scholarly journals The unphosphorylated form of the PAQosome core subunit RPAP3 binds ribosomal preassembly complexes to modulate ribosome biogenesis.

2021 ◽  
Author(s):  
Maxime Pinard ◽  
Philippe Cloutier ◽  
Christian Poitras ◽  
Marie-Soleil Gauthier ◽  
Benoit Coulombe
Keyword(s):  
Posttranslational Modifications ◽  
Ribosome Biogenesis ◽  
Quaternary Structure ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Hek293 Cells ◽  
Selection Mechanism ◽  
Ribosomal Assembly ◽  
Kinase Ck2

The PAQosome (Particle for Arrangement of Quaternary structure) is a twelve-subunit HSP90 co-chaperone involved in the biogenesis of several human protein complexes. Two mechanisms of client selection have previously been identified, namely the selective recruitment of specific adaptors and the differential use of homologous core subunits. Here, we describe a third client selection mechanism by showing that RPAP3, one of the core PAQosome subunits, is phosphorylated at several Ser residues in HEK293 cells. Affinity purification coupled with mass spectrometry (AP-MS) using expression of tagged RPAP3 with single phospho-null mutations at Ser116, Ser119 or Ser121 reveals binding of the unphosphorylated form to several proteins involved in ribosome biogenesis. In vitro phosphorylation assays indicate that the kinase CK2 phosphorylates these RPAP3 residues. This finding is supported by data showing that pharmacological inhibition of CK2 enhances binding of RPAP3 to ribosome preassembly factors in AP-MS experiments. Moreover, silencing of PAQosome subunits interferes with ribosomal assembly factors' interactome. Altogether, these results indicate that RPAP3 phosphate group addition/removal at specific residues modulates binding to subunits of preribosomal complexes and allows speculating that PAQosome posttranslational modifications is a mechanism of client selection.

scholarly journals Arabidopsis mTERF9 protein promotes chloroplast ribosomal assembly and translation by establishing ribonucleoprotein interactions in vivo

2020 ◽  
Author(s):  
Louis-Valentin Méteignier ◽  
Rabea Ghandour ◽  
Aude Zimmerman ◽  
Lauriane Kuhn ◽  
Jörg Meurer ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Higher Plants ◽  
Physiological Effects ◽  
Ribosomal Assembly ◽  
Transcription Termination Factor ◽  
Protein Interactome ◽  
70S Ribosome ◽  
Insight Into

ABSTRACTThe mitochondrial transcription termination factor proteins are nuclear-encoded nucleic acid binders defined by degenerate tandem helical-repeats of ~30 amino acids. They are found in metazoans and plants where they localize to mitochondria or chloroplasts. In higher plants, the mTERF family comprises ~30 members and several of these have been linked to plant development and response to abiotic stress. However, knowledge of the molecular basis underlying these physiological effects is scarce. We show that the Arabidopsis mTERF9 protein promotes the accumulation of the 16S and 23S rRNAs in chloroplasts, and interacts predominantly with the 16S rRNA in vivo and in vitro. Furthermore, mTERF9 is found in large complexes containing ribosomes and polysomes in chloroplasts. The comprehensive analysis of mTERF9 in vivo protein interactome identified many subunits of the 70S ribosome whose assembly is compromised in the null mterf9 mutant, putative ribosome biogenesis factors and CPN60 chaperonins. Protein interaction assays in yeast revealed that mTERF9 directly interact with these proteins. Our data demonstrate that mTERF9 integrates protein-protein and protein-RNA interactions to promote chloroplast ribosomal assembly and translation. Besides extending our knowledge of mTERF functional repertoire in plants, these findings provide an important insight into the chloroplast ribosome biogenesis.

scholarly journals Physical and functional interactome atlas of human receptor tyrosine kinases

2021 ◽  
Author(s):  
Kari Salokas ◽  
Tiina Öhman ◽  
Xiaonan Liu ◽  
Iftekhar Chowdhury ◽  
Lisa Gawriyski ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Cell Communication ◽  
Cell Surface Receptor ◽  
Kinase Assay ◽  
Surface Receptor ◽  
Molecular Context

Much cell-to-cell communication is facilitated by cell surface receptor tyrosine kinases (RTKs). These proteins phosphorylate their downstream cytoplasmic substrates in response to stimuli such as growth factors. Despite their central roles, the functions of many RTKs are still poorly understood. To resolve the lack of systematic knowledge, we used three complementary methods to map the molecular context and substrate profiles of RTKs. We used affinity purification coupled to mass spectrometry (AP-MS) to characterize stable binding partners and RTK-protein complexes, proximity-dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates. To identify how kinase interactions depend on kinase activity, we also used kinase-deficient mutants. Our data represent a comprehensive, systemic mapping of RTK interactions and substrates. This resource adds information regarding well-studied RTKs, offers insights into the functions of less well-studied RTKs, and highlights RTK-RTK interactions and shared signaling pathways.

