The protein histidine phosphatase LHPP is a tumour suppressor.

Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.

A PIM-CHK1 signaling pathway regulates PLK1 phosphorylation and function during mitosis.

Although the kinase CHK1 is a key player in the DNA damage response (DDR), several studies have recently provided evidence of DDR-independent roles of CHK1, in particular following phosphorylation of its S280 residue. Here, we demonstrate that CHK1 S280 phosphorylation is cell cycle-dependent and peaks during mitosis. We found that this phosphorylation was catalyzed by the kinase PIM2, whose protein expression was also increased during mitosis. Importantly, we identified polo-like kinase 1 (PLK1) as a direct target of CHK1 during mitosis. Genetic or pharmacological inhibition of CHK1 reduced the activating phosphorylation of PLK1 on T210, and recombinant CHK1 was able to phosphorylate T210 of PLK1 in vitro Accordingly, S280-phosphorylated CHK1 and PLK1 exhibited similar specific mitotic localizations, and PLK1 was co-immunoprecipitated with S280-phosphorylated CHK1 from mitotic cell extracts. Moreover, CHK1-mediated phosphorylation of PLK1 was dependent on S280 phosphorylation by PIM2. Inhibition of PIM proteins reduced cell proliferation and mitotic entry, which was rescued by expressing a T210D phosphomimetic mutant of PLK1. Altogether, these data identify a new PIM-CHK1-PLK1 phosphorylation cascade that regulates different mitotic steps independently of the CHK1 DDR function.This article has an associated First Person interview with the first author of the paper.

NME/NM23/NDPK and Histidine Phosphorylation.

The NME (Non-metastatic) family members, also known as NDPKs (nucleoside diphosphate kinases), were originally identified and studied for their nucleoside diphosphate kinase activities. This family of kinases is extremely well conserved through evolution, being found in prokaryotes and eukaryotes, but also diverges enough to create a range of complexity, with homologous members having distinct functions in cells. In addition to nucleoside diphosphate kinase activity, some family members are reported to possess protein-histidine kinase activity, which, because of the lability of phosphohistidine, has been difficult to study due to the experimental challenges and lack of molecular tools. However, over the past few years, new methods to investigate this unstable modification and histidine kinase activity have been reported and scientific interest in this area is growing rapidly. This review presents a global overview of our current knowledge of the NME family and histidine phosphorylation, highlighting the underappreciated protein-histidine kinase activity of NME family members, specifically in human cells. In parallel, information about the structural and functional aspects of the NME family, and the knowns and unknowns of histidine kinase involvement in cell signaling are summarized.

The Potential Functional Roles of NME1 Histidine Kinase Activity in Neuroblastoma Pathogenesis.

Neuroblastoma is the most common extracranial solid tumor in childhood. Gain of chromosome 17q material is found in >60% of neuroblastoma tumors and is associated with poor patient prognosis. The NME1 gene is located in the 17q21.3 region, and high NME1 expression is correlated with poor neuroblastoma patient outcomes. However, the functional roles and signaling activity of NME1 in neuroblastoma cells and tumors are unknown. NME1 and NME2 have been shown to possess histidine (His) kinase activity. Using anti-1- and 3-pHis specific monoclonal antibodies and polyclonal anti-pH118 NME1/2 antibodies, we demonstrated the presence of pH118-NME1/2 and multiple additional pHis-containing proteins in all tested neuroblastoma cell lines and in xenograft neuroblastoma tumors, supporting the presence of histidine kinase activity in neuroblastoma cells and demonstrating the potential significance of histidine kinase signaling in neuroblastoma pathogenesis. We have also demonstrated associations between NME1 expression and neuroblastoma cell migration and differentiation. Our demonstration of NME1 histidine phosphorylation in neuroblastoma and of the potential role of NME1 in neuroblastoma cell migration and differentiation suggest a functional role for NME1 in neuroblastoma pathogenesis and open the possibility of identifying new therapeutic targets and developing novel approaches to neuroblastoma therapy.

Histidine kinases and the missing phosphoproteome from prokaryotes to eukaryotes.

Protein phosphorylation is the most common type of post-translational modification in eukaryotes. The phosphoproteome is defined as the complete set of experimentally detectable phosphorylation sites present in a cell's proteome under various conditions. However, we are still far from identifying all the phosphorylation sites in a cell mainly due to the lack of information about phosphorylation events involving residues other than Ser, Thr and Tyr. Four types of phosphate-protein linkage exist and these generate nine different phosphoresidues-pSer, pThr, pTyr, pHis, pLys, pArg, pAsp, pGlu and pCys. Most of the effort in studying protein phosphorylation has been focused on Ser, Thr and Tyr phosphorylation. The recent development of 1- and 3-pHis monoclonal antibodies promises to increase our understanding of His phosphorylation and the kinases and phosphatases involved. Several His kinases are well defined in prokaryotes, especially those involved in two-component system (TCS) signaling. However, in higher eukaryotes, NM23, a protein originally characterized as a nucleoside diphosphate kinase, is the only characterized protein-histidine kinase. This ubiquitous and conserved His kinase autophosphorylates its active site His, and transfers this phosphate either onto a nucleoside diphosphate or onto a protein His residue. Studies of NM23 protein targets using newly developed anti-pHis antibodies will surely help illuminate the elusive His phosphorylation-based signaling pathways. This review discusses the role that the NM23/NME/NDPK phosphotransferase has, how the addition of the pHis phosphoproteome will expand the phosphoproteome and make His phosphorylation part of the global phosphorylation world. It also summarizes why our understanding of phosphorylation is still largely restricted to the acid stable phosphoproteome, and highlights the study of NM23 histidine kinase as an entrée into the world of histidine phosphorylation.

