NIPP1 maintains EZH2 phosphorylation and promoter occupancy at proliferation-related target genes

The histone methyltransferase EZH2 regulates cell proliferation and differentiation by silencing Polycomb group target genes. NIPP1, a nuclear regulator of serine/threonine protein phosphatase 1 (PP1), has been implicated in the regulation of EZH2 occupancy at target loci, but the underlying mechanism is not understood. Here, we demonstrate that the phosphorylation of EZH2 by cyclin-dependent kinases at Thr416 creates a docking site for the ForkHead-associated domain of NIPP1. Recruited NIPP1 enables the net phosphorylation of EZH2 by inhibiting its dephosphorylation by PP1. Accordingly, a NIPP1-binding mutant of EZH2 is hypophosphorylated, and the knockdown of NIPP1 results in a reduced phosphorylation of endogenous EZH2. Conversely, the loss of PP1 is associated with a hyperphosphorylation of EZH2. A genome-wide promoter-binding profiling in HeLa cells revealed that the NIPP1-binding mutant shows a deficient association with about a third of the Polycomb target genes, and these are enriched for functions in proliferation. Our data identify PP1 as an EZH2 phosphatase and demonstrate that the phosphorylation-regulated association of EZH2 with proliferation-related targets depends on associated NIPP1.     

IL-1 induces rapid phosphorylation of the IL-1 receptor

The IL-1R on murine T cells is a Mr = 80,000 plasma membrane glycoprotein. cDNA cloning and transfection experiments have shown that this is an integral membrane protein, which binds both IL-1 alpha and IL-1 beta and transduces the IL-1 signal. A mAb, RM-5, which binds an epitope on the receptor which is distinct from the IL-1 binding site has been produced in rats. RM-5 has been used to immunoprecipitate the IL-1R from 32P-orthophosphate labeled CHO cells which express approximately 100,000 functional, murine rIL-1R/cell. Phosphorylation of the receptor was observed as early as 1 min after the addition of IL-1 and continued for periods of up to 30 min. Phosphorylation increases as the concentration of IL-1 increases from 10(-13) to 10(-8) M. Potassium hydroxide hydrolysis of the phosphorylated IL-1R shows that more than 90% of the phosphate is incorporated into serine or threonine. Thus, one of the earliest events after IL-1 binding to the IL-1R is activation of a serine/threonine protein kinase and phosphorylation of the IL-1R itself.     

A rapid method for promoter exchange in Aspergillus nidulans using recombinant PCR

Recombinant PCR has been used to generate linear fragments for promoter replacement by transformation in Aspergillus nidulans. A cassette vector carrying the pyr-4 non-homologous selectable marker and conditional promoter Pr-alcA was constructed for use as a template for PCR, and is suitable for testing the function of essential genes. Two genes involved in polar growth, cotA and bemA, were used to assess the system. Efficient targeting was possible with both genes using approximately 500bp of flanking homologous sequence. Depending on yield, the linear PCR product could be used directly for transformation, or after first cloning into a suitable vector. bemA, a putative homologue of the Saccharomyces cerevisiae BEM1 gene was identified through sequence comparison. In A. nidulans, this protein appears to have a similar role to the yeast Bem1p, which acts as a scaffold protein involved in the establishment of cell polarity.     

Role of Hcn1 and its phosphorylation in fission yeast anaphase-promoting complex/cyclosome function

The anaphase-promoting complex/cyclosome (APC/C) is a conserved multisubunit ubiquitin ligase required for the degradation of key cell cycle regulators. The APC/C becomes active at the metaphase/anaphase transition and remains active during G(1) phase. One mechanism linked to activation of the APC/C is phosphorylation. Although many sites of mitotic phosphorylation have been identified in core components of the APC/C, the consequence of any individual phosphorylation event has not been elucidated in vivo. In this study, we show that Hcn1 is an essential core component of the fission yeast APC/C and is critical for maintaining complex integrity. Moreover, Hcn1 is a phosphoprotein in vivo. Phosphorylation of Hcn1 occurs at a single Cdk1 site in vitro and in vivo. Mutation of this site to alanine, but not aspartic acid, compromises APC/C function and leads to a specific defect in the completion of cell division.