Nucleolar Nek11 is a novel target of Nek2A in G1/S-arrested cells

We previously reported that Nek11, a member of the NIMA (never-in-mitosis A) family of kinases, is activated in G(1)/S-arrested cells. We provide herein several lines of evidence for a novel interaction between Nek11 and Nek2A. Both Nek11 and Nek2A, but not Nek2B, were detected at nucleoli, and the Nek2A-specific C-terminal end (amino acids 399-445) was responsible for nucleolar localization. Endogenous Nek11 coimmunoprecipitated with endogenous Nek2A, and non-catalytic regions of each kinase were involved in the complex formation. Nek11L interacted with phosphorylated Nek2A but barely with the kinase-inactive Nek2A (K37R) mutant. In addition, both Nek2A autophosphorylation activity and the Nek11L-Nek2A complex formation increased in G(1)/S-arrested cells. These results indicate that autophosphorylation of Nek2A could stimulate its interaction with Nek11L at the nucleolus. Moreover, Nek2 directly phosphorylated Nek11 in the C-terminal non-catalytic region and elevated Nek11 kinase activity. The non-catalytic region of Nek11 showed autoinhibitory activity through intramolecular interaction with its N-terminal catalytic domain. Nek2 dissociated this autoinhibitory interaction. Altogether, our studies demonstrate a unique mechanism of Nek11 activation by Nek2A in G(1)/S-arrested cells and suggest a novel possibility for nucleolar function of the NIMA family.     

Jmjd3 activates Mash1 gene in RA‐induced neuronal differentiation of P19 cells

Covalent modifications of histone tails have fundamental roles in chromatin structure and function. Tri‐methyl modification on lysine 27 of histone H3 (H3K27me3) usually correlates with gene repression that plays important roles in cell lineage commitment and development. Mash1 is a basic helix‐loop‐helix regulatory protein that plays a critical role in neurogenesis, where it expresses as an early marker. In this study, we have shown a decreased H3K27me3 accompanying with an increased demethylase of H3K27me3 (Jmjd3) at the promoter of Mash1 can elicit a dramatically efficient expression of Mash1 in RA‐treated P19 cells. Over‐expression of Jmjd3 in P19 cells also significantly enhances the RA‐induced expression and promoter activity of Mash1. By contrast, the mRNA expression and promoter activity of Mash1 are significantly reduced, when Jmjd3 siRNA or dominant negative mutant of Jmjd3 is introduced into the P19 cells. Chromatin immunoprecipitation assays show that Jmjd3 is efficiently recruited to a proximal upstream region of Mash1 promoter that is overlapped with the specific binding site of Hes1 in RA‐induced cells. Moreover, the association between Jmjd3 and Hes1 is shown in a co‐Immunoprecipitation assay. It is thus likely that Jmjd3 is recruited to the Mash1 promoter via Hes1. Our results suggest that the demethylase activity of Jmjd3 and its mediator Hes1 for Mash1 promoter binding are both required for Jmjd3 enhanced efficient expression of Mash1 gene in the early stage of RA‐induced neuronal differentiation of P19 cells. J. Cell. Biochem. 110: 1457–1463, 2010. © 2010 Wiley‐Liss, Inc.     

Oxidation resistance 1 is a novel senolytic target

The selective depletion of senescent cells (SCs) by small molecules, termed senolytic agents, is a promising therapeutic approach for treating age‐related diseases and chemotherapy‐ and radiotherapy‐induced side effects. Piperlongumine (PL) was recently identified as a novel senolytic agent. However, its mechanism of action and molecular targets in SCs was unknown and thus was investigated. Specifically, we used a PL‐based chemical probe to pull‐down PL‐binding proteins from live cells and then mass spectrometry‐based proteomic analysis to identify potential molecular targets of PL in SCs. One prominent target was oxidation resistance 1 (OXR1), an important antioxidant protein that regulates the expression of a variety of antioxidant enzymes. We found that OXR1 was upregulated in senescent human WI38 fibroblasts. PL bound to OXR1 directly and induced its degradation through the ubiquitin‐proteasome system in an SC‐specific manner. The knockdown of OXR1 expression by RNA interference significantly increased the production of reactive oxygen species in SCs in conjunction with the downregulation of antioxidant enzymes such as heme oxygenase 1, glutathione peroxidase 2, and catalase, but these effects were much less significant when OXR1 was knocked down in non‐SCs. More importantly, knocking down OXR1 selectively induced apoptosis in SCs and sensitized the cells to oxidative stress caused by hydrogen peroxide. These findings provide new insights into the mechanism by which SCs are highly resistant to oxidative stress and suggest that OXR1 is a novel senolytic target that can be further exploited for the development of new senolytic agents.     

Immunohistochemical and biochemical analyses of GD3, GT1b,and GQ1b gangliosides during neural differentiation of P19 EC cells

In an earlier study, we showed that expressions of GD3, GT1b, and GQ1b gangliosides in P19 embryonic carcinoma (EC) cells were enhanced during their neural differentiation induced by retinoic acid. We now further demonstrated that this increase of the b-series gangliosides is due to an increase in their corresponding synthases (sialyltransferase-II, -IV, and -V) in the Golgi. Of the three gangliosides studied, GQ1b appeared to be the best candidate for monitoring such differentiation process. We also used fluorescence-labeled monoclonal antibodies and confocal fluorescence microscopy to obtain direct visual information about the relationship of gangliosides and neural specific proteins in neuron development. Again, GQ1b is the most interesting as it localizes with synaptophysin and neural cell adhesion molecules (NCAMs) on synaptic boutons or dendritic spines in RA-induced neurons (R/N). This suggests that GQ1b could be used as a marker for synapse formation during construction of the neural network.