Karen E. Alexander

Global mitotic phosphorylation of C2H2 zinc finger protein linker peptides.

Cessation of transcriptional activity is a hallmark of cell division. Many biochemical pathways have been shown and proposed over the past few decades to explain the silence of this phase. In particular, many individual transcription factors have been shown to be inactivated by phosphorylation. In this report, we show the simultaneous phosphorylation and mitotic redistribution of a whole class of modified transcription factors. C2H2 zinc finger proteins (ZFPs) represent the largest group of gene expression regulators in the human genome. Despite their diversity, C2H2 ZFPs display striking conservation of small linker peptides joining their adjacent zinc finger modules. These linkers are critical for DNA binding activity. It has been proposed that conserved phosphorylation of these linker peptides could be a common mechanism for the inactivation of the DNA binding activity of C2H2 ZFPs, during mitosis. Using a novel antibody, raised against the phosphorylated form of the most conserved linker peptide sequence, we are able to visualize the massive and simultaneous mitotic phosphorylation of hundreds of these proteins. We show that this wave of phosphorylation is tightly synchronized, starting in mid-prophase right after DNA condensation and before the breakdown of the nuclear envelope. This global phosphorylation is completely reversed in telophase. In addition, the exclusion of the phospho-linker signal from condensed DNA clearly demonstrates a common mechanism for the mitotic inactivation of C2H2 ZFPs.

The Transcription Factor YY1 Is a Substrate for Polo-Like Kinase 1 at the G2/M Transition of the Cell Cycle

Yin-Yang 1 (YY1) is an essential multifunctional zinc-finger protein. It has been shown over the past two decades to be a critical regulator of a vast array of biological processes, including development, cell proliferation and differentiation, DNA repair, and apoptosis. YY1 exerts its functions primarily as a transcription factor that can activate or repress gene expression, dependent on its spatial and temporal context. YY1 regulates a large number of genes involved in cell cycle transitions, many of which are oncogenes and tumor-suppressor genes. YY1 itself has been classified as an oncogene and was found to be upregulated in many cancer types. Unfortunately, our knowledge of what regulates YY1 is very minimal. Although YY1 has been shown to be a phosphoprotein, no kinase has ever been identified for the phosphorylation of YY1. Polo-like kinase 1 (Plk1) has emerged in the past few years as a major cell cycle regulator, particularly for cell division. Plk1 has been shown to play important roles in the G/M transition into mitosis and for the proper execution of cytokinesis, processes that YY1 has been shown to regulate also. Here, we present evidence that Plk1 directly phosphorylates YY1 in vitro and in vivo at threonine 39 in the activation domain. We show that this phosphorylation is cell cycle regulated and peaks at G2/M. This is the first report identifying a kinase for which YY1 is a substrate.

Phosphorylation of the Transcription Factor YY1 by CK2α Prevents Cleavage by Caspase 7 during Apoptosis

ABSTRACT In this report, we describe the phosphorylation of Yin Yang 1 (YY1) in vitro and in vivo by CK2α (casein kinase II), a multifunctional serine/threonine protein kinase. YY1 is a ubiquitously expressed multifunctional zinc finger transcription factor implicated in regulation of many cellular and viral genes. The products of these genes are associated with cell growth, the cell cycle, development, and differentiation. Numerous studies have linked YY1 to tumorigenesis and apoptosis. YY1 is a target for cleavage by caspases in vitro and in vivo as well, but very little is known about the mechanisms that regulate its cleavage during apoptosis. Here, we identify serine 118 in the transactivation domain of YY1 as the site of CK2α phosphorylation, proximal to a caspase 7 cleavage site. CK2α inhibitors, as well as knockdown of CK2α by small interfering RNA, reduce S118 phosphorylation in vivo and enhance YY1 cleavage under apoptotic conditions, whereas increased CK2α activity by overexpression in vivo elevates S118 phosphorylation. A serine-to-alanine substitution at serine 118 also increases the cleavage of YY1 during apoptosis compared to wild-type YY1. Taken together, we have discovered a regulatory link between YY1 phosphorylation at serine 118 and regulation of its cleavage during programmed cell death.

