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
• aminokyselinové motivy MeSH
• cilie metabolismus   MeSH
• fosforylace MeSH
• fosfoserin metabolismus   MeSH
• fosfothreonin metabolismus   MeSH
• HEK293 buňky MeSH
• kaseinkinasa I metabolismus   MeSH
• lidé MeSH
• multiproteinové komplexy metabolismus   MeSH
• organogeneze * MeSH
• protein-serin-threoninkinasy chemie   metabolismus   MeSH
• substrátová specifita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• MeSH
• fibroblasty metabolismus   MeSH
• fosforylace MeSH
• fosfothreonin metabolismus   MeSH
• invazivní růst nádoru MeSH
• kontraktilní svazky metabolismus   MeSH
• lidé MeSH
• myši nahé MeSH
• nádory metabolismus   patologie   MeSH
• podozomy metabolismus   MeSH
• pohyb buněk MeSH
• proliferace buněk MeSH
• proteinkinasa C metabolismus   MeSH
• pseudopodia metabolismus   MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• substrátový protein asociovaný s Crk metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• anafázi podporující komplex metabolismus   MeSH
• Arabidopsis cytologie   genetika   MeSH
• biologické modely MeSH
• chromozomy rostlin genetika   MeSH
• cykliny genetika   metabolismus   MeSH
• dominantní geny MeSH
• fosforylace MeSH
• fosfothreonin metabolismus   MeSH
• genetická epistáze MeSH
• genetické testování MeSH
• meióza * MeSH
• mutace genetika   MeSH
• podjednotky proteinů metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• tetraploidie MeSH
• tubulin metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• adrenalin farmakologie   MeSH
• bílé tukové buňky enzymologie   MeSH
• buňky 3T3-L1 MeSH
• dioxoly farmakologie   MeSH
• fluoridy farmakologie   MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• fosfothreonin metabolismus   MeSH
• imunoprecipitace MeSH
• krysa rodu rattus MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• myši MeSH
• pertusový toxin farmakologie   MeSH
• proteinkinasy aktivované AMP metabolismus   MeSH
• proteiny vázající GTP metabolismus   MeSH
• sloučeniny hliníku farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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
• adenokarcinom patologie   MeSH
• aparát dělícího vřeténka metabolismus   ultrastruktura   MeSH
• autoantigeny chemie   fyziologie   MeSH
• biologický transport MeSH
• centromera metabolismus   MeSH
• chromozomální proteiny, nehistonové chemie   fyziologie   MeSH
• cytokineze fyziologie   MeSH
• fluorescenční mikroskopie MeSH
• fosforylace MeSH
• fosfoserin metabolismus   MeSH
• fosfothreonin metabolismus   MeSH
• histony metabolismus   MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• MFC-7 buňky cytologie   metabolismus   MeSH
• mitóza fyziologie   MeSH
• nádorové proteiny chemie   fyziologie   MeSH
• nádory prsu patologie   MeSH
• posttranslační úpravy proteinů MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The ability to separate the isoforms of human tumour suppressor protein p53 expressed in insect cells using heparin-Sepharose correlates with differences in the isoelectric point of p53, demonstrating that p53 can be heterogeneously modified and providing support for the use of insect cells as a model system for identifying novel signalling pathways that target p53. One p53 isoform that was reduced in its binding to the monoclonal antibody DO-1 could be stimulated in its binding to DO-1 by prior incubation with protein phosphatases, suggesting the presence of a previously unidentified N-terminal phosphorylation site capable of masking the DO-1 epitope. A synthetic peptide from the N-terminal domain of p53 containing phosphate at Ser(20) inhibited DO-1 binding, thus identifying the phosphorylation site responsible for DO-1 epitope masking. Monoclonal antibodies overlapping the DO-1 epitope were developed that are specific for phospho-Thr(18) (adjacent to the DO-1 epitope) and phospho-Ser(20) (within the DO-1 epitope) to determine whether direct evidence could be obtained for novel phosphorylation sites in human p53. A monoclonal antibody highly specific for phospho-Ser(20) detected significant phosphorylation of human p53 expressed in insect cells, whereas the relative proportion of p53 modified at Thr(18) was substantially lower. The relevance of these two novel phosphorylation sites to p53 regulation in human cells was made evident by the extensive phosphorylation of human p53 at Thr(18) and Ser(20) in a panel of human breast cancers with a wild-type p53 status. Phospho-Ser(20) or phospho-Thr(18) containing p53 peptides are as effective as the phospho-Ser(15) peptide at reducing mdm2 (mouse double minute 2) protein binding, indicating that the functional effects of these phosphorylation events might be to regulate the binding of heterologous proteins to p53. These results provide evidence in vivo for two novel phosphorylation sites within p53 at Ser(20) and Thr(18) that can affect p53 protein-protein interactions and indicate that some human cancers might have amplified one or more Ser(20) and Thr(18) kinase signalling cascades to modulate p53 activity.

• MeSH
• buněčné linie MeSH
• epitopy imunologie   metabolismus   MeSH
• fosfatasy metabolismus   MeSH
• fosforylace MeSH
• fosfoserin imunologie   metabolismus   MeSH
• fosfothreonin imunologie   metabolismus   MeSH
• izoelektrický bod MeSH
• jaderné proteiny * MeSH
• lidé MeSH
• monoklonální protilátky imunologie   MeSH
• nádorový supresorový protein p53 genetika   imunologie   metabolismus   MeSH
• nádory prsu imunologie   metabolismus   MeSH
• peptidové fragmenty chemická syntéza   imunologie   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• protein - isoformy genetika   imunologie   metabolismus   MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny * metabolismus   MeSH
• rekombinantní proteiny imunologie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce MeSH
• specificita protilátek MeSH
• Spodoptera MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH