The yeast carboxyl-terminal repeat domain kinase CTDK-I is a divergent cyclin-cyclin-dependent kinase complex.

Saccharomyces cerevisiae CTDK-I is a protein kinase complex that specifically and efficiently hyperphosphorylates the carboxyl-terminal repeat domain (CTD) of RNA polymerase II and is composed of three subunits of 58, 38, and 32 kDa. The kinase is essential in vivo for normal phosphorylation of the CTD and for normal growth and differentiation. We have now cloned the genes for the two smaller kinase subunits, CTK2 and CTK3, and found that they form a unique, divergent cyclin-cyclin-dependent kinase complex with the previously characterized largest subunit protein CTK1, a cyclin-dependent kinase homolog. The CTK2 gene encodes a cyclin-related protein with limited homology to cyclin C, while CTK3 shows no similarity to other known proteins. Copurification of the three gene products with each other and CTDK-I activity by means of conventional chromatography and antibody affinity columns has verified their participation in the complex in vitro. In addition, null mutations of each of the genes and all combinations thereof conferred very similar growth-impaired, cold-sensitive phenotypes, consistent with their involvement in the same function in vivo. These characterizations and the availability of all of the genes encoding CTDK-I and reagents derivable from them will facilitate investigations into CTD phosphorylation and its functional consequences both in vivo and in vitro.     

酵母RNA聚合酶II羧基端结构域激酶CTDK-I及其亚基的结构与功能

RNA聚合酶Ⅱ最大亚基Rpb1的羧基端结构域（carboxyl-terminal repeat domain,CTD）是RNA聚合酶Ⅱ发挥转录延伸功能所必需的,对其执行精确的转录调节功能至关重要。酵母细胞周期蛋白依赖性激酶CTDK-I（carboxyl-terminal repeat domain kinase,CTDK-I）由CTK1、CTK2和CTK3组成,作用于RNA聚合酶Ⅱ羧基端结构域,动态磷酸化CTD的七肽重复序列（YSPTSPS）来调控转录和翻译。酵母中的特异性蛋白CTK3与特殊的细胞周期蛋白CTK2结合形成异二聚体,再与CTDK-I的催化亚基CTK1结合以调节其活性。CTK1作为细胞周期蛋白CDK（cyclin dependent kinase,CDK）的同源蛋白,其结构与功能的研究可拓展人们对CDK蛋白家族的认识;CTK2-CTK3复合物对CTK1调控机制的研究也可为细胞周期蛋白抑制剂的研发提供新的思路。本文简述了酵母CTDK-I的功能特点及其亚基的结构与功能以及亚基间的相互作用,并展望了CTDK-I复合物的研究前景。     

Involvement of p120 carboxy-terminal domain in cadherin trafficking

P120 plays an essential role in cadherin turnover. The molecular mechanism involved, however, remains only partially understood. Here, using a gene trap targeting technique, we replaced the genomic sequence of p120 with HA-tagged p120 cDNA in mouse teratocarcinoma F9 cells. In the p120 knock-in (p120KI) cells, we found that the expression level of p120 was severely reduced and that the expression level of other components of the cadherin-catenin complex was also reduced. The stable expression of various p120 mutants in p120KI cells revealed that the armadillo repeat domain of p120 is sufficient to restore the expression level of E-cadherin. In p120KI cells, internalized E-cadherin was frequently detected as large aggregates. Transient expression of wild-type p120 and mutant p120 lacking the N-terminal region induced both relocalization of E-cadherin at the cell-cell boundaries and the disappearance of cytoplasmic E-cadherin aggregates. Transient expression of mutant p120 lacking the C-terminal region, however, only induced a small increase in E-cadherin signals at the cell-cell boundary. In these cells, the cytoplasmic E-cadherin signals became brighter and the expressed mutant p120 was incorporated in the E-cadherin aggregates. These results suggested the novel function of the p120 C-terminal region in regulating the trafficking of cytoplasmic E-cadherin.     

Diversity among beta-tubulins: a carboxy-terminal domain of yeast beta-tubulin is not essential in vivo.

