RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1-5. Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein6-is phosphorylated during spliceosome activation7-10, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the Bact complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.

• MeSH
• aktivace enzymů účinky léků   MeSH
• chinolony farmakologie   MeSH
• chromatin metabolismus   MeSH
• cyklin-dependentní kinasy * antagonisté a inhibitory   metabolismus   MeSH
• fosfoproteiny * chemie   metabolismus   MeSH
• fosforylace MeSH
• malý jaderný ribonukleoprotein U2 * chemie   metabolismus   MeSH
• prekurzory RNA * genetika   metabolismus   MeSH
• sestřih RNA * účinky léků   MeSH
• spliceozomy * účinky léků   metabolismus   MeSH
• threonin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chinolony MeSH
• chromatin MeSH
• cyklin-dependentní kinasy * MeSH
• fosfoproteiny * MeSH
• malý jaderný ribonukleoprotein U2 * MeSH
• OTS964 MeSH Prohlížeč
• prekurzory RNA * MeSH
• threonin MeSH

Cannabinoid receptor 1 (CB1R), a G protein-coupled receptor, plays a fundamental role in synaptic plasticity. Abnormal activity and deregulation of CB1R signaling result in a broad spectrum of pathological conditions. CB1R signaling is regulated by receptor desensitization including phosphorylation of residues within the intracellular C terminus by G protein-coupled receptor kinases (GRKs) that may lead to endocytosis. Furthermore, CB1R signaling is regulated by the protein Src homology 3-domain growth factor receptor-bound 2-like (SGIP1) that hinders receptor internalization, while enhancing CB1R association with β-arrestin. It has been postulated that phosphorylation of two clusters of serine/threonine residues, 425 SMGDS429 and 460 TMSVSTDTS468 , within the CB1R C-tail controls dynamics of the association between receptor and its interaction partners involved in desensitization. Several molecular determinants of these events are still not well understood. We hypothesized that the dynamics of these interactions are modulated by SGIP1. Using a panel of CB1Rs mutated in the aforementioned serine and threonine residues, together with an array of Bioluminescence energy transfer-based (BRET) sensors, we discovered that GRK3 forms complexes with Gβγ subunits of G proteins that largely independent of GRK3's interaction with CB1R. Furthermore, CB1R interacts only with activated GRK3. Interestingly, phosphorylation of two specific residues on CB1R triggers GRK3 dissociation from the desensitized receptor. SGIP1 increases the association of GRK3 with Gβγ subunits of G proteins, and with CB1R. Altogether, our data suggest that the CB1R signalosome complex is dynamically controlled by sequential phosphorylation of the receptor C-tail and is also modified by SGIP1.

• Klíčová slova
• G protein-coupled receptor kinase, G protein-coupled receptors, SGIP1, cannabinoid receptor 1, phosphorylation, β-arrestin,
• MeSH
• fosforylace MeSH
• kinetika MeSH
• proteiny vázající GTP * MeSH
• receptory kanabinoidní metabolismus   MeSH
• serin metabolismus   MeSH
• threonin metabolismus   MeSH
• transportní proteiny * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny vázající GTP * MeSH
• receptory kanabinoidní MeSH
• serin MeSH
• threonin MeSH
• transportní proteiny * MeSH

The absence of state transitions in a Nt(Hn) cybrid is due to a cleavage of the threonine residue from the misprocessed N-terminus of the LHCII polypeptides. The cooperation between the nucleus and chloroplast genomes is essential for plant photosynthetic fitness. The rapid and specific interactions between nucleus-encoded and chloroplast-encoded proteins are under intense investigation with potential for applications in agriculture and renewable energy technology. Here, we present a novel model for photosynthesis research in which alien henbane (Hyoscyamus niger) chloroplasts function on the nuclear background of a tobacco (Nicotiana tabacum). The result of this coupling is a cytoplasmic hybrid (cybrid) with inhibited state transitions-a mechanism responsible for balancing energy absorption between photosystems. Protein analysis showed differences in the LHCII composition of the cybrid plants. SDS-PAGE analysis revealed a novel banding pattern in the cybrids with at least one additional 'LHCII' band compared to the wild-type parental species. Proteomic work suggested that the N-terminus of at least some of the cybrid Lhcb proteins was missing. These findings provide a mechanistic explanation for the lack of state transitions-the N-terminal truncation of the Lhcb proteins in the cybrid included the threonine residue that is phosphorylated/dephosphorylated in order to trigger state transitions and therefore crucial energy balancing mechanism in plants.

• Klíčová slova
• Cybrid, LHCII, Non-photochemical quenching, State transitions,
• MeSH
• buněčné jádro metabolismus   MeSH
• chloroplasty metabolismus   MeSH
• fosforylace MeSH
• fotosyntéza MeSH
• fotosystém II - proteinový komplex genetika   metabolismus   MeSH
• genom chloroplastový genetika   MeSH
• genom rostlinný genetika   MeSH
• proteomika MeSH
• světlosběrné proteinové komplexy genetika   metabolismus   MeSH
• tabák genetika   fyziologie   MeSH
• threonin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fotosystém II - proteinový komplex MeSH
• světlosběrné proteinové komplexy MeSH
• threonin MeSH

During mouse postnatal eye development, the embryonic hyaloid vascular network regresses from the vitreous as an adaption for high-acuity vision. This process occurs with precisely controlled timing. Here, we show that opsin 5 (OPN5; also known as neuropsin)-dependent retinal light responses regulate vascular development in the postnatal eye. In Opn5-null mice, hyaloid vessels regress precociously. We demonstrate that 380-nm light stimulation via OPN5 and VGAT (the vesicular GABA/glycine transporter) in retinal ganglion cells enhances the activity of inner retinal DAT (also known as SLC6A3; a dopamine reuptake transporter) and thus suppresses vitreal dopamine. In turn, dopamine acts directly on hyaloid vascular endothelial cells to suppress the activity of vascular endothelial growth factor receptor 2 (VEGFR2) and promote hyaloid vessel regression. With OPN5 loss of function, the vitreous dopamine level is elevated and results in premature hyaloid regression. These investigations identify violet light as a developmental timing cue that, via an OPN5-dopamine pathway, regulates optic axis clearance in preparation for visual function.

• MeSH
• cévní endotel metabolismus   MeSH
• dopamin metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• oči krevní zásobení   enzymologie   růst a vývoj   metabolismus   MeSH
• opsiny genetika   metabolismus   MeSH
• proteiny přenášející dopamin přes plazmatickou membránu antagonisté a inhibitory   chemie   metabolismus   MeSH
• retinální gangliové buňky metabolismus   účinky záření   MeSH
• sklivec metabolismus   MeSH
• světlo * MeSH
• threonin metabolismus   MeSH
• transportéry VIAAT fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• dopamin MeSH
• membránové proteiny MeSH
• OPN5 protein, mouse MeSH Prohlížeč
• opsiny MeSH
• proteiny přenášející dopamin přes plazmatickou membránu MeSH
• Slc6a3 protein, mouse MeSH Prohlížeč
• threonin MeSH
• transportéry VIAAT MeSH
• Viaat protein, mouse MeSH Prohlížeč

Phosphorylation of serine, threonine, and tyrosine is one of the most frequently occurring and crucial post-translational modifications of proteins often associated with important structural and functional changes. We investigated the direct effect of phosphorylation on the intrinsic conformational preferences of amino acids as a potential trigger of larger structural events. We conducted a comparative study of force fields on terminally capped amino acids (dipeptides) as the simplest model for phosphorylation. Our bias-exchange metadynamics simulations revealed that all model dipeptides sampled a great heterogeneity of ensembles affected by introduction of mono- and dianionic phosphate groups. However, the detected changes in populations of backbone conformers and side-chain rotamers did not reveal a strong discriminatory shift in preferences, as could be anticipated for the bulky, charged phosphate group. Furthermore, the AMBER and CHARMM force fields provided inconsistent populations of individual conformers as well as net structural trends upon phosphorylation. Detailed analysis of ensembles revealed competition between hydration and formation of internal hydrogen bonds involving amide hydrogens and the phosphate group. The observed difference in hydration free energy and potential for hydrogen bonding in individual force fields could be attributed to the different partial atomic charges used in each force field and, hence, the different parametrization strategies. Nevertheless, conformational propensities and net structural changes upon phosphorylation are difficult to extract from experimental measurements, and existing experimental data provide limited guidance for force field assessment and further development.

• MeSH
• fosforylace MeSH
• konformace proteinů MeSH
• serin metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• threonin metabolismus   MeSH
• tyrosin metabolismus   MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• serin MeSH
• threonin MeSH
• tyrosin MeSH

The transient receptor potential vanilloid 3 (TRPV3) channel is a Ca2+-permeable thermosensitive ion channel widely expressed in keratinocytes, where together with epidermal growth factor receptor (EGFR) forms a signaling complex regulating epidermal homeostasis. Proper signaling through this complex is achieved and maintained via several pathways in which TRPV3 activation is absolutely required. Results of recent studies have suggested that low-level constitutive activity of TRPV3 induces EGFR-dependent signaling that, in turn, amplifies TRPV3 via activation of the mitogen-activated protein kinase ERK in a positive feedback loop. Here, we explored the molecular mechanism that increases TRPV3 activity through EGFR activation. We used mutagenesis and whole-cell patch clamp experiments on TRPV3 channels endogenously expressed in an immortalized human keratinocyte cell line (HaCaT) and in transiently transfected HEK293T cells and found that the sensitizing effect of EGFR on TRPV3 is mediated by ERK. We observed that ERK-mediated phosphorylation of TRPV3 alters its responsiveness to repeated chemical stimuli. Among several putative ERK phosphorylation sites, we identified threonine 264 in the N-terminal ankyrin repeat domain as the most critical site for the ERK-dependent modulation of TRPV3 channel activity. Of note, Thr264 is in close vicinity to a structurally and functionally important TRPV3 region comprising an atypical finger 3 and oxygen-dependent hydroxylation site. In summary, our findings indicate that Thr264 in TRPV3 is a key ERK phosphorylation site mediating EGFR-induced sensitization of the channel to stimulate signaling pathways involved in regulating skin homeostasis.

• Klíčová slova
• TRP channels, ankyrin repeat domain, epidermal growth factor receptor (EGFR), extracellular-signal-regulated kinase (ERK), keratinocyte, phosphorylation, transient receptor potential channels (TRP channels),
• MeSH
• cymeny MeSH
• epidermální růstový faktor metabolismus   MeSH
• erbB receptory agonisté   metabolismus   MeSH
• fosforylace účinky léků   MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• kationtové kanály TRPV agonisté   chemie   genetika   metabolismus   MeSH
• keratinocyty účinky léků   enzymologie   metabolismus   MeSH
• lidé MeSH
• MAP kinasový signální systém * účinky léků   MeSH
• metoda terčíkového zámku MeSH
• mitogenem aktivovaná proteinkinasa 3 chemie   genetika   metabolismus   MeSH
• modulátory membránového transportu farmakologie   MeSH
• monoterpeny farmakologie   MeSH
• mutace MeSH
• mutageneze cílená MeSH
• posttranslační úpravy proteinů účinky léků   MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• sloučeniny boru farmakologie   MeSH
• threonin metabolismus   MeSH
• transformované buněčné linie MeSH
• upregulace * účinky léků   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2-aminoethoxydiphenyl borate MeSH Prohlížeč
• carvacrol MeSH Prohlížeč
• cymeny MeSH
• EGFR protein, human MeSH Prohlížeč
• epidermální růstový faktor MeSH
• erbB receptory MeSH
• kationtové kanály TRPV MeSH
• mitogenem aktivovaná proteinkinasa 3 MeSH
• modulátory membránového transportu MeSH
• monoterpeny MeSH
• rekombinantní proteiny MeSH
• sloučeniny boru MeSH
• threonin MeSH
• TRPV3 protein, human MeSH Prohlížeč
• zelené fluorescenční proteiny MeSH

Phosphorylation patterns of the C-terminal domain (CTD) of largest subunit of RNA polymerase II (called the CTD code) orchestrate the recruitment of RNA processing and transcription factors. Recent studies showed that not only serines and tyrosines but also threonines of the CTD can be phosphorylated with a number of functional consequences, including the interaction with yeast transcription termination factor, Rtt103p. Here, we report the solution structure of the Rtt103p CTD-interacting domain (CID) bound to Thr4 phosphorylated CTD, a poorly understood letter of the CTD code. The structure reveals a direct recognition of the phospho-Thr4 mark by Rtt103p CID and extensive interactions involving residues from three repeats of the CTD heptad. Intriguingly, Rtt103p's CID binds equally well Thr4 and Ser2 phosphorylated CTD A doubly phosphorylated CTD at Ser2 and Thr4 diminishes its binding affinity due to electrostatic repulsion. Our structural data suggest that the recruitment of a CID-containing CTD-binding factor may be coded by more than one letter of the CTD code.

• Klíčová slova
• NMR, RNA processing, RNAPII CTD code, structural biology,
• MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• proteinkinasy metabolismus   MeSH
• proteolýza MeSH
• RNA-polymerasa II chemie   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• serin metabolismus   MeSH
• terciární struktura proteinů MeSH
• threonin chemie   metabolismus   MeSH
• transkripční faktory chemie   metabolismus   MeSH
• tyrosin metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteinkinasy MeSH
• RNA-polymerasa II MeSH
• Rtt103 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• serin MeSH
• threonin MeSH
• transkripční faktory MeSH
• tyrosin MeSH

Low temperature magnetic circular dichroism (LT MCD) spectroscopy in combination with quantum-chemical calculations are used to define the electronic structure associated with the geometric structure of the Fe(IV)═O intermediate in SyrB2 that was previously determined by nuclear resonance vibrational spectroscopy. These studies elucidate key frontier molecular orbitals (FMOs) and their contribution to H atom abstraction reactivity. The VT MCD spectra of the enzymatic S = 2 Fe(IV)═O intermediate with Br(-) ligation contain information-rich features that largely parallel the corresponding spectra of the S = 2 model complex (TMG3tren)Fe(IV)═O (Srnec, M.; Wong, S. D.; England, J; Que, L; Solomon, E. I. Proc. Natl. Acad. Sci. USA 2012, 109, 14326-14331). However, quantitative differences are observed that correlate with π-anisotropy and oxo donor strength that perturb FMOs and affect reactivity. Due to π-anisotropy, the Fe(IV)═O active site exhibits enhanced reactivity in the direction of the substrate cavity that proceeds through a π-channel that is controlled by perpendicular orientation of the substrate C-H bond relative to the halide-Fe(IV)═O plane. Also, the increased intrinsic reactivity of the SyrB2 intermediate relative to the ferryl model complex is correlated to a higher oxyl character of the Fe(IV)═O at the transition states resulting from the weaker ligand field of the halogenase.

• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• cirkulární dichroismus MeSH
• glutaráty chemie   metabolismus   MeSH
• kvantová teorie MeSH
• molekulární modely MeSH
• nehemové proteiny obsahující železo chemie   metabolismus   MeSH
• oxidoreduktasy chemie   metabolismus   MeSH
• sloučeniny železa chemie   metabolismus   MeSH
• threonin chemie   metabolismus   MeSH
• vodík chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• bakteriální proteiny MeSH
• glutaráty MeSH
• nehemové proteiny obsahující železo MeSH
• oxidoreduktasy MeSH
• sloučeniny železa MeSH
• syringomycin MeSH Prohlížeč
• threonin MeSH
• vodík MeSH

The effect of cryopreservation on the protein phosphorylation/dephosphorylation pattern of common carp (Cyprinus carpio) sperm is described. Sperm was diluted in dimethyl sulfoxide (DMSO) and ethylene glycol (EG)-based extenders, followed by equilibration, freezing, and thawing. Proteins extracted from fresh and cryopreserved spermatozoa were separated on SDS-PAGE and two-dimensional gel electrophoresis, blotted on polyvinylidene difluoride membrane, and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies. For the subsequent protein identification we used matrix-associated laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated that cryopreservation with either DMSO or EG extender significantly altered the phosphorylation state of sperm proteins on tyrosine or threonine residues. A dramatic decrease in tyrosine phosphorylation was detected in the cryopreservation procedures with DMSO extender. Endoplasmin, transketolase, and S-adenosylhomocysteine hydrolase were identified as proteins that play a key role in cellular stress responses and oxidation and/or reduction reactions. Results indicate that the phosphorylation and/or dephosphorylation modifications of sperm proteins that occur during cryopreservation could stimulate a series of biochemical effects interfering with spermatozoa function and leading to a loss of motility and fertilization ability. Our findings indicated that use of EG extender provided superior protein preservation during sperm storage.

• MeSH
• fosforylace účinky léků   MeSH
• kapři * fyziologie   MeSH
• kryoprezervace veterinární   MeSH
• kryoprotektivní látky farmakologie   MeSH
• motilita spermií účinky léků   MeSH
• proteinkinasy metabolismus   MeSH
• spermie účinky léků   fyziologie   MeSH
• threonin metabolismus   MeSH
• tyrosin metabolismus   MeSH
• uchování spermatu metody   veterinární   MeSH
• upregulace účinky léků   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kryoprotektivní látky MeSH
• proteinkinasy MeSH
• threonin MeSH
• tyrosin MeSH

The structures of fully active cyclin-dependent kinase-2 (CDK2) complexed with ATP and peptide substrate, CDK2 after the catalytic reaction, and CDK2 inhibited by phosphorylation at Thr14/Tyr15 were studied using molecular dynamics (MD) simulations. The structural details of the CDK2 catalytic site and CDK2 substrate binding box were described. Comparison of MD simulations of inhibited complexes of CDK2 was used to help understand the role of inhibitory phosphorylation at Thr14/Tyr15. Phosphorylation at Thr14/Tyr15 causes ATP misalignment for the phosphate-group transfer, changes in the Mg(2+) coordination sphere, and changes in the H-bond network formed by CDK2 catalytic residues (Asp127, Lys129, Asn132). The inhibitory phosphorylation causes the G-loop to shift from the ATP binding site, which leads to opening of the CDK2 substrate binding box, thus probably weakening substrate binding. All these effects explain the decrease in kinase activity observed after inhibitory phosphorylation at Thr14/Tyr15 in the G-loop. Interaction of the peptide substrate, and the phosphorylated peptide product, with CDK2 was also studied and compared. These results broaden hypotheses drawn from our previous MD studies as to why a basic residue (Arg/Lys) is preferred at the P(+2) substrate position.

• MeSH
• cyklin-dependentní kinasa 2 antagonisté a inhibitory   chemie   metabolismus   MeSH
• fosforylace MeSH
• katalytická doména MeSH
• lidé MeSH
• sekundární struktura proteinů MeSH
• threonin chemie   metabolismus   MeSH
• tyrosin chemie   metabolismus   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklin-dependentní kinasa 2 MeSH
• threonin MeSH
• tyrosin MeSH