A phorbol ester binding domain of protein kinase C gamma. High affinity binding to a glutathione-S-transferase/Cys2 fusion protein.
Cysteine-rich regions of protein kinase C (PKC) are implicated in diacylglycerol-dependent regulation of kinase activity. The second cysteine-rich region (residues 92-173) of PKC gamma was expressed as a fusion protein with glutathione-S-transferase in Escherichia coli and purified to homogeneity by affinity chromatography. This fusion protein displayed high affinity phorbol dibutyrate (PDBu) binding (Kd 23 nM). The phosphatidylserine dependence of PDBu binding was highly cooperative with Hill numbers (near 4.5) similar to those previously reported for PKC gamma (Burns, D. J., and Bell, R. M. (1991) J. Biol. Chem. 266, 18330-18338). The fusion protein specifically bound 4 beta-hydroxy-PDBu but not the 4 alpha-stereoisomer. Furthermore, sn-1,2-dioctanoylglycerol (diC8) stereoselectively competed for PDBu binding. The cysteine-rich region was sufficient for association of the fusion protein to liposome preparations containing phosphatidylserine and phosphatidylcholine. Association was significantly enhanced in a stereospecific manner by the presence of PDBu as well as diC8. These results establish that a single cysteine-rich domain (residues 92-173) of PKC gamma contains regions necessary and sufficient for lipid-dependent stereospecific interactions with PDBu and diC8. Furthermore, the region is sufficient to confer translocation of a fusion protein to liposomes in a PDBu- and diC8-dependent fashion. Thus, a single cysteine-rich region of PKC gamma displays many of the properties characteristic of PKC.
Quest
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AF
Section of Cell Growth, Regulation and Oncogenesis, Duke University Medical Center, Durham, North Carolina 27710.
Bardes
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ES
Bell
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RM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D003545:Cysteine; D017931:DNA Primers; D004075:Diglycerides / Q000494:pharmacology; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008969:Molecular Sequence Data; D009036:Moths; D015240:Phorbol 12,13-Dibutyrate / Q000031:analogs & derivatives / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D011493:Protein Kinase C / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D004075:Diglycerides; D007527:Isoenzymes; D008081:Liposomes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011993:Recombinant Fusion Proteins; C044295:1,2-dioctanoylglycerol; D015240:Phorbol 12,13-Dibutyrate; D005982:Glutathione Transferase; D011493:Protein Kinase C; D003545:Cysteine