Dual specificity phosphatase 7 (DUSP7) is a protein belonging to a broad group of phosphatases that can dephosphorylate phosphoserine/phosphothreonine as well as phosphotyrosine residues within the same substrate. DUSP7 has been linked to the negative regulation of mitogen activated protein kinases (MAPK), and in particular to the regulation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). MAPKs play an important role in embryonic development, where their duration, magnitude, and spatiotemporal activity must be strictly controlled by other proteins, among others by DUSPs. In this study, we focused on the effect of DUSP7 depletion on the in vitro differentiation of mouse embryonic stem (ES) cells. We showed that even though DUSP7 knock-out ES cells do retain some of their basic characteristics, when it comes to differentiation, they preferentially differentiate towards neural cells, while the formation of early cardiac mesoderm is repressed. Therefore, our data indicate that DUSP7 is necessary for the correct formation of neuroectoderm and cardiac mesoderm during the in vitro differentiation of ES cells.

• MeSH
• Dual Specificity Phosphatase 1 metabolism   MeSH
• Dual-Specificity Phosphatases genetics   metabolism   MeSH
• Phosphoserine MeSH
• Phosphothreonine MeSH
• Phosphotyrosine MeSH
• Mesoderm metabolism   MeSH
• Mitogen-Activated Protein Kinases metabolism   MeSH
• Mouse Embryonic Stem Cells * metabolism   MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Dusp7 protein, mouse MeSH Browser
• Dual Specificity Phosphatase 1 MeSH
• Dual-Specificity Phosphatases MeSH
• Phosphoserine MeSH
• Phosphothreonine MeSH
• Phosphotyrosine MeSH
• Mitogen-Activated Protein Kinases MeSH

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
• Names of Substances
• Phosphoserine MeSH
• Phosphothreonine MeSH
• Casein Kinase I MeSH
• Multiprotein Complexes MeSH
• Protein Serine-Threonine Kinases MeSH
• tau-tubulin kinase MeSH Browser

The discrete activation of individual caspases is essential during T-cell development, activation, and apoptosis. Humans carrying nonfunctional caspase-8 and caspase-8 conditional knockout mice exhibit several defects in the progression of naive CD4⁺ T cells to the effector stage. MST1, a key kinase of the Hippo signaling pathway, is often presented as a substrate of caspases, and its cleavage by caspases potentiates its activity. Several studies have focused on the involvement of MST1 in caspase activation and also reported several defects in the immune system function caused by MST1 deficiency. Here, we show the rapid activation of the MEK-ERK-MST1 axis together with the cleavage and activation of caspase-3, -6, -7, -8, and -9 after PI3K signaling blockade by the selective inhibitor GDC-0941 in Jurkat T cells. We determined the phosphorylation pattern of MST1 using a phosphoproteomic approach and identified two amino acid residues phosphorylated in an ERK-dependent manner after GDC-0941 treatment together with a novel phosphorylation site at S21 residue, which was extensively phosphorylated in an ERK-independent manner during PI3K signaling blockade. Using caspase inhibitors and the inhibition of MST1 expression using siRNA, we identified an exclusive role of the MEK-ERK-MST1 axis in the activation of initiator caspase-8, which in turn activates executive caspase-3/-7 that finally potentiate MST1 proteolytic cleavage. This mechanism forms a positive feed-back loop that amplifies the activation of MST1 together with apoptotic response in Jurkat T cells during PI3K inhibition. Altogether, we propose a novel MEK-ERK-MST1-CASP8-CASP3/7 apoptotic pathway in Jurkat T cells and believe that the regulation of this pathway can open novel possibilities in systemic and cancer therapies.

• Keywords
• AKT, ERK, Hippo/MST1, MEK, PI3K, apoptosis, caspase,
• MeSH
• Enzyme Activation drug effects   MeSH
• Apoptosis drug effects   MeSH
• Models, Biological MeSH
• Down-Regulation drug effects   MeSH
• Phosphatidylinositol 3-Kinases metabolism   MeSH
• Phosphorylation drug effects   MeSH
• Phosphothreonine metabolism   MeSH
• Hepatocyte Growth Factor chemistry   metabolism   MeSH
• Indazoles pharmacology   MeSH
• Phosphoinositide-3 Kinase Inhibitors MeSH
• Caspase Inhibitors pharmacology   MeSH
• Jurkat Cells MeSH
• Caspases metabolism   MeSH
• Humans MeSH
• MAP Kinase Signaling System drug effects   MeSH
• Mitogen-Activated Protein Kinase Kinases metabolism   MeSH
• Piperazines pharmacology   MeSH
• Proteolysis drug effects   MeSH
• Proto-Oncogene Proteins chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sulfonamides pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2-(1H-indazol-4-yl)-6-(4-methanesulfonylpiperazin-1-ylmethyl)-4-morpholin-4-ylthieno(3,2-d)pyrimidine MeSH Browser
• Phosphothreonine MeSH
• Hepatocyte Growth Factor MeSH
• Indazoles MeSH
• Phosphoinositide-3 Kinase Inhibitors MeSH
• Caspase Inhibitors MeSH
• Caspases MeSH
• macrophage stimulating protein MeSH Browser
• Mitogen-Activated Protein Kinase Kinases MeSH
• Piperazines MeSH
• Proto-Oncogene Proteins MeSH
• SCH772984 MeSH Browser
• Sulfonamides MeSH