scholarly journals Processing of the Ribosomal Ubiquitin-Like Fusion Protein FUBI-eS30/FAU is Required for 40S Maturation and Depends on USP36

2021 ◽  
Author(s):  
Jasmin van den Heuvel ◽  
Caroline Ashiono ◽  
Ludovic Gillet ◽  
Kerstin Doerner ◽  
Emanuel Wyler ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Affinity Purification ◽  
Ribosomal Subunit ◽  
Wild Type ◽  
Functional Importance ◽  
40S Ribosomal Subunit ◽  
Differential Affinity

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.

scholarly journals Processing of the ribosomal ubiquitin-like fusion protein FUBI-eS30/FAU is required for 40S maturation and depends on USP36

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jasmin van den Heuvel ◽  
Caroline Ashiono ◽  
Ludovic C Gillet ◽  
Kerstin Dörner ◽  
Emanuel Wyler ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Affinity Purification ◽  
Ribosomal Subunit ◽  
Wild Type ◽  
Functional Importance ◽  
40S Ribosomal Subunit ◽  
Differential Affinity

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.

scholarly journals Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1

2021 ◽  
Vol 22 (23) ◽  
pp. 13133
Author(s):  
Alexander Becker ◽  
Claudia Götz ◽  
Mathias Montenarh ◽  
Stephan E. Philipp
Keyword(s):  
Hek293 Cells ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Insulin Granules ◽  
Functional Consequences ◽  
Kinase Ck2 ◽  
Uptake And Metabolism

In pancreatic β-cells of the line INS-1, glucose uptake and metabolism induce the openings of Ca2+-permeable TRPM3 channels that contribute to the elevation of the intracellular Ca2+ concentration and the fusion of insulin granules with the plasma membrane. Conversely, glucose-induced Ca2+ signals and insulin release are reduced by the activity of the serine/threonine kinase CK2. Therefore, we hypothesized that TRPM3 channels might be regulated by CK2 phosphorylation. We used recombinant TRPM3α2 proteins, native TRPM3 proteins from INS-1 β-cells, and TRPM3-derived oligopeptides to analyze and localize CK2-dependent phosphorylation of TRPM3 channels. The functional consequences of CK2 phosphorylation upon TRPM3-mediated Ca2+ entry were investigated in Fura-2 Ca2+-imaging experiments. Recombinant TRPM3α2 channels expressed in HEK293 cells displayed enhanced Ca2+ entry in the presence of the CK2 inhibitor CX-4945 and their activity was strongly reduced after CK2 overexpression. TRPM3α2 channels were phosphorylated by CK2 in vitro at serine residue 1172. Accordingly, a TRPM3α2 S1172A mutant displayed enhanced Ca2+ entry. The TRPM3-mediated Ca2+ entry in INS-1 β-cells was also strongly increased in the presence of CX-4945 and reduced after overexpression of CK2. Our study shows that CK2-mediated phosphorylation controls TRPM3 channel activity in INS-1 β-cells.

scholarly journals Role for Hes1-Induced Phosphorylation in Groucho-Mediated Transcriptional Repression

2002 ◽  
Vol 22 (2) ◽  
pp. 389-399 ◽  
Author(s):  
Hugh N. Nuthall ◽  
Junaid Husain ◽  
Keith W. McLarren ◽  
Stefano Stifani
Keyword(s):  
Protein Kinase ◽  
Dna Binding ◽  
Transcriptional Repression ◽  
Protein Kinase Ck2 ◽  
Protein Complexes ◽  
Transcription Repression ◽  
Tight Association ◽  
Kinase Ck2 ◽  
Enhancer Of Split

ABSTRACT Transcriptional corepressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family regulate a number of developmental pathways in both invertebrates and vertebrates. They form transcription repression complexes with members of several DNA-binding protein families and participate in the regulation of the expression of numerous genes. Despite their pleiotropic roles, little is known about the mechanisms that regulate the functions of Gro/TLE proteins. It is shown here that Gro/TLEs become hyperphosphorylated in response to neural cell differentiation and interaction with the DNA-binding cofactor Hairy/Enhancer of split 1 (Hes1). Hyperphosphorylation of Gro/TLEs is correlated with a tight association with the nuclear compartment through interaction with chromatin, suggesting that hyperphosphorylated Gro/TLEs may mediate transcriptional repression via chromatin remodeling mechanisms. Pharmacological inhibition of protein kinase CK2 reduces the Hes1-induced hyperphosphorylation of Gro/TLEs and causes a decrease in the chromatin association of the latter. Moreover, the transcription repression activity of Gro/TLEs is reduced by protein kinase CK2 inhibition. Consistent with these observations, Gro/TLEs are phosphorylated in vitro by purified protein kinase CK2. Taken together, these results implicate protein kinase CK2 in Gro/TLE functions. They suggest further that this kinase is involved in a hyperphosphorylation mechanism activated by Hes1 that promotes the transcription repression functions of Hes1-Gro/TLE protein complexes.

scholarly journals The importance of ribosome production, and the 5S RNP–MDM2 pathway, in health and disease

2016 ◽  
Vol 44 (4) ◽  
pp. 1086-1090 ◽  
Author(s):  
Andria Pelava ◽  
Claudia Schneider ◽  
Nicholas J. Watkins
Keyword(s):  
Human Health ◽  
Ribosome Biogenesis ◽  
Protein Complexes ◽  
Ribosomal Assembly ◽  
Genes Encoding ◽  
Health And Disease ◽  
P53 Activation ◽  
Assembly Intermediate ◽  
Cellular Transcription ◽  
Rna Protein Complexes

Ribosomes are abundant, large RNA–protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease.

scholarly journals Arabidopsis mTERF9 protein promotes chloroplast ribosomal assembly and translation by establishing ribonucleoprotein interactions in vivo

2021 ◽  
Author(s):  
Louis-Valentin Méteignier ◽  
Rabea Ghandour ◽  
Aude Zimmerman ◽  
Lauriane Kuhn ◽  
Jörg Meurer ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Higher Plants ◽  
Physiological Effects ◽  
Ribosomal Assembly ◽  
Transcription Termination Factor ◽  
Protein Interactome ◽  
70S Ribosome ◽  
Insight Into

Abstract The mitochondrial transcription termination factor proteins are nuclear-encoded nucleic acid binders defined by degenerate tandem helical-repeats of ∼30 amino acids. They are found in metazoans and plants where they localize in organelles. In higher plants, the mTERF family comprises ∼30 members and several of these have been linked to plant development and response to abiotic stress. However, knowledge of the molecular basis underlying these physiological effects is scarce. We show that the Arabidopsis mTERF9 protein promotes the accumulation of the 16S and 23S rRNAs in chloroplasts, and interacts predominantly with the 16S rRNA in vivo and in vitro. Furthermore, mTERF9 is found in large complexes containing ribosomes and polysomes in chloroplasts. The comprehensive analysis of mTERF9 in vivo protein interactome identified many subunits of the 70S ribosome whose assembly is compromised in the null mterf9 mutant, putative ribosome biogenesis factors and CPN60 chaperonins. Protein interaction assays in yeast revealed that mTERF9 directly interact with these proteins. Our data demonstrate that mTERF9 integrates protein-protein and protein-RNA interactions to promote chloroplast ribosomal assembly and translation. Besides extending our knowledge of mTERF functional repertoire in plants, these findings provide an important insight into the chloroplast ribosome biogenesis.

scholarly journals Human Cytomegalovirus Protein Kinase UL97 Forms a Complex with the Tegument Phosphoprotein pp65

2007 ◽  
Vol 81 (19) ◽  
pp. 10659-10668 ◽  
Author(s):  
Jeremy P. Kamil ◽  
Donald M. Coen
Keyword(s):  
Protein Kinase ◽  
Human Cytomegalovirus ◽  
Opportunistic Infections ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Immunocompromised Host ◽  
Protein Protein Interaction ◽  
Infected Cells ◽  
Hcmv Replication

ABSTRACT UL97 is a protein kinase encoded by human cytomegalovirus (HCMV) and is an important target for antiviral drugs against this ubiquitous herpesvirus, which is a major cause of life-threatening opportunistic infections in the immunocompromised host. In an effort to better understand the function(s) of UL97 during HCMV replication, a recombinant HCMV, NTAP97, which expresses a tandem affinity purification (TAP) tag at the amino terminus of UL97, was used to obtain UL97 protein complexes from infected cells. pp65 (also known as UL83), the 65-kDa virion tegument phosphoprotein, specifically copurified with UL97 during TAP, as shown by mass spectrometry and Western blot analyses. Reciprocal coimmunoprecipitation experiments using lysates of infected cells also indicated an interaction between UL97 and pp65. Moreover, in a glutathione S-transferase (GST) pull-down experiment, purified GST-pp65 fusion protein specifically bound in vitro-translated UL97, suggesting that UL97 and pp65 do not require other viral proteins to form a complex and may directly interact. Notably, pp65 has been previously reported to form unusual aggregates during viral replication when UL97 is pharmacologically inhibited or genetically ablated, and a pp65 deletion mutant was observed to exhibit modest resistance to a UL97 inhibitor (M. N. Prichard, W. J. Britt, S. L. Daily, C. B. Hartline, and E. R. Kern, J. Virol. 79:15494-15502, 2005). A stable protein-protein interaction between pp65 and UL97 may be relevant to incorporation of these proteins into HCMV particles during virion morphogenesis, with potential implications for immunomodulation by HCMV, and may also be a mechanism by which UL97 is negatively regulated during HCMV replication.

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