Departamento de Bioquímica y Biología Molecular-A, Facultad de Veterinaria, Regional Campus of International Excellence "Campus Mare Nostrum", Universidad de Murcia, Murcia, Spain.
PLoS One. 2014 Apr 24;9(4):e95973. doi: 10.1371/journal.pone.0095973. eCollection 2014.
The C2 domain of PKCα (C2α) induces fluorescence self-quenching of NBD-PS in the presence of Ca2+, which is interpreted as the demixing of phosphatidylserine from a mixture of this phospholipid with phosphatidylcholine. Self-quenching of NBD-PS was considerably increased when phosphatidylinositol-4,5-bisphosphate (PIP2) was present in the membrane. When PIP2 was the labeled phospholipid, in the form of TopFluor-PIP2, fluorescence self-quenching induced by the C2 domain was also observed, but this was dependent on the presence of phosphatidylserine. An independent indication of the phospholipid demixing effect given by the C2α domain was obtained by using 2H-NMR, since a shift of the transition temperature of deuterated phosphatidylcholine was observed as a consequence of the addition of the C2α domain, but only in the presence of PIP2. The demixing induced by the C2α domain may have a physiological significance since it means that the binding of PKCα to membranes is accompanied by the formation of domains enriched in activating lipids, like phosphatidylserine and PIP2. The formation of these domains may enhance the activation of the enzyme when it binds to membranes containing phosphatidylserine and PIP2.
PKCα 的 C2 结构域（C2α）在 Ca2+存在的情况下诱导 NBD-PS 的荧光自猝灭，这被解释为磷脂酰丝氨酸从该磷脂与磷脂酰胆碱的混合物中分离。当膜中存在磷脂酰肌醇-4,5-二磷酸（PIP2）时，NBD-PS 的自猝灭显著增加。当 PIP2 是以 TopFluor-PIP2 的形式存在的标记磷脂时，也观察到 C2 结构域诱导的荧光自猝灭，但这取决于磷脂酰丝氨酸的存在。通过使用 2H-NMR 获得了 C2α 结构域给出的磷脂分离效应的独立指示，因为添加 C2α 结构域后观察到氘化磷脂酰胆碱的相变温度发生了位移，这仅发生在存在 PIP2 的情况下。C2α 结构域诱导的分离可能具有生理意义，因为这意味着 PKCα 与膜的结合伴随着富含激活脂质（如磷脂酰丝氨酸和 PIP2）的域的形成。当酶与含有磷脂酰丝氨酸和 PIP2 的膜结合时，这些结构域的形成可能会增强酶的激活。