Protein p130Cas constitutes an adaptor protein mainly involved in integrin signaling downstream of Src kinase. Owing to its modular structure, p130Cas acts as a general regulator of cancer cell growth and invasiveness induced by different oncogenes. However, other mechanisms of p130Cas signaling leading to malignant progression are poorly understood. Here, we show a novel interaction of p130Cas with Ser/Thr kinase PKN3, which is implicated in prostate and breast cancer growth downstream of phosphoinositide 3-kinase. This direct interaction is mediated by the p130Cas SH3 domain and the centrally located PKN3 polyproline sequence. PKN3 is the first identified Ser/Thr kinase to bind and phosphorylate p130Cas and to colocalize with p130Cas in cell structures that have a pro-invasive function. Moreover, the PKN3-p130Cas interaction is important for mouse embryonic fibroblast growth and invasiveness independent of Src transformation, indicating a mechanism distinct from that previously characterized for p130Cas. Together, our results suggest that the PKN3-p130Cas complex represents an attractive therapeutic target in late-stage malignancies.

• Keywords
• CAS, BCAR1, PKN3, SH3, Src, p130Cas,
• MeSH
• Fibroblasts metabolism   MeSH
• Phosphorylation MeSH
• Phosphothreonine metabolism   MeSH
• Neoplasm Invasiveness MeSH
• Stress Fibers metabolism   MeSH
• Humans MeSH
• Mice, Nude MeSH
• Neoplasms metabolism   pathology   MeSH
• Podosomes metabolism   MeSH
• Cell Movement MeSH
• Cell Proliferation MeSH
• Protein Kinase C metabolism   MeSH
• Pseudopodia metabolism   MeSH
• src-Family Kinases metabolism   MeSH
• Crk-Associated Substrate Protein metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphothreonine MeSH
• protein kinase N MeSH Browser
• Protein Kinase C MeSH
• src-Family Kinases MeSH
• Crk-Associated Substrate Protein MeSH

Cell cycle control must be modified at meiosis to allow two divisions to follow a single round of DNA replication, resulting in ploidy reduction. The mechanisms that ensure meiosis termination at the end of the second and not at the end of first division are poorly understood. We show here that Arabidopsis thaliana TDM1, which has been previously shown to be essential for meiotic termination, interacts directly with the Anaphase-Promoting Complex. Further, mutations in TDM1 in a conserved putative Cyclin-Dependant Kinase (CDK) phosphorylation site (T16-P17) dominantly provoked premature meiosis termination after the first division, and the production of diploid spores and gametes. The CDKA;1-CYCA1.2/TAM complex, which is required to prevent premature meiotic exit, phosphorylated TDM1 at T16 in vitro. Finally, while CYCA1;2/TAM was previously shown to be expressed only at meiosis I, TDM1 is present throughout meiosis. These data, together with epistasis analysis, lead us to propose that TDM1 is an APC/C component whose function is to ensure meiosis termination at the end of meiosis II, and whose activity is inhibited at meiosis I by CDKA;1-TAM-mediated phosphorylation to prevent premature meiotic exit. This provides a molecular mechanism for the differential decision of performing an additional round of division, or not, at the end of meiosis I and II, respectively.

• MeSH
• Anaphase-Promoting Complex-Cyclosome metabolism   MeSH
• Arabidopsis cytology   genetics   MeSH
• Models, Biological MeSH
• Chromosomes, Plant genetics   MeSH
• Cyclins genetics   metabolism   MeSH
• Genes, Dominant MeSH
• Phosphorylation MeSH
• Phosphothreonine metabolism   MeSH
• Epistasis, Genetic MeSH
• Genetic Testing MeSH
• Meiosis * MeSH
• Mutation genetics   MeSH
• Protein Subunits metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Tetraploidy MeSH
• Tubulin metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anaphase-Promoting Complex-Cyclosome MeSH
• Cyclins MeSH
• Phosphothreonine MeSH
• Protein Subunits MeSH
• Arabidopsis Proteins MeSH
• TDM1 protein, Arabidopsis MeSH Browser
• Tubulin MeSH

