Department of Biological Sciences, 217 Life Science Building, Bowling Green State University, Bowling Green, OH 43403, USA.
Department of Physics & Astronomy, 104 Overman Hall, Bowling Green State University, Bowling Green, OH 43403, USA.
Biomolecules. 2020 Sep 7;10(9):1288. doi: 10.3390/biom10091288.
Protein kinase Cs (PKCs) are activated by lipids in the plasma membrane and bind to a scaffold assembled on the epidermal growth factor (EGF) receptor (EGFR). Understanding how this complex is routed is important, because this determines whether EGFR is degraded, terminating signaling. Here, cells were preincubated in EGF-tagged gold nanoparticles, then allowed to internalize them in the presence or absence of a phorbol ester PKC activator. PKC colocalized with EGF-tagged nanoparticles within 5 min and migrated with EGFR-bearing vesicles into the cell. Two conformations of PKC-epsilon were distinguished by different primary antibodies. One, thought to be enzymatically active, was on endosomes and displayed a binding site for antibody RR (R&D). The other, recognized by Genetex green (GG), was soluble, on actin-rich structures, and loosely bound to vesicles. During a 15-min chase, EGF-tagged nanoparticles entered large, perinuclear structures. In phorbol ester-treated cells, vesicles bearing EGF-tagged nanoparticles tended to enter this endocytic recycling compartment (ERC) without the GG form. The correlation coefficient between the GG (inactive) and RR conformations on vesicles was also lower. Thus, active PKC has a Charon-like function, ferrying vesicles to the ERC, and inactivation counteracts this function. The advantage conferred on cells by aggregating vesicles in the ERC is unclear.
蛋白激酶 C（PKC）可被质膜中的脂质激活，并与表皮生长因子受体（EGFR）上组装的支架结合。了解这种复合物如何被靶向是很重要的，因为这决定了 EGFR 是否被降解，从而终止信号传递。在本研究中，细胞预先用 EGF 标记的金纳米颗粒孵育，然后在存在或不存在佛波酯 PKC 激活剂的情况下允许它们内化。PKC 在 5 分钟内与 EGF 标记的纳米颗粒共定位，并与携带 EGFR 的囊泡一起迁移到细胞内。两种 PKC-epsilon 构象可以通过不同的一抗来区分。一种被认为是具有酶活性的，位于内体上，并显示出与抗体 RR（R&D）结合的位点。另一种，被 Genetex 绿（GG）识别，是可溶性的，位于富含肌动蛋白的结构上，并且与囊泡松散结合。在 15 分钟的追踪过程中，EGF 标记的纳米颗粒进入了核周的大结构中。在佛波酯处理的细胞中，携带 EGF 标记的纳米颗粒的囊泡往往会进入这种内体再循环隔室（ERC），而没有 GG 形式。囊泡上 GG（无活性）和 RR 构象之间的相关系数也较低。因此，活性 PKC 具有类似于 Charon 的功能，将囊泡运送到 ERC，而失活则会抵消这种功能。将囊泡聚集在 ERC 中赋予细胞的优势尚不清楚。
