Primary cilia are organelles necessary for proper implementation of developmental and homeostasis processes. To initiate their assembly, coordinated actions of multiple proteins are needed. Tau tubulin kinase 2 (TTBK2) is a key player in the cilium assembly pathway, controlling the final step of cilia initiation. The function of TTBK2 in ciliogenesis is critically dependent on its kinase activity; however, the precise mechanism of TTBK2 action has so far not been fully understood due to the very limited information about its relevant substrates. In this study, we demonstrate that CEP83, CEP89, CCDC92, Rabin8, and DVL3 are substrates of TTBK2 kinase activity. Further, we characterize a set of phosphosites of those substrates and CEP164 induced by TTBK2 in vitro and in vivo. Intriguingly, we further show that identified TTBK2 phosphosites and consensus sequence delineated from those are distinct from motifs previously assigned to TTBK2. Finally, we show that TTBK2 is also required for efficient phosphorylation of many S/T sites in CEP164 and provide evidence that TTBK2-induced phosphorylations of CEP164 modulate its function, which in turn seems relevant for the process of cilia formation. In summary, our work provides important insight into the substrates-TTBK2 kinase relationship and suggests that phosphorylation of substrates on multiple sites by TTBK2 is probably involved in the control of ciliogenesis in human cells.

• MeSH
• Amino Acid Motifs MeSH
• Cilia metabolism   MeSH
• Phosphorylation MeSH
• Phosphoserine metabolism   MeSH
• Phosphothreonine metabolism   MeSH
• HEK293 Cells MeSH
• Casein Kinase I metabolism   MeSH
• Humans MeSH
• Multiprotein Complexes metabolism   MeSH
• Organogenesis * MeSH
• Protein Serine-Threonine Kinases chemistry   metabolism   MeSH
• Substrate Specificity MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Unilateral sciatic nerve compression (SNC) or complete sciatic nerve transection (CSNT), both varying degrees of nerve injury, induced activation of STAT3 bilaterally in the dorsal root ganglia (DRG) neurons of lumbar (L4-L5) as well as cervical (C6-C8) spinal cord segments. STAT3 activation was by phosphorylation at the tyrosine-705 (Y705) and serine-727 (S727) positions and was followed by their nuclear translocation. This is the first evidence of STAT3(S727) activation together with the well-known activation of STAT3(Y705) in primary sensory neurons upon peripheral nerve injury. Bilateral activation of STAT3 in DRG neurons of spinal segments anatomically both associated as well as non-associated with the injured nerve indicates diffusion of STAT3 activation inducers along the spinal cord. Increased levels of IL-6 protein in the CSF following nerve injury as well as activation and nuclear translocation of STAT3 in DRG after intrathecal injection of IL-6 shows that this cytokine, released into the subarachnoid space can penetrate the DRG to activate STAT3. Previous results on increased bilateral IL-6 synthesis and the present manifestation of STAT3 activation in remote DRG following unilateral sciatic nerve injury may reflect a systemic reaction of the DRG neurons to nerve injury.

• MeSH
• Active Transport, Cell Nucleus MeSH
• Cell Nucleus metabolism   MeSH
• Phosphorylation MeSH
• Phosphoserine metabolism   MeSH
• Phosphotyrosine metabolism   MeSH
• Rats MeSH
• Sciatic Neuropathy metabolism   pathology   MeSH
• Sensory Receptor Cells metabolism   pathology   MeSH
• Rats, Wistar MeSH
• Ganglia, Spinal metabolism   pathology   MeSH
• STAT3 Transcription Factor chemistry   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

A new, rapid, sensitive, robust, and reliable method has been developed for the qualitative analysis of phosphoserine, phosphoethanolamine, phosphoglycerol, and phosphate using gas chromatography with mass spectrometry and two-step trimethylsilylation. The method employs hexamethyldisilazane for silylation of the phosphate and hydroxyl groups in the first phase and bis(trimethylsilyl)trifluoroacetamide for silylation of the less-reactive amino groups in the second phase. This order is of key importance for the method because of the different reactivities of the two reagents and the mechanism of derivatization of the active groups of the analytes. Trimethylsilylated derivatives of the analytes were identified on the basis of their retention times and mass spectra. The probable structures of the major fragments were identified in the spectra of the trimethylsilylated derivatives and characteristic m/z fragments were selected for each analyte. Fragments with m/z 73 and 299 occurred in the spectra of all the analytes. The characteristic retention data were employed to calculate the retention indices of the individual silylated phosphorylated substances in the hydrocarbon range C12-C19 for the DB-5ms column. The method was employed to measure the polar fraction of the hydrolysate of the cytoplasmic membrane of Bacillus subtilis. The detection limits vary between 5 μg/mL (trimethylsilylated phosphate) and 72 μg/mL (trimethylsilylated phosphoethanolamine).

