Department of Physiology and Biophysics, School of Medicine, Stony Brook University, Stony Brook, NY 11794-8661, USA.
BMC Biochem. 2010 Oct 27;11:42. doi: 10.1186/1471-2091-11-42.
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.
调节非受体酪氨酸激酶 Ack1（激活的 Cdc42 相关激酶）活性的机制尚未完全阐明。Ack1 的氨基末端区域预计包含一个无菌α基序（SAM）结构域。SAM 结构域具有共同的折叠结构，并在各种蛋白质中介导蛋白质-蛋白质相互作用。在这里，我们研究了 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 blot 技术表明，缺乏 N 端的 Ack1 缺失突变体在细胞中显示出明显减少的自身磷酸化。包含 N 端和激酶结构域（NKD）的最小构建体可自身磷酸化，而单独的激酶结构域（KD）则不能。当在哺乳动物细胞中表达时，NKD 定位于质膜，而 KD 则显示出更弥散的细胞质定位。免疫共沉淀实验表明全长 Ack1 与 NKD 之间的相互作用强于全长 Ack1 与 KD 之间的相互作用，表明 N 端对于 Ack1 二聚化很重要。在脂质囊泡表面增加纯化的 Ack1 激酶结构域的局部浓度可刺激自身磷酸化和催化活性，这与活性需要二聚化和转磷酸化一致。
综上所述，数据表明 Ack1 的 N 端促进了膜定位和二聚化，从而允许自身磷酸化。