The Transcription Factor YY1 Is a Novel Substrate for Aurora B Kinase at G2/M Transition of the Cell Cycle

Yin Yang 1 (YY1) is a ubiquitously expressed and highly conserved multifunctional transcription factor that is involved in a variety of cellular processes. Many YY1-regulated genes have crucial roles in cell proliferation, differentiation, apoptosis, and cell cycle regulation. Numerous mechanisms have been shown to regulate the function of YY1, such as DNA binding affinity, subcellular localization, and posttranslational modification including phosphorylation. Polo-like kinase 1(Plk1) and Casein kinase 2α (CK2 α) were the first two kinases identified to phosphorylate YY1. In this study, we identify a third kinase. We report that YY1 is a novel substrate of the Aurora B kinase both in vitro and in vivo. Serine 184 phosphorylation of YY1 by Aurora B is cell cycle regulated and peaks at G2/M and is rapidly dephosphorylated, likely by protein phosphatase 1 (PP1) as the cells enter G1. Aurora A and Aurora C can also phosphorylate YY1 in vitro, but at serine/threonine residues other than serine 184. We present evidence that phosphorylation of YY1 in the central glycine/alanine (G/A)-rich region is important for DNA binding activity, with a potential phosphorylation/acetylation interplay regulating YY1 function. Given their importance in mitosis and overexpression in human cancers, Aurora kinases have been identified as promising therapeutic targets. Increasing our understanding of Aurora substrates will add to the understanding of their signaling pathways.

Aurora A Phosphorylation of YY1 during Mitosis Inactivates its DNA Binding Activity

Successful execution of mitotic cell division requires the tight synchronisation of numerous biochemical pathways. The underlying mechanisms that govern chromosome segregation have been thoroughly investigated. However, the mechanisms that regulate transcription factors in coordination with mitotic progression remain poorly understood. In this report, we identify the transcription factor YY1 as a novel mitotic substrate for the Aurora A kinase, a key regulator of critical mitotic events, like centrosome maturation and spindle formation. Using in vitro kinase assays, we show that Aurora A directly phosphorylates YY1 at serine 365 in the DNA-binding domain. Using a new phospho-specific antibody, we show that YY1 phosphorylation at serine 365 occurs during mitosis, and that this phosphorylation is significantly reduced upon inhibition of Aurora A. Furthermore, we show, using electrophoretic mobility shift and chromatin immunoprecipitation assays, that phosphorylation of YY1 at this site abolishes its DNA binding activity in vitro and in vivo. In conformity with this loss of binding activity, phosphorylated YY1 also loses its transctivation ability as demonstrated by a luciferase reporter assay. These results uncover a novel mechanism that implicates Aurora A in the mitotic inactivation of transcription factors.

c-Abl phosphorylation of Yin Yang 1's conserved tyrosine 254 in the spacer region modulates its transcriptional activity

Yin Yang 1 (YY1) is a multifunctional transcription factor that can activate or repress transcription depending on the promotor and/or the co-factors recruited. YY1 is phosphorylated in various signaling pathways and is critical for different biological functions including embryogenesis, apoptosis, proliferation, cell-cycle regulation and tumorigenesis. Here we report that YY1 is a substrate for c-Abl kinase phosphorylation at conserved residue Y254 in the spacer region. Pharmacological inhibition of c-Abl kinase by imatinib, nilotinib and GZD824, knock-down of c-Abl using siRNA, and the use of c-Abl kinase-dead drastically reduces tyrosine phosphorylation of YY1. Both radioactive and non-radioactive in vitro kinase assays, as well as co-immunoprecipitation in different cell lines, show that the target of c-Abl phosphorylation is tyrosine residue 254. c-Abl phosphorylation has little effect on YY1 DNA binding ability or cellular localization in asynchronous cells. However, functional studies reveal that c-Abl mediated phosphorylation of YY1 regulates YY1s transcriptional ability in vivo. In conclusion, we demonstrate the novel role of c-Abl kinase in regulation of YY1s transcriptional activity, linking YY1 regulation with c-Abl tyrosine kinase signaling pathways.