IDENTIFYING SUBSTRATES OF CYCLIN DEPENDENT KINASES BY SUBSTRATE TRAP & PURIFICAT

我们正在试图了解细胞周期蛋白和细胞周期蛋白依赖 有丝分裂中的蛋白激酶控制着细胞有丝分裂的过程。在之前的工作中，我们发现 一种名为Gin4的蛋白激酶在细胞进入有丝分裂时被激活 并且是适当地诱导特定有丝分裂事件所必需的 细胞周期蛋白Clb2。为了更多地了解Gin4在有丝分裂中的功能，我们 已经使用遗传学和生物化学的组合来识别 与Gin4功能相互作用的蛋白质。这两个都是 已有方法确定了Septin蛋白家族的成员为 在有丝分裂特异性激活Gin4中起关键作用。损失 产生一种与丢失非常相似的表型 Gin4的功能，并且Gin4的有丝分裂特异性激活是 依赖于Septin的功能。这些隔膜很可能会扮演一种 Gin4在有丝分裂特异性激活中的直接作用，因为它们 与Gin4紧密结合，并与Gin4在细胞内共同定位。 这些结果表明，Septins在早期起作用。 在胞质分裂发生之前的有丝分裂中，间隔蛋白可能扮演一个 在控制有丝分裂信号事件中的作用。细长的花蕾 对Septin突变的表型观察表明，Septins Clb2、NAP1和Gin4控制芽的途径中的功能 有丝分裂期间的生长。这种作用的进一步证据来自于 亲和层析实验，我们已经进行了 确定与Gin4激酶功能相互作用的蛋白质。 在这些实验中，我们纯化了一个全长的Gin4-GST融合蛋白 并将其偶联到珠子上，创建Gin4亲和柱。 Gin4亲和层析柱上装载了一种粗提物， 快速生长的酵母细胞，用缓冲液洗涤后的柱子 用盐的梯度洗脱。许多蛋白质结合在一起 Gin4亲和层析，但不与GST对照层析。开始，开始 对与Gin4结合的蛋白质进行表征，我们使用了MASS 光谱分析以确定洗脱出的主要蛋白质 亲和力列。我们发现其中一种蛋白质是NAP1，因为 在之前的实验中，我们展示了一个紧密的 以及Gin4和NAP1之间的特异性相互作用。另一个专业 蛋白质是Septin家族的成员，包括Cdc3，Cdc10， Cdc11、cdc12和一个新的由 酵母基因组测序计划，我们将其命名为Sep5(ORF 编号：YDL225W)。Western blotting证实CDC11与 柱子和它几乎被定量地从 当它经过Gin4-GST柱时提取。请注意，这些分隔素 共同洗脱，表明它们作为一个复合体相互关联 以同样的方式观察到了果蝇的隔膜蛋白。当一个 Dnap1菌株的粗提物被装载到Gin4亲和力上 专栏仍然发现Septins结合，表明NAP1是 Septin结合不需要。

We are seeking to understand how cyclins and cyclin-dependent kinases control the events of mitosis. In previous work, we found that a protein kinase called Gin4 is activated as cells enter mitosis and is required for the proper induction of specific mitotic events by the cyclin Clb2. To learn more about Gin4 function during mitosis, we have used a combination of genetics and biochemistry to identify proteins that functionally interact with Gin4. Both of these approaches have identified members of the septin family of proteins as playing a key role in the mitosis-specific activation of Gin4. Loss of septin function produces a phenotype that is very similar to loss of Gin4 function, and the mitosis-specific activation of Gin4 is dependent upon septin function. The septins are likely to play a direct role in the mitosis-specific activation of Gin4 because they bind tightly to Gin4 and are co-localized with Gin4 within the cell. These results demonstrate that the septins carry out functions early in mitosis before cytokinesis occurs, and that the septins may play a role in controlling mitotic signaling events. The elongated bud phenotype observed for septin mutations suggests that the septins function in the pathway used by Clb2, Nap1, and Gin4 to control bud growth during mitosis. Further evidence for such a role has come from affinity chromatography experiments that we have carried out to identify proteins that functionally interact with the Gin4 kinase. For these experiments, we purify a full length Gin4-GST fusion protein from bacteria and couple it to beads to create a Gin4 affinity column. The Gin4 affinity column is loaded with a crude extract made from rapidly growing yeast cells, and after washing with buffer the column is eluted with a gradient of salt. A number of proteins bind to the Gin4 affinity column, but not to a GST control column. To begin to characterize the proteins that bind to Gin4, we have used mass spectrometry to identify the major proteins that elute from the affinity column. We found that one of these proteins is Nap1, as expected from previous experiments in which we demonstrated a tight and specific interaction between Gin4 and Nap1. The other major proteins are members of the septin family, including Cdc3, Cdc10, Cdc11, Cdc12, and a new member of the septin family identified by the yeast genome sequencing project that we have named Sep5 (ORF designation YDL225W). Western blotting confirmed that Cdc11 binds to the column and that it is almost quantitativley depleted from the extract as it passes over the Gin4-GST column. Note that the septins co-elute, suggesting that they associate with each other as a complex in the same way that has been observed for Drosophila septins. When a crude extract from a Dnap1 strain is loaded onto a Gin4 affinity column the septins are still found to bind, demonstrating that Nap1 is not required for septin binding.