Raed Rizkallah

Regulation of the Transcription Factor YY1 in Mitosis through Phosphorylation of Its DNA-binding Domain

Yin-Yang 1 (YY1) is a ubiquitously expressed zinc finger transcription factor. It regulates a vast array of genes playing critical roles in development, differentiation, and cell cycle. Very little is known about the mechanisms that regulate the functions of YY1. It has long been proposed that YY1 is a phosphoprotein; however, a direct link between phosphorylation and the function of YY1 has never been proven. Investigation of the localization of YY1 during mitosis shows that it is distributed to the cytoplasm during prophase and remains excluded from DNA until early telophase. Immunostaining studies show that YY1 is distributed equally between daughter cells and rapidly associates with decondensing chromosomes in telophase, suggesting a role for YY1 in early marking of active and repressed genes. The exclusion of YY1 from DNA in prometaphase HeLa cells correlated with an increase in the phosphorylation of YY1 and loss of DNA-binding activity that can be reversed by dephosphorylation. We have mapped three phosphorylation sites on YY1 during mitosis and show that phosphorylation of two of these sites can abolish the DNA-binding activity of YY1. These results demonstrate a novel mechanism for the inactivation of YY1 through phosphorylation of its DNA-binding domain.

14-3-3 protein targets misfolded chaperone-associated proteins to aggresomes

Summary The aggresome is a key cytoplasmic organelle for sequestration and clearance of toxic protein aggregates. Although loading misfolded proteins cargos to dynein motors has been recognized as an important step in the aggresome formation process, the molecular machinery that mediates the association of cargos with the dynein motor is poorly understood. Here, we report a new aggresome-targeting pathway that involves isoforms of 14-3-3, a family of conserved regulatory proteins. 14-3-3 interacts with both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3), thereby recruiting chaperone-associated protein cargos to dynein motors for their transport to aggresomes. This molecular cascade entails functional dimerization of 14-3-3, which we show to be crucial for the formation of aggresomes in both yeast and mammalian cells. These results suggest that 14-3-3 functions as a molecular adaptor to promote aggresomal targeting of misfolded protein aggregates and may link such complexes to inclusion bodies observed in various neurodegenerative diseases.

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.

Temporal control of the dephosphorylation of Cdk substrates by mitotic exit pathways in budding yeast

The temporal phosphorylation of cell cycle-related proteins by cyclin-dependent kinases (Cdks) is critical for the correct order of cell cycle events. In budding yeast, CDC28 encodes the only Cdk and its association with various cyclins governs the temporal phosphorylation of Cdk substrates. S-phase Cdk substrates are phosphorylated earlier than mitotic Cdk substrates, which ensures the sequential order of DNA synthesis and mitosis. However, it remains unclear whether Cdk substrates are dephosphorylated in temporally distinct windows. Cdc14 is a conserved protein phosphatase responsible for the dephosphorylation of Cdk substrates. In budding yeast, FEAR (Cdc14 early anaphase release) and MEN (mitotic exit network) activate phosphatase Cdc14 by promoting its release from the nucleolus in early and late anaphase, respectively. Here, we show that the sequential Cdc14 release and the distinct degradation timing of different cyclins provides the molecular basis for the differential dephosphorylation windows of S-phase and mitotic cyclin substrates. Our data also indicate that FEAR-induced dephosphorylation of S-phase Cdk substrates facilitates anaphase progression, revealing an extra layer of mitotic regulation.

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.

Slk19 clusters kinetochores and facilitates chromosome bipolar attachment

Yeast kinetochore protein Slk19 is required for kinetochore clustering, and nocodazole exposure to slk19 mutant cells causes impaired kinetochore capture and delayed chromosome bipolar attachment after nocodazole washout.

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.

CHARACTERIZATION OF NEURONAL SRC KINASE PURIFIED FROM A BACTERIAL EXPRESSION SYSTEM

Neuronal Src (n-Src) is an alternative isoform of Src kinase containing a 6-amino acid insert in the SH3 domain that is highly expressed in neurons of the central nervous system (CNS). To investigate the function of n-Src, wild-type n-Src, constitutively active n-Src in which the C-tail tyrosine 535 was mutated to phenylalanine (n-Src/Y535F) and inactive n-Src in which the lysine 303 was mutated to arginine in addition to the mutation of Y535F (n-Src/K303R/Y535F), were expressed and purified from Escherichia coli BL21(DE3) cells. We found that all three types of n-Src constructs expressed at very high yields (∼500 mg/L) at 37°C, but formed inclusion bodies. In the presence of 8M urea these proteins could be solubilized, purified under denaturing conditions, and subsequently refolded in the presence of arginine (0.5M). These Src proteins were enzymatically active except for the n-Src/K303R/Y535F mutant. n-Src proteins expressed at 18°C were soluble, albeit at lower yields (∼10-20 mg/L). The lowest yields were for n-Src/Y535F (∼10 mg/L) and the highest for n-Src/K303R/Y535F (∼20 mg/L). We characterized the purified n-Src proteins expressed at 18°C. We found that altering n-Src enzyme activity either pharmacologically (e.g., application of ATP or a Src inhibitor) or genetically (mutation of Y535 or K303) was consistently associated with changes in n-Src stability: an increase in n-Src activity was coupled with a decrease in n-Src stability and vice versa. These findings, therefore, indicate that n-Src activity and stability are interdependent. Finally, the successful production of functionally active n-Src in this study indicates that the bacterial expression system may be a useful protein source in future investigations of n-Src regulation and function.

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.