Phosphorylation of PKC activation loop plays an important role in receptor-mediated translocation of PKC
Phosphorylation of PKC activation loop plays an important role in receptor-mediated translocation of PKC
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DOI：
10.1111/j.1365-2443.2005.00830.x
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发表时间：
2005-03-01
期刊：
影响因子：
2.1
通讯作者：
Sakai, N
中科院分区：
文献类型：
--
作者：
Seki, T;Matsubayashi, H;Sakai, N
蛋白激酶C（PKC）以亚型和刺激依赖性方式易位到细胞的各个区域。此后，活化的PKC磷酸化其底物并引起随后的细胞应答（PKC靶向）。3-磷酸肌醇依赖性蛋白激酶-1（PDK 1）通过磷酸化PKC激活环中的苏氨酸残基在PKC的成熟中起重要作用。为了阐明PDK 1在PKC靶向中的作用，我们表达了与GFP融合的突变体γ-或δ-PKC（γ-或δ-PKC-ALM（激活环突变体）-GFP），其激活环中的苏氨酸残基被丙氨酸取代，并在CHO细胞中将其P2 Y受体介导的易位与野生型PKC-GFP进行了比较。ATP（1 mm）诱导野生型γ-或δ-PKC-GFP从细胞质到质膜的瞬时易位和随后从膜到细胞质的再易位。γ-或δ-PKC-ALM-GFP也易位至质膜，然而，其在膜上保留的时间比野生型长。在激酶阴性的PKC突变体中也观察到类似的结果，表明PDK 1的磷酸化通过调节激酶活性影响PKC的重转位步骤。同时监测[Ca ~（2+）]（i）和甘油二酯（DG）水平与PKC转位表明PKC-ALM诱导DG的延长积累，导致PKC-ALM在质膜上的延长滞留。降低激酶活性的PKC-ALM可能延缓DG在质膜上的转化。我们目前的研究表明，激活环磷酸化在受体介导的PKC靶向中起着重要作用。
Protein kinase C (PKC) is translocated to various cellular regions in a subtype and stimulation-dependent manner. Thereafter, the activated PKC phosphorylates its substrate and causes subsequent cellular responses (PKC targeting). The 3-phosphoinositide-dependent protein kinase-1 (PDK1) has an essential role in the maturation of PKC by phosphorylating a threonine residue in the PKC activation loop. To elucidate the role of PDK1 in PKC targeting, we expressed mutant gamma- or delta-PKC fused with GFP (gamma- or delta-PKC-ALM (activation loop mutant)-GFP), whose threonine residue in the activation loop was replaced with alanine, and compared their P2Y receptor-mediated translocation with wild-type PKC-GFP in CHO cells. ATP (1 mm) induced the transient translocation of wild-type gamma- or delta-PKC-GFP from cytoplasm to plasma membrane and following retranslocation from membrane to the cytoplasm. gamma- or delta-PKC-ALM-GFP was also translocated to plasma membrane, which was, however, retained at the membrane for a longer period than wild type. Similar results were observed in kinase-negative PKC mutants, indicating that the phosphorylation by PDK1 affects the retranslocation step of PKC by regulating the kinase activity. The simultaneous monitoring of [Ca2+](i) and diacylglycerol (DG) levels with the translocation of PKC demonstrated that PKC-ALM induced the prolonged accumulation of DG, resulting in the prolonged retention of PKC-ALM at the plasma membrane. It is possible that PKC-ALM with decreased kinase activity could delay the conversion of DG at the plasma membrane. Our present study suggests that the activation loop phosphorylation plays an important role in receptor-mediated PKC targeting.