Kazanietz M G, Wang S, Milne G W, Lewin N E, Liu H L, Blumberg P M
Molecular Mechanisms of Tumor Promotion Section, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
J Biol Chem. 1995 Sep 15;270(37):21852-9. doi: 10.1074/jbc.270.37.21852.
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.
佛波酯以高亲和力与蛋白激酶C（PKC）同工酶以及两种新受体n-奇玛雅林和Unc-13结合。这些蛋白质中存在的富含半胱氨酸的区域被确定为佛波酯肿瘤启动子和亲脂性第二信使sn-二酰基甘油的结合位点。一个包含PKCδ第二个富含半胱氨酸区域的50个氨基酸的肽，在大肠杆菌中作为谷胱甘肽S-转移酶（GST）融合蛋白表达，以高亲和力（Kd = 0.8 nM）结合[3H]佛波醇12,13-二丁酸酯（PDBu）。以该富含半胱氨酸区域的cDNA为模板，通过定点诱变产生了一系列37个点突变，并对突变蛋白进行了[3H]PDBu结合的定量分析，以及在适当情况下对超效类似物[3H]苔藓抑素1结合的定量分析。突变体表现出三种行为模式之一：佛波酯结合完全消除、结合亲和力降低或结合没有明显改变。正如预期的那样，参与Zn2+配位的六个半胱氨酸中的五个以及两个组氨酸对于蛋白质与佛波酯的相互作用至关重要。此外，包括苯丙氨酸3、酪氨酸8、脯氨酸11、亮氨酸20、21和24、色氨酸21、谷氨酰胺27和缬氨酸38在内的几个位置的突变大大降低了与配体的相互作用。这些突变的影响可以从富含半胱氨酸区域的三维（NMR）结构中得到合理的解释。特别是，蛋白质的C末端部分似乎不是必需的，并且包含氨基酸20至28的环与结合活性有关。
