Ho C, Slater S J, Stagliano B A, Stubbs C D
Department of Pathology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Biochem J. 1999 Dec 1;344 Pt 2(Pt 2):451-60.
The fluorescent phorbol ester 12-N-methylanthraniloylphorbol 13-acetate [sapintoxin D (SAPD)] was used as both the activator and the probe for the activating conformational change of the C1 domain of recombinant protein kinase C (PKC)alpha. Fluorescence emission spectra and steady-state anisotropy measurements of SAPD in fully active membrane-associated PKC show that there is a relatively hydrophobic environment and restricted motional freedom characterizing the phorbol-ester-binding site. SAPD also interacts with the membrane lipids so that it was necessary to resort to time-resolved anisotropy measurements to resolve the signals corresponding to PKC-bound SAPD from that associated with buffer and lipid. In the presence of membrane lipids (unilamellar vesicles of phosphatidylcholine and phosphatidylserine, 4:1 molar ratio) and Ca(2+), at a concentration sufficient to activate the enzyme fully, a long correlation time characteristic of highly restricted motion was observed for PKC-associated SAPD. The fraction of SAPD molecules displaying this restricted motion, in comparison with the total SAPD including that in lipids and in buffer, increased with increasing concentrations of Ca(2+) and paralleled the appearance of enzyme activity, whereas the rotational correlation time remained constant. This could be rationalized as an increase in the number of active PKC conformers in the total population of PKC molecules. It therefore seems that there is a distinct conformation of the C1 activator-binding domain associated with the active form of PKC. The addition of SAPD and dioleoyl-sn-glycerol together produced an activity higher than that achievable by either activator alone both at concentrations that alone induced maximal activity for the respective activator; this higher activity was associated with a further restriction in SAPD motion. Increasing the cholesterol concentration, the phosphatidylethanolamine concentration, the sn-2 unsaturation in phosphatidylcholine and the vesicle curvature each also elevated SAPD-induced PKC activity and again increased the PKC-associated SAPD rotational correlation time. In summary, the rotational correlation time of PKC-bound SAPD, extractable from a single time-resolved fluorescence anisotropy measurement, provides a novel probe for the involvement of interactions between the C1 domain and phorbol ester in the modulation of PKC activity.
荧光佛波酯12 - N - 甲基邻氨基苯甲酰佛波醇13 - 乙酸酯[沙平毒素D（SAPD）]被用作重组蛋白激酶C（PKC）α的C1结构域激活构象变化的激活剂和探针。在完全活性的膜结合型PKC中对SAPD进行荧光发射光谱和稳态各向异性测量表明，佛波酯结合位点具有相对疏水的环境和受限的运动自由度。SAPD还与膜脂相互作用，因此有必要采用时间分辨各向异性测量来区分与PKC结合的SAPD和与缓冲液及脂质相关的信号。在存在膜脂（磷脂酰胆碱和磷脂酰丝氨酸的单层囊泡，摩尔比为4:1）和Ca(2+)且浓度足以完全激活该酶的情况下，观察到与PKC相关的SAPD具有高度受限运动的长相关时间。与包括脂质和缓冲液中的SAPD在内的总SAPD相比，表现出这种受限运动的SAPD分子比例随Ca(2+)浓度增加而增加，且与酶活性的出现平行，而旋转相关时间保持恒定。这可以解释为PKC分子总数中活性PKC构象体数量的增加。因此，似乎存在与PKC活性形式相关的C1激活剂结合结构域的独特构象。在单独诱导各自激活剂最大活性的浓度下，同时添加SAPD和二油酰 - sn - 甘油产生的活性高于单独使用任何一种激活剂所能达到的活性；这种更高的活性与SAPD运动的进一步受限有关。增加胆固醇浓度、磷脂酰乙醇胺浓度、磷脂酰胆碱中sn - 2位的不饱和度以及囊泡曲率也均提高了SAPD诱导的PKC活性，并再次增加了与PKC相关的SAPD旋转相关时间。总之，从单次时间分辨荧光各向异性测量中提取的与PKC结合的SAPD的旋转相关时间，为C1结构域与佛波酯之间的相互作用在PKC活性调节中的参与提供了一种新型探针。
