Residues in the second cysteine-rich region of protein kinase C delta relevant to phorbol ester binding as revealed by site-directed mutagenesis.
Phorbol esters bind with high affinity to protein kinase C (PKC) isozymes as well as to two novel receptors, n-chimaerin and Unc-13. The cysteine-rich regions present in these proteins were identified as the binding sites for the phorbol ester tumor promoters and the lipophilic second messenger sn-diacylglycerol. A 50-amino-acid peptide comprising the second cysteine-rich region of PKC delta, expressed in Escherichia coli as a glutathione S-transferase (GST)-fusion protein, bound [3H]phorbol 12,13-dibutyrate (PDBu) with high affinity (Kd = 0.8 nM). Using the cDNA of that cysteine-rich region as a template, a series of 37 point mutations was generated by site-directed mutagenesis, and the mutated proteins were analyzed quantitatively for binding of [3H]PDBu and, as appropriate, for binding of the ultrapotent analog [3H]bryostatin 1. Mutants displayed one of three patterns of behavior: phorbol ester binding was completely abolished, binding affinity was reduced, or binding was not significantly modified. As expected, five of the six cysteines as well as the two histidines involved in Zn2+ coordination are critical for the interaction of the protein with the phorbol esters. In addition, mutations in several positions, including phenylalanine 3, tyrosine 8, proline 11, leucines 20, 21 and 24, tryptophan 21, glutamine 27, and valine 38 drastically reduced the interaction with the ligands. The effect of these mutations can be rationalized from the three-dimensional (NMR) structure of the cysteine-rich region. In particular, the C-terminal portion of the protein does not appear to be essential, and the loop comprising amino acids 20 to 28 is implicated in the binding activity.
Kazanietz
M G
MG
Molecular Mechanisms of Tumor Promotion Section, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Wang
S
S
Milne
G W
GW
Lewin
N E
NE
Liu
H L
HL
Blumberg
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D054713:Bryostatins; D003001:Cloning, Molecular; D016384:Consensus Sequence; D003545:Cysteine; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000096:biosynthesis; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007783:Lactones / Q000378:metabolism; D018942:Macrolides; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D017354:Point Mutation; D011493:Protein Kinase C / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014316:Tritium
D000970:Antineoplastic Agents; D054713:Bryostatins; D007527:Isoenzymes; D007783:Lactones; D018942:Macrolides; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014316:Tritium; D015240:Phorbol 12,13-Dibutyrate; C046785:bryostatin 1; D005982:Glutathione Transferase; D011493:Protein Kinase C; D003545:Cysteine