The cysteine-rich sprouty translocation domain targets mitogen-activated protein kinase inhibitory proteins to phosphatidylinositol 4,5-bisphosphate in plasma membranes
The cysteine-rich sprouty translocation domain targets mitogen-activated protein kinase inhibitory proteins to phosphatidylinositol 4,5-bisphosphate in plasma membranes
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DOI：
10.1128/mcb.22.22.7953-7966.2002
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发表时间：
2002-11-01
影响因子：
5.3
通讯作者：
Guy, GR
中科院分区：
文献类型：
--
作者：
Lim, J;Yusoff, P;Guy, GR
Spry蛋白被发现是Ras/丝裂原活化蛋白激酶（MAPK）级联反应的抑制剂，而MAPK是多种酪氨酸受体激酶激活引发的发育过程的关键途径。在COS-1和Swiss 3T3细胞中，所有Spry同种异构体在激活后都会转移到质膜上，尤其是褶边。本研究表明，微量注射活性Rae诱导了Spry异构体的易位，这表明Spry易位域（SpryTD）的靶点位于活性Rac的下游。肌动蛋白聚合的靶向破坏表明，SpryTD靶点出现在细胞骨架重排的上游。越来越多的证据表明，磷脂酰肌醇4,5-二磷酸[PtdIns(4,5)P-2]可能是SpryTD的靶点。在囊泡结合试验中，人Spry2TD （hSpry2TD）与PtdIns(4,5)P-2结合。hSpry2TD与结合PtdIns(4,5)P-2的磷脂酶Cdelta的pleckstrin同源结构域共定位。在离子霉素处理的MDCK细胞中，或者通过过表达一种工程化的、绿色荧光蛋白标记的肌醇5-磷酸酶特异性地去磷酸化Ptdlns(4,5)P-2， hSpry2TD的质膜定位被消除。类似地，最近发现的一种新型Ras/MAPK抑制剂Spred含有保守的富含半胱氨酸的SpryTD，它也易位到外周膜并与Ptdlns(4,5)P-2结合。通过对Spry和Spred蛋白的比对，我们发现了一个易位缺陷点突变体hSpry2 D252。hSpry2靶向Ptdlns(4,5)P-2被证明是下调Ras/MAPK信号的必要条件。
Sprouty (Spry) proteins have been revealed as inhibitors of the Ras/mitogen-activated protein kinase (MAPK) cascade, a pathway crucial for developmental processes initiated by activation of various receptor tyrosine kinases. In COS-1 and Swiss 3T3 cells, all Spry isoforms translocate to the plasma membrane, notably ruffles, following activation. Here we show that microinjection of active Rae induced the translocation of Spry isoforms, indicating that the target of the Spry translocation domain (SpryTD) is downstream of active Rac. Targeted disruption of actin polymerization revealed that the SpryTD target appeared upstream of cytoskeletal rearrangements. Accumulated evidence indicated that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2] is the likely SpryTD target. Human Spry2TD (hSpry2TD) binds to PtdIns(4,5)P-2 in vesicle-binding assays. hSpry2TD colocalizes with the pleckstrin homology domain of phospholipase Cdelta, which binds PtdIns(4,5)P-2. The plasma membrane localization of hSpry2TD was abolished in ionomycin-treated MDCK cells or when Ptdlns(4,5)P-2 was specifically dephosphorylated by overexpression of an engineered, green fluorescent protein-tagged inositol 5-phosphatase. Similarly, Spred, a novel Ras/MAPK inhibitor recently found to contain the conserved cysteine-rich SpryTD, also translocated to peripheral membranes and bound to Ptdlns(4,5)P-2. Alignment of the Spry and Spred proteins led us to identify a translocation-defective point mutant, hSpry2 D252. Targeting of hSpry2 to Ptdlns(4,5)P-2 was shown to be essential for the down-regulation of Ras/MAPK signaling.