Kazanietz M G, Bustelo X R, Barbacid M, Kolch W, Mischak H, Wong G, Pettit G R, Bruns J D, Blumberg P M
Molecular Mechanisms of Tumor Promotion Section, National Cancer Institute, Bethesda, Maryland 20892.
J Biol Chem. 1994 Apr 15;269(15):11590-4.
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.
佛波酯结合结构域由一个富含半胱氨酸的区域组成，该区域具有一个假定的结合共有序列，包含15个氨基酸（艾哈迈德，S.，科兹马，R.，李，J.，蒙弗里斯，C.，哈登，N.，和林，L.（1991年）《生物化学杂志》280卷，233 - 241页）。在蛋白激酶Cζ（PKCζ）中，这是唯一缺乏佛波酯结合能力的PKC同工型，该区域与共有序列仅在一个残基上存在差异（基序第11位的脯氨酸）。通过对PKCζ进行定点诱变恢复该脯氨酸，并不能恢复[³H]佛波醇12,13 - 二丁酸酯或超效配体[³H]苔藓抑素1的结合，这表明即使是低亲和力的配体相互作用也不存在。此外，vav和c - raf原癌基因产物，这些蛋白质具有与PKC同工酶和其他佛波酯受体高度同源的富含半胱氨酸的区域，但它们无法结合这些配体中的任何一种。相反，所有这些富含半胱氨酸的区域都能结合锌。我们的结果表明，除了假定的共有序列中的那些氨基酸外，其他氨基酸对于配体结合也必定是必需的，并且反对佛波酯对PKCζ、Vav和c - Raf的直接调节作用。
