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Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation

机译：Akt通过磷酸化调节Rab27a结合蛋白JFC1的亚细胞定位

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Here, we show that the Rab27a-binding protein JFC1/ Slp1 (synaptotagmin-like protein) is regulated by Akt-mediated phosphorylation. Using the phosphatase and tensin homolog-null LNCaP cells and the phosphaticlylinositol 3-kinase inhibitor LY294002, we show that the phosphorylation of endogenous JFC1 is dependent on the phosphatidylinositol 3-kinase/Akt pathway. JFC1 was phosphorylated in cells expressing a constitutively active Akt, confirming that it is an Akt substrate in vivo. Direct phosphorylation of JFC1 by Akt was confirmed in vitro. Using microcapillary high-performance liquid chromatography tandem mass spectrometry, we identified five Akt-phosphorylation sites in JFC1. By mutagenesis analysis and subsequent immunoprecipitation (IP), we established that Akt phosphorylates JFC1 at serine 241. JFC1 and Rab27a colocalize in the proximity of the plasma membrane in LNCaP cells. The interaction was confirmed by IP analysis and was abolished by the point mutation W83S in JFC1. Phosphorylation did not alter the ability of JFC1 to bind to Rab27a. Instead, phosphorylation by Akt dramatically decreased when JFC1 was bound to Rab27a. Finally, we show that as a consequence of in vivo phosphorylation, JFC1 dissociates from the membrane, promoting JFC1 redistribution to the cytosol. Our results suggest that Akt regulates JFC1/Slpl function by phosphorylation and may have implications on Rab27a-containing vesicle secretion.
机译：在这里，我们显示Rab27a结合蛋白JFC1 / Slp1（突触素样蛋白）受Akt介导的磷酸化调节。使用磷酸酶和肌腱蛋白同源的LNCaP细胞和磷脂酰肌醇3-激酶抑制剂LY294002，我们表明内源性JFC1的磷酸化依赖于磷脂酰肌醇3-激酶/ Akt途径。 JFC1在表达组成性活性Akt的细胞中被磷酸化，证实它是体内的Akt底物。在体外证实了Akt对JFC1的直接磷酸化作用。使用微毛细管高效液相色谱串联质谱法，我们在JFC1中鉴定了五个Akt磷酸化位点。通过诱变分析和随后的免疫沉淀（IP），我们确定Akt在丝氨酸241处使JFC1磷酸化。JFC1和Rab27a共定位在LNCaP细胞的质膜附近。相互作用通过IP分析确认，并被JFC1中的点突变W83S取消。磷酸化并没有改变JFC1结合Rab27a的能力。相反，当JFC1与Rab27a结合时，Akt的磷酸化作用显着降低。最后，我们显示出体内磷酸化的结果，JFC1从膜上解离，从而促进JFC1重新分布到细胞质中。我们的结果表明，Akt通过磷酸化调节JFC1 / Slpl功能，并且可能对含Rab27a的囊泡分泌有影响。

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