Molecular analysis reveals localization of Saccharomyces cerevisiae protein kinase C to sites of polarized growth and Pkc1p targeting to the nucleus and mitotic spindle
Molecular analysis reveals localization of Saccharomyces cerevisiae protein kinase C to sites of polarized growth and Pkc1p targeting to the nucleus and mitotic spindle
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DOI：
10.1128/ec.4.1.36-45.2005
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发表时间：
2005-01-01
期刊：
影响因子：
--
通讯作者：
Cyert, MS
中科院分区：
文献类型：
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作者：
Denis, V;Cyert, MS
蛋白激酶C（PKC）的催化活性和细胞内定位在体内都受到高度调节。该激酶家族包含保守的调节基序，即，C1、C2和HR1结构域，将PKC亚型靶向特定的亚细胞区室并在空间上限制其活性。酿酒酵母含有一个单一的PKC同工酶，Pkc1p，其中包含所有的哺乳动物PKC中发现的调控基序。Pkc1p定位于极化生长的位点，与其维持细胞完整性的主要功能一致。我们解剖的分子基础Pkc1p本地化的表达，其每个域单独和组合的绿色荧光蛋白融合。我们发现，Rho1p结合结构域，HR1和C1，负责针对海带芽尖和细胞周边，分别。我们表明，Pkc1p的活性是需要其正常本地化的芽颈，这也取决于完整的隔蛋白环。此外，我们首次表明，酵母蛋白激酶C可以积累在细胞核中，我们确定了核出口信号以及核定位信号内的Pkc1p序列。因此，我们建议，Pkc1p穿梭进出细胞核，因此有机会获得核底物。令人惊讶的是，我们发现，删除的HR1域的结果在Pkc1p本地化的有丝分裂纺锤体和C2结构域是负责这个目标。这种新的核和纺锤体定位的Pkc1p可能提供了一个分子的解释，以前的观察表明Pkc1p在调节微管功能的作用。
The catalytic activity and intracellular localization of protein kinase C (PKC) are both highly regulated in vivo. This family of kinases contains conserved regulatory motifs, i.e., the C1, C2, and HR1 domains, which target PKC isoforms to specific subcellular compartments and restrict their activity spatially. Saccharomyces cerevisiae contains a single PKC isozyme, Pkc1p, which contains all of the regulatory motifs found in mammalian PKCs. Pkc1p localizes to sites of polarized growth, consistent with its main function in maintaining cell integrity. We dissected the molecular basis of Pkc1p localization by expressing each of its domains individually and in combinations as green fluorescent protein fusions. We find that the Rho1p-binding domains, HR1 and C1, are responsible for targeting Kelp to the bud tip and cell periphery, respectively. We demonstrate that Pkc1p activity is required for its normal localization to the bud neck, which also depends on the integrity of the septin ring. In addition, we show for the first time that yeast protein kinase C can accumulate in the nucleus, and we identify a nuclear exit signal as well as nuclear localization signals within the Pkc1p sequence. Thus, we propose that Pkc1p shuttles in and out of the nucleus and consequently has access to nuclear substrates. Surprisingly, we find that deletion of the HR1 domain results in Pkc1p localization to the mitotic spindle and that the C2 domain is responsible for this targeting. This novel nuclear and spindle localization of Pkc1p may provide a molecular explanation for previous observations that suggest a role for Pkc1p in regulating microtubule function.