Actin-binding and Cell Proliferation Activities of Angiomotin Family Members Are Regulated by Hippo Pathway-mediated Phosphorylation
Actin-binding and Cell Proliferation Activities of Angiomotin Family Members Are Regulated by Hippo Pathway-mediated Phosphorylation
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DOI：
10.1074/jbc.m113.527598
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发表时间：
2013-12-27
影响因子：
4.8
通讯作者：
Hong, Wanjin
中科院分区：
文献类型：
--
作者：
Chan, Siew Wee;Lim, Chun Jye;Hong, Wanjin
Hippo通路是否有YAP和TAZ以外的下游靶点尚不清楚。在本报告中，我们已经确定了血管运动素（Amot）家族成员作为Hippo核心激酶的新底物。Amot蛋白的n端区域包含一个保守的HXRXXS共识位点，用于lats1 /2介导的磷酸化。磷酸化特异性抗体表明，Hippo核心激酶可以介导内源性和外源性Amot家族成员的磷酸化。敲低LATS1和LATS2内源性降低了磷酸化特异性抗体检测到的Amots的磷酸化。Amot和AmotL2中HXRXXS位点的丝氨酸突变为丙氨酸，证实该位点对Hippo核心激酶介导的磷酸化至关重要。野生型和非磷酸化Amot （Amot- s175a）靶向肌动蛋白丝，而磷酸化模拟Amot （Amot- s175d）无法定位肌动蛋白。las2过表达导致Amot与肌动蛋白分离，而Amot- s175a不分离。Amot的肌动蛋白结合位点图谱显示，Amot的丝氨酸175对其肌动蛋白结合活性起重要作用。Amot- s175a促进细胞增殖，而Amot和Amot- s175d抑制细胞增殖。这些结果共同表明，Hippo通路通过直接磷酸化负向调节Amot家族成员的肌动蛋白结合活性。
Whether the Hippo pathway has downstream targets other than YAP and TAZ is unknown. In this report, we have identified angiomotin (Amot) family members as novel substrates of Hippo core kinases. The N-terminal regions of Amot proteins contain a conserved HXRXXS consensus site for LATS1/2-mediated phosphorylation. Phospho-specific antibodies showed that Hippo core kinases could mediate phosphorylation of endogenous as well as exogenous Amot family members. Knockdown of LATS1 and LATS2 endogenously reduced the phosphorylation of Amots detected by the phospho-specific antibodies. Mutation of the serine to alanine within this HXRXXS site in Amot and AmotL2 established that this site was essential for Hippo core kinase-mediated phosphorylation. Wild-type and non-phosphorylated Amot (Amot-S175A) were targeted to actin filaments, whereas phospho-mimic Amot (Amot-S175D) failed to be localized with actin. Overexpression of LATS2 caused dissociation of Amot from actin but not Amot-S175A. Mapping of the actin-binding site of Amot showed that serine 175 of Amot was important for the actin-binding activity. Amot-S175A promoted, whereas Amot and Amot-S175D inhibited, cell proliferation. These results collectively suggest that the Hippo pathway negatively regulates the actin-binding activity of Amot family members through direct phosphorylation.