Sequences of genes for beta-tubulins from many different organisms demonstrate that they encode highly conserved proteins but that these proteins diverge considerably at their carboxyl termini. The patterns of interspecies conservation of this diversity suggest that it may have functional significance. We have taken advantage of the properties of Saccharomyces cerevisiae to test this hypothesis in vivo. The sole beta-tubulin gene of this species is one of the most divergent of all beta-tubulins and encodes 12 amino acids which extend past the end of most other beta-tubulin molecules. We have constructed strains in which the only beta-tubulin gene is an allele lacking these 12 codons. We show here that this carboxy-terminal extension is not essential. The absence of these 12 amino acids had no effect on a number of microtubule-dependent functions, such as mitotic and meiotic division and mating. It did confer dominant supersensitivity to a microtubule-depolymerizing drug.     

The carboxy-terminal segment of the yeast alpha-factor receptor is a regulatory domain

The alpha-factor receptor is rapidly hyperphosphorylated on Thr and Ser residues in its hydrophilic C-terminal domain after cells are exposed to pheromone. Mutant receptors in which this domain is altered or removed are biologically active and bind alpha-factor with nearly normal affinity. However, cells expressing the mutant receptors are hypersensitive to pheromone action and appear to be defective in recovery from alpha-factor-induced growth arrest. Mutant receptors with partial C-terminal truncations undergo ligand-induced endocytosis, suggesting that down-regulation of receptor number is not the sole process for adaptation at the receptor level. A mutant receptor lacking the entire C-terminal domain (134 residues) does not display ligand-induced endocytosis. Genetic experiments indicate that the contribution of SST2 function to adaptation does not require the C-terminal domain of the receptor.     

Regulation of I(kappa)B kinase complex by phosphorylation of (gamma)-binding domain of I(kappa)B kinase (beta) by Polo-like kinase 1

IkappaB kinase (IKK) complex is a key regulator of NF-kappaB pathways. Signal-induced interaction of the IKKgamma (NEMO) subunit with the C-terminal IKKgamma/NEMO-binding domain (gammaBD) of IKKbeta is an essential interaction for IKK regulation. Underlying regulatory mechanism(s) of this interaction are not known. Phosphorylation of gammaBD has been suggested to play a regulatory role for IKK activation. However, a kinase that phosphorylates gammaBD has not been identified. In this study, we used a C-terminal fragment of IKKbeta as substrate and purified Polo-like kinase 1 (Plk1) from HeLa cell extracts by standard chromatography as a gammaBD kinase. Plk1 phosphorylates serines 733, 740, and 750 in the gammaBD of IKKbeta in vitro. Phosphorylating gammaBD with Plk1 decreased its affinity for IKKgamma in pulldown assay. We generated phosphoantibodies against serine 740 and showed that gammaBD is phosphorylated in vivo. Expressing a constitutively active Plk1 in mammalian cells reduced tumor necrosis factor (TNF)-induced IKK activation, resulting in decreased phosphorylation of endogenous IkappaBalpha and reduced NF-kappaB activation. To activate endogenous Plk1, cells were treated with nocodazole, which reduced TNF-induced IKK activation, and increased the phosphorylation of gammaBD. Knocking down Plk1 in mammalian cells restored TNF-induced IKK activation in nocodazole-treated cells. Activation of Plk1 inhibited TNF-induced expression of cyclin D1. In cells in which Plk1 was knocked down, TNFalpha increased expression of cyclin D1 and the proportion of cells in the S phase of the cell cycle. Taken together, this study shows that phosphorylation regulates the interaction of gammaBD of IKKbeta with IKKgamma and therefore plays a critical role for IKK activation. Moreover, we identify Plk1 as a gammaBD kinase, which negatively regulates TNF-induced IKK activation and cyclin D1 expression, thereby affecting cell cycle regulation. Untimely activation of cyclin D1 by TNFalpha can provide a potential mechanism for an involvement of TNFalpha in inflammation-induced cancer.