Zegzouti Hicham, Anthony Richard G, Jahchan Nadine, Bögre László, Christensen Sioux K
Department of Molecular, Cell, and Developmental Biology and Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
Proc Natl Acad Sci U S A. 2006 Apr 18;103(16):6404-9. doi: 10.1073/pnas.0510283103. Epub 2006 Apr 6.
Activity of the serine-threonine protein kinase PINOID (PID) has been implicated in the asymmetrical localization of the membrane-associated PINFORMED (PIN) family of auxin transport facilitators. However, the means by which PID regulates PIN protein distribution is unknown. We have used recombinant PID protein to dissect the regulation of PID activity in vitro. We demonstrate that intramolecular PID autophosphorylation is required for the ability of PID to phosphorylate an exogenous substrate. PID-like mammalian AGC kinases act in a phosphorylation cascade initiated by the phospholipid-associated kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1), which binds to the C-terminal hydrophobic PDK1-interacting fragment (PIF) domain found in PDK1 substrates. We find that Arabidopsis PDK1 interacts with PID, and that transphosphorylation by PDK1 increases PID autophosphorylation. We show that a PID activation loop serine is required for PDK1-dependent PID phosphorylation. This activation is rapid and requires the PIF domain. Cell extracts from flowers and seedling shoots dramatically increase PID phosphorylation in a tissue-specific manner. A PID protein variant in which the PIF domain was mutated failed to be activated by the seedling shoot extracts. PID immunoprecipitated from Arabidopsis cells in which PDK1 expression was inhibited by RNAi showed a dramatic reduction in transphosphorylation of myelin basic protein substrate. These results indicate that AtPDK1 is a potent enhancer of PID activity and provide evidence that phospholipid signaling may play a role in the signaling processes controlling polar auxin transport.
丝氨酸 - 苏氨酸蛋白激酶PID（PINOID）的活性与生长素运输促进因子膜相关PINFORMED（PIN）家族的不对称定位有关。然而，PID调节PIN蛋白分布的方式尚不清楚。我们使用重组PID蛋白在体外剖析PID活性的调节。我们证明，PID分子内自磷酸化是PID磷酸化外源底物能力所必需的。类似PID的哺乳动物AGC激酶在由磷脂相关激酶3 - 磷酸肌醇依赖性蛋白激酶1（PDK1）引发的磷酸化级联反应中起作用，PDK1与在PDK1底物中发现的C末端疏水PDK1相互作用片段（PIF）结构域结合。我们发现拟南芥PDK1与PID相互作用，并且PDK1的转磷酸化增加了PID的自磷酸化。我们表明，PDK1依赖性PID磷酸化需要PID激活环丝氨酸。这种激活是快速的，并且需要PIF结构域。花和幼苗芽的细胞提取物以组织特异性方式显著增加PID磷酸化。PIF结构域发生突变的PID蛋白变体未能被幼苗芽提取物激活。从通过RNAi抑制PDK1表达的拟南芥细胞中免疫沉淀的PID显示髓鞘碱性蛋白底物的转磷酸化显著降低。这些结果表明AtPDK1是PID活性的有效增强剂，并提供了磷脂信号可能在控制极性生长素运输的信号传导过程中起作用的证据。
