Multisite phosphorylation of oxysterol-binding protein regulates sterol binding and activation of sphingomyelin synthesis.
Multisite phosphorylation of oxysterol-binding protein regulates sterol binding and activation of sphingomyelin synthesis.
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DOI：
10.1091/mbc.e12-04-0283
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发表时间：
2012-09
影响因子：
3.3
通讯作者：
Ridgway ND
中科院分区：
文献类型：
--
作者：
Goto A;Liu X;Robinson CA;Ridgway ND
氧固醇结合蛋白(OSBP)参与内质网(ER)-高尔基体固醇的转运，但其活性如何调控尚不清楚。OSBP在多个位点被磷酸化，其中两个位点调节内质网中的甾醇结合和与囊泡相关的膜蛋白相关蛋白A的相互作用。磷酸化不影响磷脂酰肌醇4-磷酸的结合，磷脂酰肌醇4-磷酸结合可以作为一种反配体促进高尔基体中固醇的释放。氧固醇结合蛋白(OSBP)的内质网(ER)-高尔基体醇转移活性调节鞘磷脂(SM)的合成，以及高尔基体胆固醇外流后的途径。OSBP的磷酸化和ER-高尔基体定位相关，表明这种修饰调节了转移活性的方向性和/或特异性。在本文中，我们报道了两个富含丝氨酸的基序S381-S391(第1位)和S192、S195、S200(第2位)上的磷酸化特异性地控制了内质网的OSBP活性。模拟SM/胆固醇敏感的磷酸化位点1(OSBP-S5E)在体外增加了胆固醇和25-羟基胆固醇的结合能力，并增加了从脂质体中提取胆固醇的能力，但降低了转移活性。磷脂酰肌醇4-磷酸(PI(4)P)和胆固醇在OSBP上竞争一个共同的结合部位，而PI(4)P的直接结合不受位点1磷酸化的影响。在OSBP缺乏的CHO细胞中，单个位点1和位点2的磷酸化突变体支持氧固醇激活SM的合成。然而，一个双位点1/2突变体(OSBP-S381A/S3D)缺乏这种活性，并与囊泡相关膜蛋白相关蛋白A(VAP-A)在一个崩溃的ER网络中组成共定位。本研究确定了内质网中OSBP对固醇和VAP-A结合的磷酸化调节，PI(4)P是高尔基体中可以交换为固醇的替代配体。
Oxysterol-binding protein (OSBP) is involved in endoplasmic reticulum (ER)-Golgi sterol transport, but how its activity is regulated is unknown. OSBP is phosphorylated at multiple sites, two of which regulate sterol binding and interaction with vesicle-associated, membrane protein–associated protein A in the ER. Phosphorylation does not affect phosphatidylinositol 4-phosphate binding, which could serve as a counter-ligand to facilitate sterol release in the Golgi. The endoplasmic reticulum (ER)-Golgi sterol transfer activity of oxysterol-binding protein (OSBP) regulates sphingomyelin (SM) synthesis, as well as post-Golgi cholesterol efflux pathways. The phosphorylation and ER-Golgi localization of OSBP are correlated, suggesting this modification regulates the directionality and/or specificity of transfer activity. In this paper, we report that phosphorylation on two serine-rich motifs, S381-S391 (site 1) and S192, S195, S200 (site 2), specifically controls OSBP activity at the ER. A phosphomimetic of the SM/cholesterol-sensitive phosphorylation site 1 (OSBP-S5E) had increased in vitro cholesterol and 25-hydroxycholesterol–binding capacity, and cholesterol extraction from liposomes, but reduced transfer activity. Phosphatidylinositol 4-phosphate (PI(4)P) and cholesterol competed for a common binding site on OSBP; however, direct binding of PI(4)P was not affected by site 1 phosphorylation. Individual site 1 and site 2 phosphomutants supported oxysterol activation of SM synthesis in OSBP-deficient CHO cells. However, a double site1/2 mutant (OSBP-S381A/S3D) was deficient in this activity and was constitutively colocalized with vesicle-associated membrane protein–associated protein A (VAP-A) in a collapsed ER network. This study identifies phosphorylation regulation of sterol and VAP-A binding by OSBP in the ER, and PI(4)P as an alternate ligand that could be exchanged for sterol in the Golgi apparatus.