A Serine/Threonine Kinase 16-Based Phospho-Proteomics Screen Identifies WD Repeat Protein-1 As A Regulator Of Constitutive Secretion.
A Serine/Threonine Kinase 16-Based Phospho-Proteomics Screen Identifies WD Repeat Protein-1 As A Regulator Of Constitutive Secretion.
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DOI：
10.1038/s41598-018-31426-1
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发表时间：
2018-08-29
影响因子：
4.6
通讯作者：
Tuma PL
中科院分区：
文献类型：
--
作者：
López-Coral A;Striz AC;Tuma PL
极化肝细胞的质膜在功能上分为两个区域：顶侧和基底侧。我们的重点是定义极化蛋白分选新合成的膜和分泌蛋白在WIF-B细胞，极化肝细胞的一个很好的模型系统的分子基础。我们确定MAL 2（髓鞘和淋巴细胞蛋白2）及其结合伴侣丝氨酸/苏氨酸激酶16（STK 16）调节基底外侧组成性分泌。因为STK 16是组成型活性激酶，我们推断组成型磷酸化底物必须参与组成型分泌。为了鉴定STK 16底物或其他调节组成性分泌的蛋白质，我们采用了蛋白质组学方法。在2D凝胶上分离来自表达野生型或激酶死亡（E202 A）STK 16的细胞的核后上清液，并用针对磷酸丝氨酸/苏氨酸残基的抗体进行免疫印迹。从E202 A表达细胞中鉴定出16个斑点，这些斑点可重复地显示免疫反应性降低。从这些斑点中，鉴定出28种蛋白质为可能的STK 16底物。在这28种可能的底物中，其中25%编码预测的STK 16磷酸化共有位点，其中WD重复包含蛋白-1（WDR 1）编码两个这样的位点。基于这一发现以及肝分泌需要肌动蛋白重塑的发现，我们进一步证实了WDR 1是一种调节分泌的磷蛋白。
The plasma membrane of polarized hepatocytes is functionally divided into two domains: the apical and basolateral. Our focus is to define the molecular basis of polarized protein sorting of newly-synthesized membrane and secretory proteins in WIF-B cells, an excellent model system for polarized hepatocytes. We determined that MAL2 (myelin and lymphocyte protein 2) and its binding partner, serine/threonine kinase 16 (STK16) regulate basolateral constitutive secretion. Because STK16 is a constitutively active kinase, we reasoned that constitutively phosphorylated substrates must participate in constitutive secretion. To identify either STK16 substrates or other proteins that regulate constitutive secretion, we took a proteomics approach. Post-nuclear supernatants from cells expressing wild type or a kinase-dead (E202A) STK16 were separated on 2D gels and immunoblotted with antibodies against phospho-serine/threonine residues. Sixteen spots were identified from E202A-expressing cells that reproducibly displayed decreased immunoreactivity. From these spots, 28 proteins were identified as possible STK16 substrates. Out of these 28 possible substrates, 25% of them encode predicted STK16 phosphorylation consensus sites, with WD repeat containing protein-1 (WDR1) encoding two such sites. Based on this finding and on the finding that actin remodeling is required for hepatic secretion, we further confirmed that WDR1 is a phosphoprotein that regulates secretion.
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DOI：
10.1016/j.str.2007.10.026
发表时间：
2008-01
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Eswaran J;Bernad A;Ligos JM;Guinea B;Debreczeni JE;Sobott F;Parker SA;Najmanovich R;Turk BE;Knapp S
通讯作者：
Knapp S
DOI：
10.1038/nsb0195-28
发表时间：
1995-01-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
AMBERG, DC;BASART, E;BOTSTEIN, D
通讯作者：
BOTSTEIN, D
WIF-B细胞：用于研究肝细胞极性的体外模型。
影响因子：
7.8
作者：
Ihrke, G;Neufeld, E B;Meads, T;Shanks, M R;Cassio, D;Laurent, M;Schroer, T A;Pagano, R E;Hubbard, A L
通讯作者：
Hubbard, A L
影响因子：
4.8
作者：
Garuti, R;Jones, C;Hobbs, HH
通讯作者：
Hobbs, HH
影响因子：
13.5
作者：
Hayes, JH;Soroka, CJ;Boyer, JL
通讯作者：
Boyer, JL