Dynamic sequestration of the recycling compartment by classical protein kinase C
Dynamic sequestration of the recycling compartment by classical protein kinase C
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DOI：
10.1074/jbc.m512540200
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发表时间：
2006-08-04
影响因子：
4.8
通讯作者：
Hannun, Yusuf A.
中科院分区：
文献类型：
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作者：
Idkowiak-Baldys, Jolanta;Becker, Kevin P.;Hannun, Yusuf A.
先前已经表明，在用佛波醇酯持续刺激（30-60分钟）后，蛋白激酶C（PKC）α和β II被隔离在核外区，即中心体周围。PKC的激活也导致转铁蛋白的螯合，表明PKC在调节内吞作用和再循环组分的螯合中的作用。在这项工作中，我们的特点是作为一个PKC依赖的子集的回收室的中心周围。我们证明，在持续刺激PKC，蛋白质（CD 59，小窝蛋白）和可能也脂质（Bodipy-GM 1）货物成为隔离在PKC依赖的方式。这种隔离显示出严格的温度要求，并且在低于32 ℃时受到抑制。用佛波醇肉豆蔻酸酯乙酸酯处理细胞60分钟导致形成独特的膜结构。高渗蔗糖以及钾耗竭（网格蛋白依赖性内吞作用的抑制剂），但不是由制霉菌素或菲律宾菌素，抑制网格蛋白独立的途径，阻断PKC的隔离和中心体周围的形成。有趣的是，还观察到一些通过网格蛋白非依赖性途径（CD 59，Bodipy-GM 1，caveolin）内化的分子也在持续的PKC激活后被隔离到中心周围，这表明PKC作用于内吞货物内化位点的远端。总之，这些结果表明，PKC调节螯合回收分子进入这个区室，中心周围。
It has been previously shown that upon sustained stimulation (30-60 min) with phorbol esters, protein kinase C(PKC) alpha and beta II become sequestered in a juxtanuclear region, the pericentrion. The activation of PKC also results in sequestration of transferrin, suggesting a role for PKC in regulating endocytosis and sequestration of recycling components. In this work we characterize the pericentrion as a PKC-dependent subset of the recycling compartment. We demonstrate that upon sustained stimulation of PKC, both protein (CD59, caveolin) and possibly also lipid (Bodipy-GM1) cargo become sequestered in a PKC-dependent manner. This sequestration displayed a strict temperature requirement and was inhibited below 32 C. Treatment of cells with phorbol myristate acetate for 60 min led to the formation of a distinct membrane structure. PKC sequestration and pericentrion formation were blocked by hypertonic sucrose as well as by potassium depletion (inhibitors of clathrin-dependent endocytosis) but not by nystatin or filipin, which inhibit clathrin-independent pathways. Interestingly, it was also observed that some molecules that internalize through clathrin-independent pathways (CD59, Bodipy-GM1, caveolin) also sequestered to the pericentrion upon sustained PKC activation, suggesting that PKC acted distal to the site of internalization of endocytic cargo. Together these results suggest that PKC regulates sequestration of recycling molecules into this compartment, the pericentrion.