remove metal ions from GAL4 and the human glucocorticoid receptor, phorbol ester binding was inhibited. These data provide evidence for the role of a zinc-dependent structure in phorbol ester binding.
authors.affiliation: Institute of Molecular and Cell Biology, National University of Singapore.
authors.forename: S
authors.initials: S
authors.lastname: Ahmed
authors.forename: R
authors.initials: R
authors.lastname: Kozma
authors.forename: J
authors.initials: J
authors.lastname: Lee
authors.forename: C
authors.initials: C
authors.lastname: Monfries
authors.forename: N
authors.initials: N
authors.lastname: Harden
authors.forename: L
authors.initials: L
authors.lastname: Lim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D020848:Chimerin 1; D003001:Cloning, Molecular; D003545:Cysteine; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D019852:Diacylglycerol Kinase; D006801:Humans; D008667:Metalloproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D015032:Zinc / Q000378:metabolism*
chemical_list: D020848:Chimerin 1; D008667:Metalloproteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D015240:Phorbol 12,13-Dibutyrate; D004247:DNA; D010770:Phosphotransferases; D019852:Diacylglycerol Kinase; D011493:Protein Kinase C; D015032:Zinc; D003545:Cysteine