Nuclear receptor phosphorylation in xenobiotic signal transduction

Nuclear pregnane X receptor (PXR, NR1I2) and constitutive active/androstane receptor (CAR, NR1I3) are nuclear receptors characterized in 1998 by their capability to respond to xenobiotics and activate cytochrome P450 (CYP) genes. An anti-epileptic drug, phenobarbital (PB), activates CAR and its target CYP2B genes, whereas PXR is activated by drugs such as rifampicin and statins for the CYP3A genes. Inevitably, both nuclear receptors have been investigated as ligand-activated nuclear receptors by identifying and characterizing xenobiotics and therapeutics that directly bind CAR and/or PXR to activate them. However, PB, which does not bind CAR directly, presented an alternative research avenue for an indirect ligand-mediated nuclear receptor activation mechanism: phosphorylation-mediated signal regulation. This review summarizes phosphorylation-based mechanisms utilized by xenobiotics to elicit cell signaling. First, the review presents how PB activates CAR (and other nuclear receptors) through a conserved phosphorylation motif located between two zinc fingers within its DNA-binding domain. PB-regulated phosphorylation at this motif enables nuclear receptors to form communication networks, integrating their functions. Next, the review discusses xenobiotic-induced PXR activation in the absence of the conserved DNA-binding domain phosphorylation motif. In this case, phosphorylation occurs at a motif located within the ligand-binding domain to transduce cell signaling that regulates hepatic energy metabolism. Finally, the review delves into the implications of xenobiotic-induced signaling through phosphorylation in disease development and progression.

CBL–CIPK module-mediated phosphoregulation: facts and hypothesis

Calcium (Ca2+) signaling is a versatile signaling network in plant and employs very efficient signal decoders to transduce the encoded message. The CBL–CIPK module is one of the sensor-relay decoders that have probably evolved with the acclimatization of land plant. The CBLs are unique proteins with non-canonical Ca2+ sensing EF-hands, N-terminal localization motif and a C-terminal phosphorylation motif. The partner CIPKs are Ser/Thr kinases with kinase and regulatory domains. Phosphorylation plays a major role in the functioning of the module. As the module has a functional kinase to transduce signal, it employs phosphorylation as a preferred mode for modulation of targets as well as its interaction with CBL. We analyze the data on the substrate regulation by the module from the perspective of substrate phosphorylation. We have also predicted some of the probable sites in the identified substrates that may be the target of the CIPK mediated phosphorylation. In addition, phosphatases have been implicated in reversing the CIPK mediated phosphorylation of substrates. Therefore, we have also presented the role of phosphatases in the modulation of the CBL–CIPK and its targets. We present here an overview of the phosphoregulation mechanism of the CBL–CIPK module.

Widespread arginine phosphorylation in human cells - a novel protein PTM revealed by mass spectrometry

ABSTRACTArginine phosphorylation (pArg) is recently discovered as a ubiquitous protein N- phosphorylation in bacteria. However, its prevalence and roles in mammalian cells remain largely unknown due to the lack of established workflow and the inherent lability of the phosphoramidate (P-N) bond. Emerging evidence suggests that N-phosphorylation may extensively exist in eukaryotes and play crucial roles. We report an experimental phosphoproteomic workflow, which for the first time allowed to reveal the widespread occurrence of pArg in human cells by mass spectrometry. By virtue of this approach, we identified 152 high-confidence pArg sit]es derived from 118 proteins. Remarkably, the discovered phosphorylation motif and gene ontology of pArg hint a possible cellular function of arginine phosphorylation by regulating the favorability of propeptide convertase substrate. The generated extensive data set should enable a better understanding of the biological functions of eukaryotic pArg in the future.Abstract Figure

Citron kinase interacts with LATS2 and inhibits its activity by occluding its hydrophobic phosphorylation motif

The inhibitory effect of large tumor suppressor kinase (LATS1/2) on the activity of the oncoprotein yes-associated protein (YAP) is crucial to maintain tissue homeostasis. Proteomic studies have identified several new regulators of this process. Recently, citron kinase (CIT) was listed as a potential binding candidate of Hippo-related components, suggesting a new connection between CIT and the Hippo pathway. Aside from CIT’s role in cytokinesis, the molecular crosstalk between CIT and the Hippo pathway is largely unknown. Here, we demonstrate a role for CIT as a scaffold protein linking LATS2 and YAP. More importantly, CIT interacts with LATS2 to directly suppress LATS2 phosphorylation at the hydrophobic motif—targeted by MST1, leading to LATS2 inactivation and YAP activation. By studying their genetic interactions, we found that Sticky, the CIT homolog in Drosophila melanogaster, functions with Warts to control Drosophila eye development. Together, our study confirms citron kinase as a novel regulator of the Hippo pathway.

FPD: A comprehensive phosphorylation database in fungi

Protein phosphorylation, one of the most classic post-translational modification, plays a critical role in the diverse cellular processes including cell cycle, growth and signal transduction pathways. However, the available information of phosphorylation in fungi is limited. Here we provided a Fungi Phosphorylation Database (FPD) that comprises high-confidence in vivo phosphosites identified by MS-based proteomics in various fungal species. This comprehensive phosphorylation database contains 62,272 non-redundant phosphorylation sites in 11,222 proteins across eight organisms, including Aspergillus flavus, Aspergillus nidulans, Fusarium graminearum, Magnaporthe oryzae, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Cryptococcus neoformans. A fungi-specific phosphothreonine motif and several conserved phosphorylation motif were discovered by comparatively analyzing the pattern of phosphorylation sites in fungi, plants and animals.Database URL: http://bis.zju.edu.cn/FPD/index.php

qpMerge: Merging different peptide isoforms using a motif centric strategy

Accurate quantification and enumeration of peptide motifs is hampered by redundancy in peptide identification. A single phosphorylation motif may be split across charge states, alternative modifications (e.g. acetylation and oxidation), and multiple miss-cleavage sites which render the biological interpretation of MS data a challenge. In addition motif redundancy can affect quantitative and statistical analysis and prevent a realistic comparison of peptide numbers between datasets. In this study, we present a merging tool set developed for the Galaxy workflow environment to achieve a non-redundant set of quantifications for phospho-motifs. We present a Galaxy workflow to merge three exemplar dataset, and observe reduced phospho-motif redundancy and decreased replicate variation. The qpMerge tools provide a straightforward and reusable approach to facilitating phospho-motif analysis.The source-code and wiki documentation is publically available at http://sourceforge.net/projects/ppmerge. The galaxy pipeline used in the exemplar analysis can be found at http://www.myexperiment.org/workflows/4186.