Casein kinase II phosphorylates the eukaryote-specific C-terminal domain of topoisomerase II in vivo.

The decatenation activity of DNA topoisomerase II is essential for viability as eukaryotic cells traverse mitosis. Phosphorylation has been shown to stimulate topoisomerase II activity in vitro. Here we show that topoisomerase II is a phosphoprotein in yeast and that the level of incorporated phosphate is significantly higher at mitosis than in G1. Comparison of tryptic phosphopeptide maps reveals that the major phosphorylation sites in vivo are targets for casein kinase II. Incorporation of phosphate into topoisomerase II is nearly undetectable at the non-permissive temperature in a conditional casein kinase II mutant. The sites modified by casein kinase II are located in the extreme C-terminal domain of topoisomerase II. This domain is absent in prokaryotic and highly divergent among eukaryotic type II topoisomerases, and may serve to regulate functions of topoisomerase II that are unique to eukaryotic cells.     

A rice homolog of Cdk7/MO15 phosphorylates both cyclin-dependent protein kinases and the carboxy-terminal domain of RNA polymerase II

The activation of cyclin-dependent protein kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop by a CDK-activating kinase (CAK). The R2 protein of rice is very similar to CAKs of animals and fission yeast at the amino acid level but phosphorylation by R2 has not yet been demonstrated. When R2 was overexpressed in a CAK-deficient mutant of budding yeast, it suppressed the temperature sensitivity of the mutation. Immunoprecipitates of rice proteins with the anti-R2 antibody phosphorylated human CDK2, one of the rice CDKs (Cdc2Os1), and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II of Arabidopsis. Mutational analysis indicated that R2 phosphorylated the threonine residue within the T-loop of CDK2 and Cdc2Os1. R2 was found mainly in two protein complexes which had molecular masses of 190 kDa and 70 kDa, respectively, whilst the CDK- and CTD-kinase activities associated with R2 were identified in a complex of 105 kDa. These results indicate that R2 is closely related to CAKs of animals and fission yeast in terms of its phosphorylation activity and, moreover, that this CAK of rice is distinct from a CAK of the dicotyledonous plant Arabidopsis.     

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。     

Casein kinase 2 associates with and phosphorylates dishevelled.

The dishevelled (dsh) gene of Drosophila melanogaster encodes a phosphoprotein whose phosphorylation state is elevated by Wingless stimulation, suggesting that the phosphorylation of Dsh and the kinase(s) responsible for this phosphorylation are integral parts of the Wg signaling pathway. We found that immunoprecipitated Dsh protein from embryos and from cells in tissue culture is associated with a kinase activity that phosphorylates Dsh in vitro. Purification and peptide sequencing of a 38 kDa protein co-purifying with this kinase activity showed it to be identical to Drosophila Casein Kinase 2 (CK2). Tryptic phosphopeptide mapping indicates that identical peptides are phosphorylated by CK2 in vitro and in vivo, suggesting that CK2 is at least one of the kinases that phosphorylates Dsh. Overexpression of Dfz2, a Wingless receptor, also stimulated phosphorylation of Dsh, Dsh-associated kinase activity, and association of CK2 with Dsh, thus suggesting a role for CK2 in the transduction of the Wg signal.     

Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II

RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation.     

An encephalitozoon cuniculi ortholog of the RNA polymerase II carboxyl-terminal domain (CTD) serine phosphatase Fcp1

Fcp1 is an essential protein serine phosphatase that dephosphorylates Ser2 or Ser5 of the RNA polymerase II carboxyl-terminal domain (CTD) heptad repeat Y(1)S(2)P(3)T(4)S(5)P(6)S(7). The CTD of the microsporidian parasite Encephalitozoon cuniculi consists of 15 heptad repeats, which approximates the minimal CTD length requirement for cell viability in yeast. Here we show that E. cuniculi encodes a minimized 411-aa Fcp1-like protein (EcFcp1), which consists of a DxDx(T/V) phosphatase domain and a BRCA1 carboxyl terminus (BRCT) domain but lacks the large N- and C-terminal domains found in fungal and metazoan Fcp1 enzymes. Nonetheless, EcFcp1 can function in lieu of Saccharomyces cerevisiae Fcp1 to sustain yeast cell growth. Recombinant EcFcp1 is a monomeric enzyme with intrinsic phosphatase activity against nonspecific (p-nitrophenyl phosphate) and specific (CTD-PO(4)) substrates. EcFcp1 dephosphorylates CTD positions Ser2 and Ser5 with similar efficacy in vitro. We exploit synthetic CTD Ser2-PO(4) and Ser5-PO(4) peptides to define minimized substrates for EcFcp1 and to illuminate the importance of CTD primary structure in Ser2 and Ser5 phosphatase activity.