Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase.
Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase.
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DOI：
10.1042/bj20140889
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发表时间：
2014-11-01
期刊：
影响因子：
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通讯作者：
Alessi DR
中科院分区：
文献类型：
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作者：
Bago R;Malik N;Munson MJ;Prescott AR;Davies P;Sommer E;Shpiro N;Ward R;Cross D;Ganley IG;Alessi DR
Vps 34（vacuolar protein sorting 34）III类PI 3 K（phosphoinositide 3-kinase）使内体膜上的PtdIns（phosphatidylinositol）磷酸化，产生PtdIns（3）P，PtdIns（3）P通过募集具有PtdIns（3）P结合PX（phox同源性）和FYVE结构域的蛋白亚组的能力来调节膜运输过程。在本研究中，我们描述了一种高度选择性和有效的Vps 34抑制剂，称为VPS 34-IN 1，在体外以25 nM IC 50抑制Vps 34，但不显著抑制340种蛋白激酶或25种脂质激酶的活性，包括I类和II类PI 3 K的所有亚型。给予细胞VPS 34-IN 1可诱导特异性PtdIns（3）P结合探针在1分钟内从核内体膜上快速扩散，且呈剂量依赖性，而不影响I类PI 3 K调节Akt的能力。此外，我们探讨了SGK 3（血清和糖皮质激素调节激酶-3），唯一已知通过其N-末端PX结构域与PtdIns（3）P特异性相互作用的蛋白激酶，是否可能受Vps 34控制。破坏PtdIns（3）P结合的突变通过抑制T环[PDK 1（磷酸肌醇依赖性激酶1）位点]和疏水基序（哺乳动物雷帕霉素靶位点）残基的磷酸化来消除SGK 3激酶活性。VPS 34-IN 1在1分钟内诱导SGK 3磷酸化快速丧失约50-60%。VPS 34-IN 1不抑制不具有PtdIns（3）P-结合PX结构域的SGK 2同种型的活性。此外，不抑制Vps 34的I类PI 3 K抑制剂（GDC-0941和BKM 120）抑制SGK 3活性约40%。将VPS 34-IN 1和GDC-0941组合使SGK 3活性降低约80- 90%。这些数据表明SGK 3磷酸化，因此活性由两个PtdIns（3）P库控制。第一个是通过内体处的Vps 34磷酸化PtdIns产生的。第二种是由于I类PI 3 K产物PtdIns（3，4，5）P3通过PtdIns 5-磷酸酶[SHIP 1/2（含Src同源2-结构域的肌醇磷酸酶1/2）]和PtdIns 4-磷酸酶[INPP 4 B（肌醇多磷酸4-磷酸酶II型）]的顺序作用转化为PtdIns（3）P。VPS 34-IN 1将是一个有用的探针来描绘VPS 34的生理作用。监测SGK 3磷酸化和活性可用作Vps 34活性的生物标志物，其方式与Akt用于探测细胞I类PI 3 K活性的方式类似。组合I类（GDC-0941）和III类（VPS 34-IN 1）PI 3 K抑制剂可用作更好地分析难以捉摸的II类PI 3 K的作用和调节的策略。我们表征VPS 34-IN，一种有效的和选择性的III类Vps 34 PI 3 K抑制剂。使用VPS 34-IN 1，我们证明由Vps 34产生的PtdIns（3）P控制SGK 3蛋白激酶的磷酸化和活性。
The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates PtdIns (phosphatidylinositol) at endosomal membranes to generate PtdIns(3)P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing PtdIns(3)P-binding PX (phox homology) and FYVE domains. In the present study, we describe a highly selective and potent inhibitor of Vps34, termed VPS34-IN1, that inhibits Vps34 with 25 nM IC50 in vitro, but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific PtdIns(3)P-binding probe from endosome membranes, within 1 min, without affecting the ability of class I PI3K to regulate Akt. Moreover, we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3), the only protein kinase known to interact specifically with PtdIns(3)P via its N-terminal PX domain, might be controlled by Vps34. Mutations disrupting PtdIns(3)P binding ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop [PDK1 (phosphoinositide-dependent kinase 1) site] and hydrophobic motif (mammalian target of rapamycin site) residues. VPS34-IN1 induced a rapid ~50–60% loss of SGK3 phosphorylation within 1 min. VPS34-IN1 did not inhibit activity of the SGK2 isoform that does not possess a PtdIns(3)P-binding PX domain. Furthermore, class I PI3K inhibitors (GDC-0941 and BKM120) that do not inhibit Vps34 suppressed SGK3 activity by ~40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ~80–90%. These data suggest SGK3 phosphorylation and hence activity is controlled by two pools of PtdIns(3)P. The first is produced through phosphorylation of PtdIns by Vps34 at the endosome. The second is due to the conversion of class I PI3K product, PtdIns(3,4,5)P3 into PtdIns(3)P, via the sequential actions of the PtdIns 5-phosphatases [SHIP1/2 (Src homology 2-domain-containing inositol phosphatase 1/2)] and PtdIns 4-phosphatase [INPP4B (inositol polyphosphate 4-phosphatase type II)]. VPS34-IN1 will be a useful probe to delineate physiological roles of the Vps34. Monitoring SGK3 phosphorylation and activity could be employed as a biomarker of Vps34 activity, in an analogous manner by which Akt is used to probe cellular class I PI3K activity. Combining class I (GDC-0941) and class III (VPS34-IN1) PI3K inhibitors could be used as a strategy to better analyse the roles and regulation of the elusive class II PI3K. We characterize VPS34-IN, a potent and selective inhibitor of class III Vps34 PI3K. Using VPS34-IN1, we demonstrate that PtdIns(3)P, produced by Vps34 controls phosphorylation and activity of the SGK3 protein kinase.