• MeSH
• Bacillus subtilis chemistry   MeSH
• Cell Membrane chemistry   MeSH
• Chromatography, Gas methods   MeSH
• Ethanolamines analysis   MeSH
• Phosphates analysis   MeSH
• Phosphoserine analysis   MeSH
• Mass Spectrometry methods   MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH

Fructose-rich diet induces metabolic changes similar to those observed in metabolic syndrome. Among other matrix metalloproteinases, MMP-9 has an important role in adverse cardiac remodelling and might have a role in the development of cardiovascular disorders associated with metabolic syndrome. The changes of MMP-9 expression could be mediated via the NFκB pathway. In this study we investigated the effect of fructose-rich diet on MMP-9 expression in the heart of male and female rats, along with the effect of fructose-rich diet and oestradiol on MMP-9 expression in ovariectomized females. We further assessed the effect of fructose-rich diet and oestradiol on NFκB activation, measured as the level of p65 phosphorylation at Ser 276. The results showed that the diet regime did not affect the heart mass. Higher MMP-9 gene expression was found in cardiac tissue of male rats fed the fructose-rich diet than in females on the same diet regime. In ovariectomized females, fructose-rich diet upregulated MMP-9 protein and mRNA expression in the heart, as well as phosphorylation of the p65 subunit of NFκB at Ser 276. Oestradiol replacement therapy reverted these changes in the heart of ovariectomized females. This study has shown that oestradiol could revert the early molecular changes in MMP-9 expression induced by fructose-rich diet that occurred before cardiac hypertrophy development by decreasing phosphorylation of the NFκB p65 subunit at Ser 276.

• MeSH
• Diet adverse effects   MeSH
• Estradiol pharmacology   MeSH
• Phosphorylation drug effects   MeSH
• Phosphoserine metabolism   MeSH
• Fructose adverse effects   MeSH
• Matrix Metalloproteinase 9 genetics   metabolism   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Myocardium enzymology   pathology   MeSH
• NF-kappa B metabolism   MeSH
• Ovariectomy MeSH
• Rats, Wistar MeSH
• Gene Expression Regulation, Enzymologic drug effects   MeSH
• Body Weight drug effects   MeSH
• Transcription Factor RelA metabolism   MeSH
• Organ Size drug effects   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

L-serine is one of the proteinogenic amino acids and participates in several essential processes in all organisms. In plants, the light-dependent photorespiratory and the light-independent phosphoserine pathways contribute to serine biosynthesis. In cyanobacteria, the light-dependent photorespiratory pathway for serine synthesis is well characterized, but the phosphoserine pathway has not been identified. Here, we investigated three candidate genes for enzymes of the phosphoserine pathway in Synechocystis sp. PCC 6803. Only the gene for the D-3-phosphoglycerate dehydrogenase is correctly annotated in the genome database, whereas the 3-phosphoserine transaminase and 3-phosphoserine phosphatase (PSP) proteins are incorrectly annotated and were identified here. All enzymes were obtained as recombinant proteins and showed the activities necessary to catalyse the three-step phosphoserine pathway. The genes coding for the phosphoserine pathway were found in most cyanobacterial genomes listed in CyanoBase. The pathway seems to be essential for cyanobacteria, because it was impossible to mutate the gene coding for PSP in Synechocystis sp. PCC 6803 or in Synechococcus elongatus PCC 7942. A model approach indicates a 30-60% contribution of the phosphoserine pathway to the overall serine pool. Hence, this study verified that cyanobacteria, similar to plants, use the phosphoserine pathway in addition to photorespiration for serine biosynthesis.

