Regulation of Ack1 localization and activity by the amino-terminal SAM domain.
Regulation of Ack1 localization and activity by the amino-terminal SAM domain.
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DOI：
10.1186/1471-2091-11-42
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发表时间：
2010-10-27
期刊：
影响因子：
--
通讯作者：
Miller WT
中科院分区：
文献类型：
--
作者：
Prieto-Echagüe V;Gucwa A;Brown DA;Miller WT
调节非受体酪氨酸激酶Ack1（活化的cdc42相关激酶）活性的机制尚不清楚。Ack1的氨基末端区域预计包含一个不育α基序（SAM）结构域。SAM结构域具有共同的折叠，并介导多种蛋白质的蛋白质相互作用。在这里，我们讨论了Ack1 SAM结构域在激酶活性中的重要性。我们使用免疫荧光和Western blotting显示，缺乏n端Ack1缺失突变体在细胞中表现出显著降低的自磷酸化。由n端和激酶结构域（NKD）组成的最小结构被自磷酸化，而单独的激酶结构域（KD）没有被自磷酸化。当在哺乳动物细胞中表达时，NKD定位于质膜，而KD则显示出更分散的细胞质定位。共免疫沉淀实验显示全长Ack1与NKD之间的相互作用比全长Ack1与KD之间的相互作用更强，表明n端对Ack1二聚化很重要。增加脂质囊泡表面纯化Ack1激酶结构域的局部浓度可刺激自磷酸化和催化活性，这与二聚化和反式磷酸化对活性的要求一致。总的来说，这些数据表明Ack1的n端促进膜定位和二聚化，从而允许自磷酸化。
The mechanisms that regulate the activity of the nonreceptor tyrosine kinase Ack1 (activated Cdc42-associated kinase) are poorly understood. The amino-terminal region of Ack1 is predicted to contain a sterile alpha motif (SAM) domain. SAM domains share a common fold and mediate protein-protein interactions in a wide variety of proteins. Here, we addressed the importance of the Ack1 SAM domain in kinase activity. We used immunofluorescence and Western blotting to show that Ack1 deletion mutants lacking the N-terminus displayed significantly reduced autophosphorylation in cells. A minimal construct comprising the N-terminus and kinase domain (NKD) was autophosphorylated, while the kinase domain alone (KD) was not. When expressed in mammalian cells, NKD localized to the plasma membrane, while KD showed a more diffuse cytosolic localization. Co-immunoprecipitation experiments showed a stronger interaction between full length Ack1 and NKD than between full length Ack1 and KD, indicating that the N-terminus was important for Ack1 dimerization. Increasing the local concentration of purified Ack1 kinase domain at the surface of lipid vesicles stimulated autophosphorylation and catalytic activity, consistent with a requirement for dimerization and trans-phosphorylation for activity. Collectively, the data suggest that the N-terminus of Ack1 promotes membrane localization and dimerization to allow for autophosphorylation.
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影响因子：
11.2
作者：
Hoare, Sarasiia;Hoare, Kishalay;May, W. Stratford, Jr.
通讯作者：
May, W. Stratford, Jr.
DOI：
10.1006/bbrc.2001.5004
发表时间：
2001-06-08
影响因子：
3.1
作者：
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KNIGHT, WB
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64.8
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通讯作者：
LIM, L
影响因子：
13.8
作者：
Kim, CA;Bowie, JU
通讯作者：
Bowie, JU