The mycoparasite Pythium oligandrum induces legume pathogen resistance and shapes rhizosphere microbiota without impacting mutualistic interactions.

Pythium oligandrum is a soil-borne oomycete associated with rhizosphere and root tissues. Its ability to enhance plant growth, stimulate plant immunity and parasitize fungal and oomycete preys has led to the development of agricultural biocontrol products. Meanwhile, the effect of P. oligandrum on mutualistic interactions and more generally on root microbial communities has not been investigated. Here, we developed a biological system comprising P. oligandrum interacting with two legume plants, Medicago truncatula and Pisum sativum. P. oligandrum activity was investigated at the transcriptomics level through an RNAseq approach, metabolomics and finally metagenomics to investigate the impact of P. oligandrum on root microbiota. We found that P. oligandrum promotes plant growth in these two species and protects them against infection by the oomycete Aphanomyces euteiches, a devastating legume root pathogen. In addition, P. oligandrum up-regulated more than 1000 genes in M. truncatula roots including genes involved in plant defense and notably in the biosynthesis of antimicrobial compounds and validated the enhanced production of M. truncatula phytoalexins, medicarpin and formononetin. Despite this activation of plant immunity, we found that root colonization by P. oligandrum did not impaired symbiotic interactions, promoting the formation of large and multilobed symbiotic nodules with Ensifer meliloti and did not negatively affect the formation of arbuscular mycorrhizal symbiosis. Finally, metagenomic analyses showed the oomycete modifies the composition of fungal and bacterial communities. Together, our results provide novel insights regarding the involvement of P. oligandrum in the functioning of plant root microbiota.

Subcellular Localization of Histidine Phosphorylated Proteins Through Indirect Immunofluorescence.

Immunofluorescence (IF) takes advantage of biological and physical mechanisms to identify proteins in cell or tissue samples, exploiting the specificity of antibodies and stimulated fluorescence light emission. Here, we describe an immunofluorescence staining method for the identification of histidine phosphorylated proteins that uses neutral/alkaline conditions and targeted reagents to overcome the chemical lability of histidine phosphorylation. This method describes how 1- and 3-phosphohistidine (pHis) monoclonal antibodies can be used to reveal the localization of proteins containing these elusive phosphoramidate bonds in cells. Standard procedures and materials for IF staining with adherent and nonadherent cells are described.

Empirical Evidence of Cellular Histidine Phosphorylation by Immunoblotting Using pHis mAbs.

Immunoblotting is a ubiquitous immunological technique that aids in detecting and quantifying proteins (including those of lower abundance) and their posttranslational modifications such as phosphorylation, acetylation, ubiquitylation, and sumoylation. The technique involves electrophoretically separating proteins on an SDS-PAGE gel, transferring them onto a PVDF (or nitrocellulose) membrane and probing with specific antibodies. Here we describe an immunoblotting technique for detecting cellular phosphohistidine, a labile posttranslational modification, by optimizing experimental conditions such that the labile phosphohistidine signal is conserved throughout the experiment.

Control of Pim2 kinase stability and expression in transformed human haematopoietic cells.

The oncogenic Pim2 kinase is overexpressed in several haematological malignancies, such as multiple myeloma and acute myeloid leukaemia (AML), and constitutes a strong therapeutic target candidate. Like other Pim kinases, Pim2 is constitutively active and is believed to be essentially regulated through its accumulation. We show that in leukaemic cells, the three Pim2 isoforms have dramatically short half-lives although the longer isoform is significantly more stable than the shorter isoforms. All isoforms present a cytoplasmic localization and their degradation was neither modified by broad-spectrum kinase or phosphatase inhibitors such as staurosporine or okadaic acid nor by specific inhibition of several intracellular signalling pathways including Erk, Akt and mTORC1. Pim2 degradation was inhibited by proteasome inhibitors but Pim2 ubiquitination was not detected even by blocking both proteasome activity and protein de-ubiquitinases (DUBs). Moreover, Pyr41, an ubiquitin-activating enzyme (E1) inhibitor, did not stabilize Pim2, strongly suggesting that Pim2 was degraded by the proteasome without ubiquitination. In agreement, we observed that purified 20S proteasome particles could degrade Pim2 molecule in vitro. Pim2 mRNA accumulation in UT7 cells was controlled by erythropoietin (Epo) through STAT5 transcription factors. In contrast, the translation of Pim2 mRNA was not regulated by mTORC1. Overall, our results suggest that Pim2 is only controlled by its mRNA accumulation level. Catalytically active Pim2 accumulated in proteasome inhibitor-treated myeloma cells. We show that Pim2 inhibitors and proteasome inhibitors, such as bortezomib, have additive effects to inhibit the growth of myeloma cells, suggesting that Pim2 could be an interesting target for the treatment of multiple myeloma.

Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia.

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD-induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD(+) cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy.

Tyrosine kinase inhibitors induce down-regulation of c-Kit by targeting the ATP pocket.

The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-ß-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket.