scholarly journals Phosphorylation of Steroidogenic Factor 1 Is Mediated by Cyclin-Dependent Kinase 7

2008 ◽  
Vol 22 (1) ◽  
pp. 91-104 ◽  
Author(s):  
Aurélia E. Lewis ◽  
Marte Rusten ◽  
Erling A. Hoivik ◽  
Elisabeth L. Vikse ◽  
Magnus L. Hansson ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Cdk Inhibitor ◽  
Structural Studies ◽  
Cyclin Dependent Kinase ◽  
Steroidogenic Factor 1 ◽  
Phosphorylation Level ◽  
Inactive Form ◽  
And Function ◽  
Cdk Activating Kinase

Abstract The nuclear receptor steroidogenic factor-1 (SF1) is critical for development and function of steroidogenic tissues. Posttranslational modifications are known to influence the transcriptional capacity of SF1, and it was previously demonstrated that serine 203 is phosphorylated. In this paper we report that serine 203 is phosphorylated by a cyclin-dependent kinase 7 (CDK7)-mediated process. As part of the CDK-activating kinase complex, CDK7 is a component of the basal transcription factor TFIIH, and phosphorylation of SF1 as well as SF1-dependent transcription was clearly reduced in cells carrying a mutation that renders the CDK-activating kinase complex unable to interact with the TFIIH core. Coimmunoprecipitation analyses revealed that SF1 and CDK7 reside in the same complex, and kinase assays demonstrated that immunoprecipitated CDK7 and purified TFIIH phosphorylate SF1 in vitro. The CDK inhibitor roscovitine blocked phosphorylation of SF1, and an inactive form of CDK7 repressed the phosphorylation level and the transactivation capacity of SF1. Structural studies have identified phosphoinositides as potential ligands for SF1. Interestingly, we found that mutations designed to block phospholipid binding dramatically decreased the level of SF1 phosphorylation. Together our results suggest a connection between ligand occupation and phosphorylation and association with the basic transcriptional machinery, indicating an intricate regulation of SF1 transactivation.

scholarly journals Posttranslational Modifications and the Immunogenicity of Biotherapeutics

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Roy Jefferis
Keyword(s):  
Posttranslational Modifications ◽  
Drug Product ◽  
Molecular Forms ◽  
Phagocytic Cells ◽  
Production Platform ◽  
The Individual ◽  
And Function ◽  
Antigen Presenting

Whilst the amino acid sequence of a protein is determined by its gene sequence, the final structure and function are determined by posttranslational modifications (PTMs), including quality control (QC) in the endoplasmic reticulum (ER) and during passage through the Golgi apparatus. These processes are species and cell specific and challenge the biopharmaceutical industry when developing a production platform for the generation of recombinant biologic therapeutics. Proteins and glycoproteins are also subject to chemical modifications (CMs) bothin vivoandin vitro. The individual is naturally tolerant to molecular forms of self-molecules but nonself variants can provoke an immune response with the generation of anti-drug antibodies (ADA); aggregated forms can exhibit enhanced immunogenicity and QC procedures are developed to avoid or remove them. Monoclonal antibody therapeutics (mAbs) are a special case because their purpose is to bind the target, with the formation of immune complexes (ICs), a particular form of aggregate. Such ICs may be removed by phagocytic cells that have antigen presenting capacity. These considerations may frustrate the possibility of ameliorating the immunogenicity of mAbs by rigorous exclusion of aggregates from drug product. Alternate strategies for inducing immunosuppression or tolerance are discussed.

scholarly journals A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.

1997 ◽  
Vol 17 (12) ◽  
pp. 7362-7374 ◽  
Author(s):  
J A Diehl ◽  
C J Sherr
Keyword(s):  
Cyclin D1 ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Phosphorylation Site ◽  
Dominant Negative ◽  
Cyclin Dependent Kinase ◽  
Linker Peptide ◽  
Cdk Activating Kinase

Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.

scholarly journals Site-specific phosphorylation of Huntingtin exon 1 recombinant proteins enabled by the discovery of novel kinases

2020 ◽  
Author(s):  
Anass Chiki ◽  
Jonathan Ricci ◽  
Ramanath Hegde ◽  
Luciano A. Abriata ◽  
Andreas Reif ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Proof Of Concept ◽  
Wild Type ◽  
Expression And Purification ◽  
Sumo Fusion ◽  
Exon 1 ◽  
Health And Disease ◽  
Experimental Approaches ◽  
And Function

AbstractPosttranslational modifications (PTMs) within the first 17 amino acids (Nt17) of exon1 of the Huntingtin protein (Httex1) play important roles in modulating its cellular properties and functions in health and disease. In particular, phosphorylation of threonine and serine residues (T3, S13, and/or S16) has been shown to inhibit Htt aggregation in vitro and inclusion formation in cellular and animal models of Huntington’s disease (HD). In this manuscript, we describe a new and simple methodology for producing milligram quantities of highly pure wild type or mutant Httex1 proteins that are site-specifically phosphorylated at T3 or at both S13 and S16. This advance was enabled by 1) the discovery and validation of novel kinases that efficiently phosphorylate Httex1 at S13 and S16 (TBK1), at T3 (GCK) or T3 and S13 (TNIK and HGK); and, 2) the development of an efficient methodology for producing recombinant native Httex1 proteins using a SUMO-fusion expression and purification strategy. As proof of concept, we demonstrate how this method can be applied to produce Httex1 proteins that are both site- specifically phosphorylated and fluorescently labeled or isotopically labeled. Together, these advances should increase access to these valuable tools and expand the range of methods and experimental approaches that can be used to elucidate the mechanisms by which phosphorylation influences Httex1 structure, aggregation, interactome and function(s) in health and disease.

scholarly journals LXRα Phosphorylation in Cardiometabolic Disease: Insight From Mouse Models

Endocrinology ◽  
2020 ◽  
Vol 161 (7) ◽  
Author(s):  
Maud Voisin ◽  
Matthew C Gage ◽  
Natalia Becares ◽  
Elina Shrestha ◽  
Edward A Fisher ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Posttranslational Modifications ◽  
Physiological Response ◽  
Metabolic Diseases ◽  
Cardiometabolic Disease ◽  
Nonalcoholic Fatty Liver ◽  
Environmental Signals ◽  
Powerful Means ◽  
And Function

Abstract Posttranslational modifications, such as phosphorylation, are a powerful means by which the activity and function of nuclear receptors such as LXRα can be altered. However, despite the established importance of nuclear receptors in maintaining metabolic homeostasis, our understanding of how phosphorylation affects metabolic diseases is limited. The physiological consequences of LXRα phosphorylation have, until recently, been studied only in vitro or nonspecifically in animal models by pharmacologically or genetically altering the enzymes enhancing or inhibiting these modifications. Here we review recent reports on the physiological consequences of modifying LXRα phosphorylation at serine 196 (S196) in cardiometabolic disease, including nonalcoholic fatty liver disease, atherosclerosis, and obesity. A unifying theme from these studies is that LXRα S196 phosphorylation rewires the LXR-modulated transcriptome, which in turn alters physiological response to environmental signals, and that this is largely distinct from the LXR-ligand–dependent action.

scholarly journals CDK5 influences the organization of the Circadian Machinery in peripheral clocks

2021 ◽  
Author(s):  
Jürgen Ripperger ◽  
Urs Albrecht ◽  
Andrea Brenna
Keyword(s):  
Posttranslational Modifications ◽  
Spatial Dynamics ◽  
Mouse Embryonic Fibroblast ◽  
Cellular Level ◽  
Cyclin Dependent Kinase ◽  
Biochemical Processes ◽  
Peripheral Clocks ◽  
Embryonic Fibroblast ◽  
Cyclin Dependent Kinase 5

AbstractCircadian rhythms are self-sustained physiological changes that drive rhythmicity within the 24-hours cycles. Posttranslational modifications (PMTs), such as protein phosphorylation, acetylation, sumoylation, and ubiquitination, are biochemical processes that modify protein structure and functions, ensuring circadian rhythm precision. For example, phosphorylation is considered the most important hallmark of rhythmicity from cyanobacteria to mammals. Cyclin-dependent kinase 5 (CDK5) has been shown to regulate the mammalian SCN’s circadian clock via phosphorylation of PER2. Here, we show that CDK5 influences the clock machinery assembling, using immortalized mouse embryonic fibroblast as an in vitro model for studying the peripheral clock. In fact, the circadian period at the cellular level is lengthened. Furthermore, the clock-controlled gene’s expression amplitude is dampened in Cdk5 ko cell lines, while the phase is delayed about 4 hours.Taken together, we show in vitro that CDK5 is critically involved in regulating the peripheral clocks, influencing their temporal and spatial dynamics.

scholarly journals Studying bacterial chemosensory array with CryoEM

2021 ◽  
Author(s):  
Zhuan Qin ◽  
Peijun Zhang
Keyword(s):  
State Of The Art ◽  
Lipid Monolayer ◽  
Structural Studies ◽  
Future Directions ◽  
In Vitro Systems ◽  
Bacterial Ghosts ◽  
And Function ◽  
Lipid Nanodiscs

Bacteria direct their movement in respond to gradients of nutrients and other stimuli in the environment through the chemosensory system. The behavior is mediated by chemosensory arrays that are made up of thousands of proteins to form an organized array near the cell pole. In this review, we briefly introduce the architecture and function of the chemosensory array and its core signaling unit. We describe the in vivo and in vitro systems that have been used for structural studies of chemosensory array by cryoEM, including reconstituted lipid nanodiscs, 2D lipid monolayer arrays, lysed bacterial ghosts, bacterial minicells and native bacteria cells. Lastly, we review recent advances in structural analysis of chemosensory arrays using state-of-the-art cryoEM and cryoET methodologies, focusing on the latest developments and insights with a perspective on current challenges and future directions.

scholarly journals Transcriptional knockout of steroidogenic factor 1 in vivo in Oreochromis niloticus increased weight and suppressed gonad development using antisense RNA

2021 ◽  
Author(s):  
Zhe Cao ◽  
Jun Qiang ◽  
Jun Zhu ◽  
Hong Li ◽  
Yi Tao ◽  
...  
Keyword(s):  
Oreochromis Niloticus ◽  
Antisense Rna ◽  
Nile Tilapia ◽  
Gonad Development ◽  
External Genitalia ◽  
Cloning And Expression ◽  
Steroidogenic Factor 1 ◽  
And Function

Abstract Steroidogenic factor 1 (sf1) is an important regulator of gonad development and function in mammals. However, study of sf1 in fish is limited to cloning and expression and in vitro experiments. Using antisense RNA we knockout transcription of the sf1 gene in Nile tilapia Oreochromis niloticus, and obtain experimental fish in vivo. We demonstrate that antisense RNA can silence sf1 transcription and protein expression, and report suppression of sf1 transcription to affect gonad development and external genitalia formation in Nile tilapia. We also report disfunction of retinal metabolism and fatty acid metabolism to be important causes of weight gain and gonad abnormality with sf1 suppression. The feasibility of using antisense RNA for gene editing in fish is verified, and a new way of studying gene function and performing biological breeding is presented.

Localization and regulation of the cdk-activating kinase (Cak1p) from budding yeast

1998 ◽  
Vol 111 (24) ◽  
pp. 3585-3596 ◽  
Author(s):  
P. Kaldis ◽  
Z.W. Pitluk ◽  
I.A. Bany ◽  
D.A. Enke ◽  
M. Wagner ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Budding Yeast ◽  
Subcellular Fractionation ◽  
Eukaryotic Cell ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Cycles ◽  
Cdk Activating Kinase

Eukaryotic cell cycles are controlled by the activities of cyclin-dependent kinases (cdks). The major cdk in budding yeast, Saccharomyces cerevisiae, is Cdc28p. Activation of Cdc28p requires phosphorylation on threonine 169 and binding to a cyclin. Thr-169 is phosphorylated by the cdk-activating kinase (CAK), Cak1p, which was recently identified as the physiological CAK in budding yeast. Here we present our further characterization of yeast Cak1p. We have found that Cak1p is dispersed throughout the cell as shown by immunofluorescence; biochemical subcellular fractionation confirmed that most of the Cak1p is found in the cytoplasm. Cak1p is a monomeric enzyme in crude yeast lysates. Mutagenesis of potential sites of activating phosphorylation had little effect on the activity of Cak1p in vitro or in vivo. Furthermore, Cak1p contains no posttranslational modifications detectable by two-dimensional isoelectric focusing. We found that Cak1p is a stable protein during exponential growth but that its expression decreases considerably when cells enter stationary phase. In contrast, Cak1p levels oscillate dramatically during meiosis, reflecting regulation at both the transcriptional and post-translational level. The localization and regulation of Cak1p are in contrast to those of the known vertebrate CAK, p40(MO15).

scholarly journals Cdk2-dependent phosphorylation of p21 regulates the role of Cdk2 in cisplatin cytotoxicity

2011 ◽  
Vol 300 (5) ◽  
pp. F1171-F1179 ◽  
Author(s):  
Rawad Hodeify ◽  
Adel Tarcsafalvi ◽  
Judit Megyesi ◽  
Robert L. Safirstein ◽  
Peter M. Price
Keyword(s):  
Mass Spectrometry ◽  
Cell Death ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Cdk2 Activity ◽  
Adenoviral Transduction ◽  
A Site

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. We found that an 18-kDa protein identified by mass spectrometry as p21WAF1/Cip1 was phosphorylated by Cdk2 starting 12 h after cisplatin exposure. The analysis showed it was phosphorylated at serine 78, a site not previously identified. The adenoviral transduction of p21 before cisplatin exposure protects from cytotoxicity by inhibiting Cdk2. Although cisplatin causes induction of endogenous p21, the protection is inefficient. We hypothesized that phosphorylation of p21 at serine 78 could affect its role as a Cdk inhibitor, and thereby lessen its ability to protect from cisplatin cytotoxicity. To investigate the effect of serine 78 phosphorylation on p21 activity, we replaced serine 78 with aspartic acid, creating the phosphomimic p21S78D. Mutant p21S78D was an inefficient inhibitor of Cdk2 and was inefficient at protecting TKPTS cells from cisplatin-induced cell death. We conclude that phosphorylation of p21 by Cdk2 limits the effectiveness of p21 to inhibit Cdk2, which is the mechanism for continued cisplatin cytotoxicity even after the induction of a protective protein.

scholarly journals Phosphorylation of Sic1, a Cyclin-dependent Kinase (Cdk) Inhibitor, by Cdk Including Pho85 Kinase Is Required for Its Prompt Degradation

1998 ◽  
Vol 9 (9) ◽  
pp. 2393-2405 ◽  
Author(s):  
Masafumi Nishizawa ◽  
Masaoki Kawasumi ◽  
Marie Fujino ◽  
Akio Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Cdk Inhibitor ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cdk Phosphorylation ◽  
Cdc28 Kinase

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 andCLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable inpho85Δ cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.

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