Regulation of polo-like kinase 1 by DNA damage and PP2A/B55α

In addition to governing mitotic progression, Plk1 also suppresses the activation of the G2 DNA damage checkpoint and promotes checkpoint recovery. Previous studies have shown that checkpoint activation after DNA damage requires inhibition of Plk1, but the underlying mechanism of Plk1 regulation was unknown. In this study we show that the specific phosphatase activity toward Plk1 Thr-210 in interphase Xenopus egg extracts is predominantly PP2A-dependent, and this phosphatase activity is upregulated by DNA damage. Consistently, PP2A associates with Plk1 and the association increases after DNA damage. We further revealed that B55α, a targeting subunit of PP2A and putative tumor suppressor, mediates PP2A/Plk1 association and Plk1 dephosphorylation. B55α and PP2A association is greatly strengthened after DNA damage in an ATM/ATR and checkpoint kinase-dependent manner. Collectively, we report a phosphatase-dependent mechanism that responds to DNA damage and regulates Plk1 and checkpoint recovery.     

ATP analog-sensitive Pat1 protein kinase for synchronous fission yeast meiosis at physiological temperature

To study meiosis, synchronous cultures are often indispensable, especially for physical analyses of DNA and proteins. A temperature-sensitive allele of the Pat1 protein kinase (pat1-114) has been widely used to induce synchronous meiosis in the fission yeast Schizosaccharomyces pombe, but pat1-114-induced meiosis differs from wild-type meiosis, and some of these abnormalities might be due to higher temperature needed to inactivate the Pat1 kinase. Here, we report an ATP analog-sensitive allele of Pat1 [Pat1(L95A), designated pat1-as2] that can be used to generate synchronous meiotic cultures at physiological temperature. In pat1-as2 meiosis, chromosomes segregate with higher fidelity, and spore viability is higher than in pat1-114 meiosis, although recombination is lower by a factor of 2–3 in these mutants than in starvation-induced pat1⁺ meiosis. Addition of the mat-Pc gene improved chromosome segregation and spore viability to nearly the level of starvation-induced meiosis. We conclude that pat1-as2 mat-Pc cells offer synchronous meiosis with most tested properties similar to those of wild-type meiosis.     

Viral oncoprotein LMP1 disrupts p53-induced cell cycle arrest and apoptosis through modulating K63-linked ubiquitination of p53

Disruption of the gatekeeper p53 tumor suppressor is involved in various virus-associated tumorigeneses, with aberrant ubiquitination as the major cause of p53 abnormalities in virus-associated tumors. Of note, wild-type p53 is accumulated in Epstein-Barr virus (EBV)-associated tumors, especially in nasopharyngeal carcinoma (NPC). We have previously identified that p53 is accumulated and phosphorylated by EBV oncoprotein latent membrane protein 1 (LMP1) in NPC. Here, we further found that LMP1 promoted p53 accumulation via two distinct ubiquitin modifications. LMP1 promoted p53 stability and accumulation by suppressing K48-linked ubiquitination of p53 mediated by E3 ligase MDM2, which is associated with its phosphorylation at Ser20, while increasing the levels of total cellular ubiquitinated p53. LMP1 also induced K63-linked ubiquitination of p53 by interacting with tumor necrosis factor receptor-associated factor 2 (TRAF2), thus contributing to p53 accumulation. Furthermore, LMP1 rescued tumor cell apoptosis and cell cycle arrest mediated by K63-linked ubiquitination of p53. Collectively, these results demonstrate aberrant ubiquitin modifications of p53 and its biological functions by viral protein LMP1, which has broad implications to the pathogenesis of multiple EBV-associated tumors.     

