The NDR/LATS family kinase Cbk1 directly controls transcriptional asymmetry.
The NDR/LATS family kinase Cbk1 directly controls transcriptional asymmetry.
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DOI：
10.1371/journal.pbio.0060203
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发表时间：
2008-08-19
期刊：
影响因子：
9.8
通讯作者：
Weiss, Eric L.
中科院分区：
文献类型：
--
作者：
Mazanka, Emily;Alexander, Jess;Yeh, Brian J.;Charoenpong, Patrick;Lowery, Drew M.;Yaffe, Michael;Weiss, Eric L.
细胞命运可以通过基因表达调节剂的不对称分离来确定。在芽殖酵母酿酒酵母（Saccharomyces cerevisiae）中，转录因子Ace 2在子细胞核中特异性地积累，在那里它驱动在母细胞中不表达的基因的转录。NDR/LATS家族蛋白激酶Cbk 1是Ace 2分离和功能所必需的。使用肽扫描阵列，我们确定了Cbk 1的磷酸化共有基序，这是该家族酶的第一个无偏方法，表明它是一种嗜碱性激酶，对组氨酸−5的磷酸化位点具有不寻常的偏好。我们发现Cbk 1磷酸化了Ace 2中的这些位点，这些修饰对Ace 2的分配和功能至关重要。使用标记有GFP变体的蛋白质，我们发现Ace 2从各向同性分布移动到子细胞核定位，早在胞质分裂之前，并且细胞核必须进入子细胞以发生Ace 2积累。我们发现，与Ace 2不同，Cbk 1仅限于子细胞。通过体内和体外实验，我们发现两个关键的Cbk 1磷酸化阻断了Ace 2与核输出机制的相互作用，而第三个远端修饰最有可能增加转录因子的活性。我们的研究结果表明，Gbk 1直接控制Ace 2，通过三个磷酸化位点调节转录因子的活性以及与核输出机制的相互作用。此外，Cbk 1表现出一种新的特异性，可能是保守的相关激酶从酵母到后生动物。cbk 1在功能上仅限于子细胞，不能从女儿扩散到母亲。除了提供Ace 2分离的机制外，这些发现还表明，通过调节蛋白激酶的空间分离，各向同性分布的细胞命运决定子可以在胞质连续细胞中不对称分配。细胞可以通过分离分子来分化，这些分子将特定基因组的表达导向分裂产生的两个细胞之一。这通常通过这些分子的直接机械运动或不对称锚定发生，这些分子在分裂后作用以影响基因表达。在这项研究中，我们定义了一个不同的机制，芽殖酵母转录调节Ace 2是不对称分配。我们发现，Ace 2移动从均匀分布到强大的积累在女儿的细胞核，而母亲和女儿的细胞仍然是连接的，并直接控制这种分离的酶Cbk 1通过连接磷酸盐的特定位点Ace 2。我们还表明，Cbk 1是限制在子细胞。使用生物化学和活细胞实验，我们表明，Cbk 1介导的修改激活Ace 2和阻断其与核输出机制的相互作用，将其困在子细胞核。除了证明Cbk 1与多细胞生物中的相关酶具有显著的生化相似性外，我们的分析还表明，通过局部修饰酶的直接作用，可以使均匀分布的基因表达调节因子在连接的细胞中不对称地活跃。一种保守的蛋白激酶，Cbk-1，通过直接阻断转录因子Ace 2的核输出，在胞质连接的细胞中产生不同的基因表达程序。
Cell fate can be determined by asymmetric segregation of gene expression regulators. In the budding yeast Saccharomyces cerevisiae, the transcription factor Ace2 accumulates specifically in the daughter cell nucleus, where it drives transcription of genes that are not expressed in the mother cell. The NDR/LATS family protein kinase Cbk1 is required for Ace2 segregation and function. Using peptide scanning arrays, we determined Cbk1′s phosphorylation consensus motif, the first such unbiased approach for an enzyme of this family, showing that it is a basophilic kinase with an unusual preference for histidine −5 to the phosphorylation site. We found that Cbk1 phosphorylates such sites in Ace2, and that these modifications are critical for Ace2′s partitioning and function. Using proteins marked with GFP variants, we found that Ace2 moves from isotropic distribution to the daughter cell nuclear localization, well before cytokinesis, and that the nucleus must enter the daughter cell for Ace2 accumulation to occur. We found that Cbk1, unlike Ace2, is restricted to the daughter cell. Using both in vivo and in vitro assays, we found that two critical Cbk1 phosphorylations block Ace2′s interaction with nuclear export machinery, while a third distal modification most likely acts to increase the transcription factor's activity. Our findings show that Cbk1 directly controls Ace2, regulating the transcription factor's activity and interaction with nuclear export machinery through three phosphorylation sites. Furthermore, Cbk1 exhibits a novel specificity that is likely conserved among related kinases from yeast to metazoans. Cbk1 is functionally restricted to the daughter cell, and cannot diffuse from the daughter to the mother. In addition to providing a mechanism for Ace2 segregation, these findings show that an isotropically distributed cell fate determinant can be asymmetrically partitioned in cytoplasmically contiguous cells through spatial segregation of a regulating protein kinase. Cells can differentiate by segregating molecules that direct expression of specific sets of genes to one of the two cells produced by division. This generally occurs by direct mechanical movement or asymmetric anchoring of these molecules, which act after division to influence gene expression. In this study, we define a different mechanism by which the budding yeast transcription regulator Ace2 is asymmetrically partitioned. We show that Ace2 moves from uniform distribution to strong accumulation in the daughter nucleus while mother and daughter cells are still connected, and that the enzyme Cbk1 directly controls this segregation by attaching phosphate to specific sites on Ace2. We also demonstrate that Cbk1 is restricted to the daughter cell. Using both biochemical and live-cell experiments, we show that the Cbk1-mediated modifications activate Ace2 and block its interaction with nuclear export machinery, trapping it in the daughter cell nucleus. In addition to demonstrating Cbk1′s remarkable biochemical similarity to related enzymes in multicellular organisms, our analysis shows that a uniformly distributed regulator of gene expression can be made asymmetrically active in connected cells through the direct action of a localized modifying enzyme. A conserved protein kinase, Cbk-1, produces different gene expression programs in cytoplasmically connected cells by directly blocking nuclear export of the transcription factor Ace2.
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