Responses of adipose cells to adrenoceptor regulation, including that of β-adrenoceptor (AR), and the signalling machinery involved in these responses are not sufficiently understood; information that is helpful for elucidating the adrenoceptor (adrenergic and β-AR)-responsive machinery is insufficient. We examined phospho-Thr-172 AMPK production in mouse-derived 3T3-L1 adipocytes treated with epinephrine or CL316243 (a β3-AR agonist) for 15 min. We also examined MAPK activation or G protein-associated PI3K activation or -associated PI3K p85 complex formation in rat epididymal (white) adipocytes treated with CL316243 for 15 min or aluminum fluoride (a G-protein signalling activator) for 20 min. Furthermore, we examined the effect of PTX (a trimeric G-protein inactivator) on p85 complex formation induced by aluminum fluoride treatment. Western blot analysis revealed that epinephrine or CL316243 treatment increased the phospho- Thr-172 AMPK (an active form of AMPK) level in 3T3-L1 adipocytes. Activated kinase analysis with a specific substrate showed that CL316243 or aluminum fluoride treatment activated MAPK in rat adipocytes. Immunoprecipitation experiments with a G-protein β subunit (Gβ) antibody showed that treatment of rat adipocytes with CL316243 activated PI3K and increased the PI3K p85 level in the Gβ antibody immunoprecipitates. Such an increase in the p85 level was similarly elicited by aluminum fluoride treatment in a PTX-sensitive manner. Our results provide possible clues for clarifying the signalling machinery involved in adrenoceptor responses, including those of β3-AR, in mouse-derived adipocytes and rat white adipocytes. Our findings advance the understanding of responses to adrenoceptor regulation in adipose cells and of the cellular signalling machinery present in the cells.

• MeSH
• Epinephrine pharmacology   MeSH
• Adipocytes, White enzymology   MeSH
• 3T3-L1 Cells MeSH
• Dioxoles pharmacology   MeSH
• Fluorides pharmacology   MeSH
• Phosphatidylinositol 3-Kinases metabolism   MeSH
• Phosphothreonine metabolism   MeSH
• Immunoprecipitation MeSH
• Rats MeSH
• Mitogen-Activated Protein Kinases metabolism   MeSH
• Mice MeSH
• Pertussis Toxin pharmacology   MeSH
• AMP-Activated Protein Kinases metabolism   MeSH
• GTP-Binding Proteins metabolism   MeSH
• Aluminum Compounds pharmacology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Epinephrine MeSH
• aluminum fluoride MeSH Browser
• Dioxoles MeSH
• disodium (R,R)-5-(2-((2-(3-chlorophenyl)-2-hydroxyethyl)-amino)propyl)-1,3-benzodioxole-2,3-dicarboxylate MeSH Browser
• Fluorides MeSH
• Phosphatidylinositol 3-Kinases MeSH
• Phosphothreonine MeSH
• Mitogen-Activated Protein Kinases MeSH
• Pertussis Toxin MeSH
• AMP-Activated Protein Kinases MeSH
• GTP-Binding Proteins MeSH
• Aluminum Compounds 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
• Centromere Protein A MeSH
• Pregnancy MeSH
• Check Tag
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Autoantigens MeSH
• CENPA protein, human MeSH Browser
• Chromosomal Proteins, Non-Histone MeSH
• Phosphoserine MeSH
• Phosphothreonine MeSH
• Histones MeSH
• Neoplasm Proteins MeSH
• Centromere Protein A MeSH

The existence of phosphoprotein phosphatase (PPP) in aerial mycelium of S. granaticolor was demonstrated. Using inhibitors of serine and/or threonine PPP and specifically labeled substrate it was found that the PPP is of the serine and/or threonine type.

• MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Phosphoserine metabolism   MeSH
• Phosphothreonine metabolism   MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Phosphoprotein Phosphatases analysis   antagonists & inhibitors   metabolism   MeSH
• Streptomyces enzymology   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphoserine MeSH
• Phosphothreonine MeSH
• Enzyme Inhibitors MeSH
• Phosphoprotein Phosphatases MeSH

We have cloned and sequenced the tuf1 gene from a kirromycin-producing strain of Streptomyces collinus. The gene encodes a polypeptide of 396 amino acid residues with a molecular weight of 43,849. The protein shows 97% identity with EF-Tu1 of S. coelicolor and is sensitive to kirromycin. EF-Tu-dependent translation of poly(U) was reduced to 50% in the presence of 0.25 microM kirromycin. Using high resolution two-dimensional electrophoresis and specific immunodetection with monoclonal antibodies we found that the EF-Tu1 is phosphorylated on threonine and that serine is the second phosphate-accepting amino acid. EF-Tu1 phosphorylated on threonine and serine residues was detected among the S150 supernatant proteins of vegetative cells, aerial mycelium and spores. The level of phosphorylated EF-Tu1 varied during the growth and differentiation.

• MeSH
• Drug Resistance, Microbial MeSH
• DNA, Bacterial chemistry   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Peptide Elongation Factor Tu antagonists & inhibitors   metabolism   MeSH
• Escherichia coli MeSH
• Phosphorylation MeSH
• Phosphoserine metabolism   MeSH
• Phosphothreonine metabolism   MeSH
• Immunosorbent Techniques MeSH
• Isoelectric Focusing MeSH
• Molecular Sequence Data MeSH
• Pyridones pharmacology   MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA * MeSH
• Streptomyces genetics   growth & development   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Bacterial MeSH
• Peptide Elongation Factor Tu MeSH
• Phosphoserine MeSH
• Phosphothreonine MeSH
• mocimycin MeSH Browser
• Pyridones MeSH