• MeSH
• Enzyme Activation MeSH
• Phosphoglycerate Dehydrogenase genetics   metabolism   MeSH
• Phosphoserine metabolism   MeSH
• Metabolic Networks and Pathways * MeSH
• Molecular Sequence Data MeSH
• Gene Expression Regulation, Enzymologic MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Serine metabolism   MeSH
• Substrate Specificity MeSH
• Light * MeSH
• Synechocystis physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Serine 7 of centromere protein A (CENP-A) is a very important mitosis-specific phosphorylation site. In this study, we demonstrate the subcellular distribution of Ser7 phosphorylated CENP-A during mitosis in MCF-7 cells. The Ser7 phosphorylation of CENP-A was observed beginning at prophase at centromeres. Upon progression of mitosis, the fluorescence signals emerged in the central region of the metaphase plate and were maintained until anaphase at centromeres. At late anaphase, the fluorescence signals moved to the midzone gradually and transferred from the centromere to the midbody completely at telophase. They were compacted into the centre of the midbody in a thin cylinder consisting of a sandglass-like "mitotic machine" with microtubules and condensed chromosome. We also found that Ser10 phosphorylated H3 and Thr11 phosphorylated H3 were co-localized at the midbody in two bell-like symmetrical bodies with Ser7 phosphorylated CENP-A during the terminal stage of cytokinesis. Midbody isolation and immunoblotting experiments also indicated that Ser7 phosphorylated CENP-A are components of the midbody. These findings suggest that Ser7 phosphorylated CENP-A acts as a chromosomal passenger protein and may play an important role in cytokinesis.

• MeSH
• Adenocarcinoma pathology   MeSH
• Spindle Apparatus metabolism   ultrastructure   MeSH
• Autoantigens chemistry   physiology   MeSH
• Biological Transport MeSH
• Centromere metabolism   MeSH
• Chromosomal Proteins, Non-Histone chemistry   physiology   MeSH
• Cytokinesis physiology   MeSH
• Microscopy, Fluorescence MeSH
• Phosphorylation MeSH
• Phosphoserine metabolism   MeSH
• Phosphothreonine metabolism   MeSH
• Histones metabolism   MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• MCF-7 Cells cytology   metabolism   MeSH
• Mitosis physiology   MeSH
• Neoplasm Proteins chemistry   physiology   MeSH
• Breast Neoplasms pathology   MeSH
• Protein Processing, Post-Translational MeSH
• Pregnancy MeSH
• Check Tag
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The integrity of the human genome is constantly threatened by exogenous or endogenous genotoxic agents that cause DNA damage. The ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are considered as the most deleterious forms of DNA damage which could lead to genomic instability and to cancer development, if left unrepaired. The DNA damage response (DDR) is comprised of a network of proteins that cooperate to regulate cell cycle progression and repair of DNA lesions. Our understanding of molecular basis of repair processes and of functions of repair proteins, as well as understanding of chromatin modifications may provide new possibilities in improvement of cancer management. Phosphorylation of histone variant H2AX at serine 139 (γ-H2AX) and formation of γ-H2AX repair foci seems to be the most sensitive DNA damage marker in the chromatin flanking the free DNA double-stranded ends in DSBs. Monitoring of γ-H2AX levels can serve for early indication of cancer development, as biomarker of cancer therapy efficiency or as a biodosimetric marker of radiation exposure.

• MeSH
• Biomarkers MeSH
• Chromatin genetics   metabolism   MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• DNA radiation effects   MeSH
• DNA Breaks, Double-Stranded radiation effects   MeSH
• Financing, Organized MeSH
• Phosphorylation MeSH
• Phosphoserine metabolism   MeSH
• Histones genetics   metabolism   MeSH
• Radiation, Ionizing MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• Neoplasms diagnosis   MeSH
• DNA Repair MeSH
• Tyrosine metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Review MeSH

PROBLEM: The present work was undertaken to investigate the occurence of autoantibodies to eight various phospholipids in time of urgent termination of the pregnancy (sectio caesarea) in patients in reproductive age with severe preeclamptic symptoms. METHOD OF STUDY: Autoantibodies against annexin V, ph-serine, ph-ethanolamine, ph-inositol, ph-DL-glycerol, cardiolipin, beta2-glycoprotein I (beta2-GPI), and phosphatidic acid were studied by ELISA methods. RESULTS: Increased levels of IgA-beta2-glycoprotein I, IgG-beta2-glycoprotein I, IgG- anti-ph-serine, and IgG-anticardiolipin were found in sera of preeclamptic women in the time of urgent sectio caesarea when compared to the control group with physiological pregnancy. CONCLUSION: Supposed increase in various antiphospholipid antibodies (aPLs) levels due to the stress during the short time of admission and a need for a quick medical decision to terminate the pregnancy was not unambiguously proven, but our results are evidently influenced by the current urgent life-saving treatment.

• MeSH
• Antibodies, Antiphospholipid blood   MeSH
• beta 2-Glycoprotein I immunology   MeSH
• Adult MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Phosphoserine immunology   MeSH
• Humans MeSH
• Placental Circulation immunology   MeSH
• Abortion, Therapeutic MeSH
• Pre-Eclampsia blood   immunology   physiopathology   therapy   MeSH
• Pregnancy MeSH
• Thrombosis MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The ATM kinase is a tumour suppressor and a key activator of genome integrity checkpoints in mammalian cells exposed to ionizing radiation (IR) and other insults that elicit DNA double-strand breaks (DSBs). In response to IR, autophosphorylation on serine 1981 causes dissociation of ATM dimers and initiates cellular ATM kinase activity. Here, we show that the kinetics and magnitude of ATM Ser1981 phosphorylation after exposure of human fibroblasts to low doses (2 Gy) of IR are altered in cells deficient in Nbs1, a substrate of ATM and a component of the MRN (Mre11-Rad50-Nbs1) complex involved in processing/repair of DSBs and ATM-dependent cell cycle checkpoints. Timely phosphorylation of both ATM Ser1981 and the ATM substrate Smc1 after IR were rescued via retrovirally mediated reconstitution of Nbs1-deficient cells by wild-type Nbs1 or mutants of Nbs1 defective in the FHA domain or nonphosphorylatable by ATM, but not by Nbs1 lacking the Mre11-interaction domain. Our data indicate that apart from its role downstream of ATM in the DNA damage checkpoint network, the MRN complex serves also as a modulator/amplifier of ATM activity. Although not absolutely required for ATM activation, the MRN nuclease complex may help reach the threshold activity of ATM necessary for optimal genome maintenance and prevention of cancer.

• MeSH
• Gene Amplification MeSH
• Ataxia Telangiectasia Mutated Proteins MeSH
• DNA-Binding Proteins MeSH
• Phosphorylation MeSH
• Phosphoserine metabolism   MeSH
• Radiation, Ionizing MeSH
• Nuclear Proteins chemistry   metabolism   deficiency   MeSH
• Kinetics MeSH
• Humans MeSH
• Tumor Suppressor Proteins MeSH
• Neoplasms genetics   prevention & control   MeSH
• DNA Damage genetics   MeSH
• Protein Serine-Threonine Kinases * metabolism   radiation effects   MeSH
• Cell Cycle Proteins chemistry   metabolism   MeSH
• Ataxia Telangiectasia MeSH
• Binding Sites MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH

p53 protein activity as a transcription factor can be activated in vivo by antibodies that target its C-terminal negative regulatory domain suggesting that cellular enzymes that target this domain may play a role in stimulating p53-dependent gene expression. A phospho-specific monoclonal antibody to the C-terminal Ser(315) phospho-epitope was used to determine whether phosphorylation of endogenous p53 at Ser(315) can be detected in vivo, whether steady-state Ser(315) phosphorylation increases or decreases in an irradiated cell, and whether this phosphorylation event activates or inhibits p53 in vivo. A native phospho-specific IgG binding assay was developed for quantitating the extent of p53 phosphorylation at Ser(315) where one, two, three, or four phosphates/tetramer could be defined after in vitro phosphorylation by cyclin-dependent protein kinases. Using this assay, near-stoichiometric Ser(315) phosphorylation of endogenous p53 protein was detected in vivo after UV irradiation of MCF7 and A375 cells, coinciding with elevated p53-dependent transcription. Transfection of the p53 gene with an alanine mutation at the Ser(315) site into Saos-2 cells gave rise to a form of p53 protein with a substantially reduced specific activity as a transcription factor. The treatment of cells with the cyclin-dependent protein kinase inhibitor Roscovitine promoted a reduction in the specific activity of endogenous p53 or ectopically expressed p53. These results indicate that the majority of p53 protein has been phosphorylated at Ser(315) after irradiation damage and identify a cyclin-dependent kinase pathway that plays a role in stimulating p53 function.

• MeSH
• Transcriptional Activation MeSH
• Point Mutation MeSH
• DNA metabolism   MeSH
• Phosphorylation MeSH
• Phosphoserine metabolism   MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Humans MeSH
• Antibodies, Monoclonal immunology   MeSH
• Tumor Cells, Cultured MeSH
• Tumor Suppressor Protein p53 genetics   immunology   metabolism   MeSH
• Purines pharmacology   MeSH
• Transcription Factors immunology   metabolism   MeSH
• Ultraviolet Rays MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH