GABAB receptors assemble from GABAB1 and GABAB2 subunits. GABAB2 additionally associates with auxiliary KCTD subunits (named after their K(+) channel tetramerization-domain). GABAB receptors couple to heterotrimeric G-proteins and activate inwardly-rectifying K(+) channels through the βγ subunits released from the G-protein. Receptor-activated K(+) currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K(+) currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G-protein βγ subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast desensitization of GABAB receptor-activated K(+) currents while protein kinase-A inhibition accelerates fast desensitization. Protein kinase-A fails to regulate fast desensitization in KCTD12 knock-out mice or knock-in mice with a serine-892 to alanine mutation, thus demonstrating that serine-892 phosphorylation regulates KCTD12-induced desensitization in vivo. Fast current desensitization is accelerated in hippocampal neurons carrying the serine-892 to alanine mutation, showing that tonic serine-892 phosphorylation normally limits KCTD12-induced desensitization. Tonic serine-892 phosphorylation is in turn promoted by assembly of receptors with KCTD12. This cross-regulation of serine-892 phosphorylation and KCTD12 activity sharpens the response during repeated receptor activation.

• MeSH
• Alanine genetics   metabolism   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Potassium metabolism   MeSH
• Phosphorylation MeSH
• Hippocampus cytology   metabolism   MeSH
• Cells, Cultured MeSH
• Patch-Clamp Techniques MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Neurons metabolism   MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• GTP-Binding Proteins metabolism   MeSH
• Receptors, GABA-B genetics   metabolism   MeSH
• Receptors, GABA genetics   metabolism   MeSH
• Serine genetics   metabolism   MeSH
• Amino Acid Substitution MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Phosphorylation, or dephosphorylation, is one of the most frequent post-translational modifications regulating protein-protein activity in eukaryotic cells. Whereas mature spermatozoa (as specialized cells) are transcriptionally inactive and do not synthesize new proteins, phosphorylation of sperm proteins is very important for the regulation of the sperm function. Although the post-testicular maturation of spermatozoa is a process common to all mammals, comparative studies showed significant differences in sperm surface proteins and the mechanisms of protein modification during the epididymal maturation. In our study, the evaluation of tyrosine phosphorylation, represented by the fluorescent patterns of used anti-phosphotyrosine antibodies (P-Tyr-01 and 4G10), in spermatozoa isolated from different regions of the epididymis - caput, corpus and cauda - was performed. Although in general both antibodies detected almost the same reaction patterns, we observed some dissimilarity associated with the binding specificity of the antibodies and also the segment-dependent manner of phosphorylated protein localization. These data were filled up by immunohistochemical analysis of testes and epididymides cryosections. Additionally, our phosphoproteomic study focused on evaluation of the changes in the pattern of tyrosine-phosphorylated proteins during the post-testicular maturation of bull spermatozoa (PY20 antibody). To summarize the results, an increasing trend of tyrosine phosphorylation of proteins during the maturation of bull sperm in the epididymis was consistently observed in all the methods/experiments.

• MeSH
• Epididymis cytology   MeSH
• Fluorescence MeSH
• Phosphorylation MeSH
• Phosphotyrosine metabolism   MeSH
• Proteins metabolism   MeSH
• Cattle MeSH
• Spermatozoa cytology   metabolism   MeSH
• Testis cytology   MeSH
• Sperm Maturation * MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequences of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale.

• MeSH
• Antioxidants metabolism   MeSH
• Flavin-Adenine Dinucleotide chemistry   MeSH
• Flavoproteins metabolism   MeSH
• Phosphorylation MeSH
• Humans MeSH
• Mutation MeSH
• NAD(P)H Dehydrogenase (Quinone) * metabolism   MeSH
• Neoplasms * genetics   MeSH
• Proteasome Endopeptidase Complex metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

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
• Enzyme Activation drug effects   MeSH
• Quinolones pharmacology   MeSH
• Chromatin metabolism   MeSH
• Cyclin-Dependent Kinases * antagonists & inhibitors   metabolism   MeSH
• Phosphoproteins * chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Ribonucleoprotein, U2 Small Nuclear * chemistry   metabolism   MeSH
• RNA Precursors * genetics   metabolism   MeSH
• RNA Splicing * drug effects   MeSH
• Spliceosomes * drug effects   metabolism   MeSH
• Threonine metabolism   MeSH
• Publication type
• Journal Article MeSH

Spermatozoa of externally fertilizing freshwater fish possess several different modes of motility activation. Spermatozoa of common carp (Cyprinus carpio L.) are activated by hypoosmolality, whereas spermatozoa of sterlet (Acipenser ruthenus) require Ca2+ and low concentration of K+ for motility activation. Intracellular signaling differs between these two species as well, particularly in terms of utilization of secondary messengers (cAMP and Ca2+), and kinase activities. The current study was performed in order to determine the importance of protein phosphorylation and protein kinases for activation of sperm motility in carp and sterlet. Treatment with kinase inhibitors indicates that protein kinases A and C (PKA and PKC) participate in spermatozoa motility of both species. Immunodetection of phospho-(Ser/Thr) PKA substrates shows that phosphorylated proteins are localized differently in spermatozoa of carp and sterlet. Strong phosphorylation of PKC substrate was observed in flagella of sterlet spermatozoa, whereas in carp sperm, PKC substrates were lightly phosphorylated in the midpiece and flagella. Motility activation induced either phosphorylation or dephosphorylation on serine, threonine and tyrosine residues of numerous proteins in carp and sterlet spermatozoa. Proteomic methods were used to identify proteins whose phosphorylation state changes upon the initiation of sperm motility. Numerous mitochondrial and glycolytic enzymes were identified in spermatozoa of both species, as well as axonemal proteins, heat shock proteins, septins and calcium-binding proteins. Our results contribute to an understanding of the roles of signaling molecules, protein kinases and protein phosphorylation in motility activation and regulation of two valuable fish species, C. carpio and A. ruthenus.

• MeSH
• Phosphorylation MeSH
• Carps * metabolism   MeSH
• Sperm Motility physiology   MeSH
• Protein Kinase C metabolism   MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• Proteomics MeSH
• Fish Proteins metabolism   MeSH
• Fishes * metabolism   MeSH
• Signal Transduction MeSH
• Spermatozoa metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The Bcl-2 protein is one of the key components of biochemical pathways controlling programmed cell death. The function of this protein can be regulated by posttranslational modifications. Phosphorylation of Bcl-2 has been considered to be significantly associated with cell cycle arrest in the G2/M phase of the cell cycle, and with cell death caused by defects of microtubule dynamics. This study shows that phosphorylation of Bcl-2 can be induced by heavy metals due to activation of the Jun N-terminal kinase pathway that is not linked to the G2/M cell cycle arrest. Furthermore, we demonstrate that hyperphosphorylated Bcl-2 protein is a more potent inhibitor of zinc-induced cell death than its hypophosphorylated mutant form. These data suggest that regulation of Bcl-2 protein function by phosphorylation is an important part of cell responses to stress.

• MeSH
• Apoptosis drug effects   MeSH
• Electrophoresis MeSH
• Financing, Organized MeSH
• Phosphorylation drug effects   MeSH
• Stress, Physiological drug effects   MeSH
• JNK Mitogen-Activated Protein Kinases metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Protein Processing, Post-Translational drug effects   MeSH
• Proto-Oncogene Proteins c-bcl-2 metabolism   MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• Signal Transduction drug effects   MeSH
• Metals, Heavy pharmacology   MeSH
• Zinc pharmacology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH

Purpose: Lacking effective targeted therapies, triple-negative breast cancer (TNBCs) is highly aggressive and metastatic disease, and remains clinically challenging breast cancer subtype to treat. Despite the survival dependency on the proteasome pathway genes, FDA-approved proteasome inhibitors induced minimal clinical response in breast cancer patients due to weak proteasome inhibition. Hence, developing effective targeted therapy using potent proteasome inhibitor is required. Methods: We evaluated anti-cancer activity of a potent proteasome inhibitor, marizomib, in vitro using breast cancer lines and in vivo using 4T1.2 murine syngeneic model, MDA-MB-231 xenografts, and patient-derived tumor xenografts. Global proteome profiling, western blots, and RT-qPCR were used to investigate the mechanism of action for marizomib. Effect of marizomib on lung and brain metastasis was evaluated using syngeneic 4T1BR4 murine TNBC model in vivo. Results: We show that marizomib inhibits multiple proteasome catalytic activities and induces a better anti-tumor response in TNBC cell lines and patient-derived xenografts alone and in combination with the standard-of-care chemotherapy. Mechanistically, we show that marizomib is a dual inhibitor of proteasome and oxidative phosphorylation (OXPHOS) in TNBCs. Marizomib reduces lung and brain metastases by reducing the number of circulating tumor cells and the expression of genes involved in the epithelial-to-mesenchymal transition. We demonstrate that marizomib-induced OXPHOS inhibition upregulates glycolysis to meet the energetic demands of TNBC cells and combined inhibition of glycolysis with marizomib leads to a synergistic anti-cancer activity. Conclusions: Our data provide a strong rationale for a clinical evaluation of marizomib in primary and metastatic TNBC patients.