Polo-like kinase-activating kinases

The events of cell division are regulated by a complex interplay between kinases and phosphatases. Cyclin-dependent kinases (Cdks), polo-like kinases (Plks) and Aurora kinases play central roles in this process. Polo kinase (Plk1 in humans) regulates a wide range of events in mitosis and cytokinesis. To ensure the accuracy of these processes, polo activity itself is subject to complex regulation. Phosphorylation of polo in its T loop (or activation loop) increases its kinase activity several-fold. It has been shown that Aurora A kinase, with its co-factor Bora, activates Plk1 in G₂, and that this is essential for recovery from cell cycle arrest induced by DNA damage. In a recent article published in PLoS Biology, we report that Drosophila polo is activated by Aurora B kinase at centromeres, and that this is crucial for polo function in regulating chromosome dynamics in prometaphase. Our results suggest that this regulatory pathway is conserved in humans. Here, we propose a model for the collaboration between Aurora B and polo in the regulation of kinetochore attachment to microtubules in early mitosis. Moreover, we suggest that Aurora B could also function to activate Polo/Plk1 in cytokinesis. Finally, we discuss recent findings and open questions regarding the activation of polo and polo-like kinases by different kinases in mitosis, cytokinesis and other processes.     

Serotonin and the search for the anatomical substrate of aggression

Abstract

All species of animals display aggression in order to obtain resources such as territories, mates, or food. Appropriate displays of aggression rely on the correct identification of a potential competitor, an evaluation of the environmental signals, and the physiological state of the animal. With a hard-wired circuitry involving fixed numbers of neurons, neuromodulators like serotonin offer adaptive flexibility in behavioral responses without changing the “hard-wiring”. In a recent report, we combined intersectional genetics, quantitative behavioral assays and morphological analyses to identify single serotonergic neurons that modulate the escalation of aggression. We found anatomical target areas within the brain where these neurons appear to form synaptic contacts with 5HT1A receptor-expressing neurons, and then confirmed the likelihood of those connections on a functional level. In this Extra View article, we offer an extended discussion of these recent findings and elaborate on how they can link a cellular and functional mapping of an aggression-regulating circuit at a single-cell resolution level.     

Determination of the biochemical properties of full-length human PIF1 ATPase

The PIF1 helicase family performs many cellular functions. To better understand the functions of the human PIF1 helicase, we characterized the biochemical properties of its ATPase. PIF1 is very sensitive to temperature, whereas it is not affected by pH, and the ATPase activity of human PIF1 is dependent on the divalent cations Mg²⁺ and Mn²⁺ but not Ca²⁺ and Zn²⁺. Inhibition was observed when single-stranded DNA was coated with RPA or SSB. Moreover, the ATPase activity of PIF1 proportionally decreased with decreasing oligonucleotide length due to a decreased binding ability. A minimum of 10 oligonucleotide bases are required for PIF1 binding and the hydrolysis of ATP. The analysis of the biochemical properties of PIF1 together with numerous genetic observations should aid in the understanding of its cellular functions.     

Emerging roles of Orai3 in pathophysiology

Calcium (Ca²⁺) is a ubiquitous second messenger that regulates a plethora of physiological functions. Deregulation of calcium homeostasis has been reported in a wide variety of pathological conditions including cardiovascular disorders, cancer and neurodegenerative diseases. One of the most ubiquitous pathways involved in regulated Ca²⁺ influx into cells is the store-operated Ca²⁺ entry (SOCE) pathway. In 2006, Orai1 was identified as the channel protein that mediates SOCE in immune cells. Orai1 has two mammalian homologs, Orai2 and Orai3. Although Orai1 has been the most widely studied Orai isoform, Orai3 has recently received significant attention. Under native conditions, Orai3 was demonstrated to be an important component of store-independent arachidonate-regulated Ca²⁺ (ARC) entry in HEK293 cells, and more recently of a store-independent leukotrieneC₄-regulated Ca²⁺ (LRC) entry pathway in vascular smooth muscle cells. Recent studies have shown upregulation of Orai3 in estrogen receptor-expressing breast cancers and a critical role for Orai3 in breast cancer development in immune-compromised mice. Orai3 upregulation was also shown to contribute to vascular smooth muscle remodeling and neointimal hyperplasia caused by vascular injury. Furthermore, Orai3 has been shown to contribute to proliferation of effector T-lymphocytes under oxidative stress. In this review, we will discuss the role of Orai3 in reported pathophysiological conditions and will contribute ideas on the potential role of Orai3 in native Ca²⁺ signaling pathways and human disease.