Zinc finger domains and phorbol ester pharmacophore. Analysis of binding to mutated form of protein kinase C zeta and the vav and c-raf proto-oncogene products.
The phorbol ester binding domain consists of a cysteine-rich region with a postulated consensus sequence for binding that includes 15 amino acids (Ahmed, S., Kozma, R., Lee, J., Monfries, C., Harden, N., and Lim, L. (1991) Biochem. J. 280, 233-241). In PKC zeta, the only PKC isoform lacking phorbol ester binding, this region differs in a single residue from the consensus (proline in position 11 of the motif). Restoration of this proline by site-directed mutagenesis of PKC zeta does not restore binding of either [3H]phorbol 12,13-dibutyrate or of the ultrapotent ligand [3H]bryostatin 1, suggesting that even a low affinity ligand interaction is absent. In addition, the vav and c-raf proto-oncogene products, proteins that possess cysteine-rich regions with high homology to PKC isozymes and other phorbol ester receptors, are unable to bind any of these ligands. Instead, all of these cysteine-rich regions bind zinc. Our results suggest that other amino acids besides those postulated for the consensus must be necessary for ligand binding and argue against direct modulation of PKC zeta, Vav, and c-Raf by phorbol esters.
Kazanietz
M G
MG
Molecular Mechanisms of Tumor Promotion Section, National Cancer Institute, Bethesda, Maryland 20892.
Bustelo
X R
XR
Barbacid
M
M
Kolch
W
W
Mischak
H
H
Wong
G
G
Pettit
G R
GR
Bruns
J D
JD
Blumberg
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D054713:Bryostatins; D018797:Cell Cycle Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism; D007783:Lactones / Q000378:metabolism; D018942:Macrolides; D051379:Mice; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011493:Protein Kinase C / Q000096:biosynthesis / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D051567:Proto-Oncogene Proteins c-vav; D011519:Proto-Oncogenes; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers
D054713:Bryostatins; D018797:Cell Cycle Proteins; D017931:DNA Primers; D007527:Isoenzymes; D007783:Lactones; C000715470:MAS1 protein, human; D018942:Macrolides; D009838:Oligodeoxyribonucleotides; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; D011994:Recombinant Proteins; C496618:VAV1 protein, human; C496619:Vav1 protein, mouse; D015240:Phorbol 12,13-Dibutyrate; C046785:bryostatin 1; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D015032:Zinc