• MeSH
• Apoptosis drug effects   genetics   MeSH
• Epithelial-Mesenchymal Transition drug effects   genetics   MeSH
• Proteasome Inhibitors therapeutic use   MeSH
• Lactones therapeutic use   MeSH
• Humans MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Oxidative Phosphorylation drug effects   MeSH
• Cell Proliferation drug effects   genetics   MeSH
• Proteasome Endopeptidase Complex drug effects   metabolism   MeSH
• Antineoplastic Agents therapeutic use   MeSH
• Pyrroles therapeutic use   MeSH
• Triple Negative Breast Neoplasms drug therapy   genetics   metabolism   MeSH
• Xenograft Model Antitumor Assays MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Recruitment of appropriate RNA processing factors to the site of transcription is controlled by post-translational modifications of the C-terminal domain (CTD) of RNA polymerase II (RNAP II). Here, we report the solution structure of the Ser5 phosphorylated (pSer5) CTD bound to Nrd1. The structure reveals a direct recognition of pSer5 by Nrd1 that requires the cis conformation of the upstream pSer5-Pro6 peptidyl-prolyl bond of the CTD. Mutations at the complex interface diminish binding affinity and impair processing or degradation of noncoding RNAs. These findings underpin the interplay between covalent and noncovalent changes in the CTD structure that constitute the CTD code.

• MeSH
• Phosphorylation MeSH
• Models, Molecular MeSH
• RNA, Untranslated metabolism   MeSH
• Proline metabolism   MeSH
• RNA-Binding Proteins chemistry   metabolism   MeSH
• RNA Polymerase II metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae cytology   enzymology   genetics   metabolism   MeSH
• Serine metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Cell Survival MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase-mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1's role in HR, with potential clinical implications for cancer treatment.

• MeSH
• Time Factors MeSH
• Chromatin metabolism   MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Phosphorylation MeSH
• Phthalazines pharmacology   MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Homologous Recombination * MeSH
• Protein Interaction Domains and Motifs MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• Ovarian Neoplasms drug therapy   enzymology   genetics   pathology   MeSH
• Poly(ADP-ribose) Polymerase Inhibitors pharmacology   MeSH
• Piperazines pharmacology   MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Cell Cycle Proteins metabolism   MeSH
• Proto-Oncogene Proteins metabolism   MeSH
• Rad51 Recombinase genetics   metabolism   MeSH
• Chromatin Assembly and Disassembly * MeSH
• RNA Interference MeSH
• Signal Transduction drug effects   MeSH
• Transfection MeSH
• Carrier Proteins genetics   metabolism   MeSH
• Protein Binding MeSH
• Dose-Response Relationship, Drug MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cyclin-dependent kinases regulate the cell cycle and transcription in higher eukaryotes. We have determined the crystal structure of the transcription kinase Cdk13 and its Cyclin K subunit at 2.0 Å resolution. Cdk13 contains a C-terminal extension helix composed of a polybasic cluster and a DCHEL motif that interacts with the bound ATP. Cdk13/CycK phosphorylates both Ser5 and Ser2 of the RNA polymerase II C-terminal domain (CTD) with a preference for Ser7 pre-phosphorylations at a C-terminal position. The peptidyl-prolyl isomerase Pin1 does not change the phosphorylation specificities of Cdk9, Cdk12, and Cdk13 but interacts with the phosphorylated CTD through its WW domain. Using recombinant proteins, we find that flavopiridol inhibits Cdk7 more potently than it does Cdk13. Gene expression changes after knockdown of Cdk13 or Cdk12 are markedly different, with enrichment of growth signaling pathways for Cdk13-dependent genes. Together, our results provide insights into the structure, function, and activity of human Cdk13/CycK.

• MeSH
• Cyclin-Dependent Kinases genetics   metabolism   MeSH
• Phosphorylation MeSH
• Humans MeSH
• Signal Transduction MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH