scholarly journals Protein network study of human AF4 reveals its central role in RNA Pol II-mediated transcription and in phosphorylation-dependent regulatory mechanisms

2011 ◽  
Vol 438 (1) ◽  
pp. 121-131 ◽  
Author(s):  
Gabriella Esposito ◽  
Armando Cevenini ◽  
Alessandro Cuomo ◽  
Francesca de Falco ◽  
Dario Sabbatino ◽  
...  
Keyword(s):  
Fragile X ◽  
Regulatory Mechanisms ◽  
Transcriptional Elongation ◽  
Interacting Proteins ◽  
Cyclin Dependent Kinase ◽  
Multiprotein Complex ◽  
Physiological Mechanisms ◽  
Rna Pol Ii ◽  
Proteomic Approach ◽  
Mixed Lineage Leukaemia

AF4 belongs to a family of proteins implicated in childhood lymphoblastic leukaemia, FRAXE (Fragile X E site) mental retardation and ataxia. AF4 is a transcriptional activator that is involved in transcriptional elongation. Although AF4 has been implicated in MLL (mixed-lineage leukaemia)-related leukaemogenesis, AF4-dependent physiological mechanisms have not been clearly defined. Proteins that interact with AF4 may also play important roles in mediating oncogenesis, and are potential targets for novel therapies. Using a functional proteomic approach involving tandem MS and bioinformatics, we identified 51 AF4-interacting proteins of various Gene Ontology categories. Approximately 60% participate in transcription regulatory mechanisms, including the Mediator complex in eukaryotic cells. In the present paper we report one of the first extensive proteomic studies aimed at elucidating AF4 protein cross-talk. Moreover, we found that the AF4 residues Thr220 and Ser212 are phosphorylated, which suggests that AF4 function depends on phosphorylation mechanisms. We also mapped the AF4-interaction site with CDK9 (cyclin-dependent kinase 9), which is a direct interactor crucial for the function and regulation of the protein. The findings of the present study significantly expand the number of putative members of the multiprotein complex formed by AF4, which is instrumental in promoting the transcription/elongation of specific genes in human cells.

scholarly journals Emerging multifaceted roles of BAP1 complexes in biological processes

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Aileen Patricia Szczepanski ◽  
Lu Wang
Keyword(s):  
Transcriptional Repression ◽  
Target Genes ◽  
Regulatory Mechanisms ◽  
Biological Processes ◽  
Multiprotein Complex ◽  
Downstream Target ◽  
Evolutionarily Conserved ◽  
Complex Forms ◽  
Polycomb Repressive Complex 1

AbstractHistone H2AK119 mono-ubiquitination (H2AK119Ub) is a relatively abundant histone modification, mainly catalyzed by the Polycomb Repressive Complex 1 (PRC1) to regulate Polycomb-mediated transcriptional repression of downstream target genes. Consequently, H2AK119Ub can also be dynamically reversed by the BAP1 complex, an evolutionarily conserved multiprotein complex that functions as a general transcriptional activator. In previous studies, it has been reported that the BAP1 complex consists of important biological roles in development, metabolism, and cancer. However, identifying the BAP1 complex’s regulatory mechanisms remains to be elucidated due to its various complex forms and its ability to target non-histone substrates. In this review, we will summarize recent findings that have contributed to the diverse functional role of the BAP1 complex and further discuss the potential in targeting BAP1 for therapeutic use.

Abstract 1470: The Kinase Activity of Cyclin-Dependent Kinase-9 is Required for Phosphorylation of p300 and its Histone Acetyltransferase Activity during Cardiomyocyte Hypertrophy

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Yoichi Sunagawa ◽  
Tatsuya Morimoto ◽  
Tomohide Takaya ◽  
Teruhisa Kawamura ◽  
Hiromichi Wada ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Kinase Activity ◽  
Histone Acetyltransferase ◽  
Single Amino Acid ◽  
Cardiomyocyte Hypertrophy ◽  
Cyclin Dependent Kinase ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Functional Relationships ◽  
Cdk9 Kinase Activity

Introduction: A zinc finger protein GATA4 is one of the factors involved in transcriptional regulation during myocardial cell hypertrophy and forms a complex with an intrinsic histone acetyltransferase (HAT), p300. HAT activity of p300 is required for acetylation and the transcriptional activity of GATA4 as well as for cardiomyocyte hypertrophy and the development of heart failure in vivo . By tandem affinity purification and mass spectrometric analyses, we identified cyclin-dependent kinase-9 (Cdk9), a component of positive transcription elongation factor b, as a novel GATA4-binding partner. Cdk9 also formed a complex with p300 as well as GATA4. However, the precise functional relationships among p300, GATA4, and Cdk9 remain unknown. Methods and Results: A series of GST pull-down assays revealed that the N-terminal of Cdk9 bound to the N-terminal zinc finger domain of GATA4 and C/H-3 domain of p300, respectively. By chromatin immunoprecipitation and DNA pull-down assay, we showed that GATA4 recruited Cdk9 onto GATA elements within the endothelin-1 promoter. Immuno-precipitation followed by Western blotting demonstrated that intact p300 induced not only the acetylation of GATA4, but also the interaction between GATA4 and Cdk9. Furthermore, p300 induced the hyperphosphorylation of RNA Pol II, suggesting that p300 is involved in the regulation of Cdk9 kinase activity. All of these effects were inhibited by the co-expression of a dominant-negative form (DN-) of p300. Conversely, Cdk9 induced not only the hyperphos-phorylation of RNA Pol II, but also the phosphorylation of p300. Notably, Cdk9 induced the acetylation and DNA binding of GATA4. However, DN-Cdk9, which loses its kinase activity by a single amino acid substitution, was unable to achieve these, suggesting the requirement of Cdk9 kinase activity for p300 HAT activity. Finally, a Cdk9 kinase inhibitor, DRB, inhibited phenylephrine-induced hypertrophic responses as well as the acetylation of GATA4 in cardiomyocytes. Conclusion: These findings demonstrate that Cdk9 is required for the phosphorylation of p300 and its HAT activity, and forms a functional complex with p300/GATA4 during cardiomyocyte hypertrophy.

scholarly journals SLC36A1-mTORC1 signaling drives acquired resistance to CDK4/6 inhibitors

2019 ◽  
Vol 5 (9) ◽  
pp. eaax6352 ◽  
Author(s):  
Akihiro Yoshida ◽  
Yiwen Bu ◽  
Shuo Qie ◽  
John Wrangle ◽  
E. Ramsay Camp ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Fragile X ◽  
Acquired Resistance ◽  
Mammalian Target Of Rapamycin ◽  
Cyclin Dependent Kinase ◽  
Solute Carrier Family ◽  
Potential Therapeutic Target ◽  
Mtorc1 Signaling ◽  
Solute Carrier

The cyclin-dependent kinase 4/6 (CDK4/6) kinase is dysregulated in melanoma, highlighting it as a potential therapeutic target. CDK4/6 inhibitors are being evaluated in trials for melanoma and additional cancers. While beneficial, resistance to therapy is a concern, and the molecular mechanisms of such resistance remain undefined. We demonstrate that reactivation of mammalian target of rapamycin 1 (mTORC1) signaling through increased expression of the amino acid transporter, solute carrier family 36 member 1 (SLC36A1), drives resistance to CDK4/6 inhibitors. Increased expression of SLC36A1 reflects two distinct mechanisms: (i) Rb loss, which drives SLC36A1 via reduced suppression of E2f; (ii) fragile X mental retardation syndrome–associated protein 1 overexpression, which promotes SLC36A1 translation and subsequently mTORC1. Last, we demonstrate that a combination of a CDK4/6 inhibitor with an mTORC1 inhibitor has increased therapeutic efficacy in vivo, providing an important avenue for improved therapeutic intervention in aggressive melanoma.

scholarly journals Emerging crosstalk between two signaling pathways coordinates K+ and Na+ homeostasis in the halophyte Hordeum brevisubulatum

2020 ◽  
Vol 71 (14) ◽  
pp. 4345-4358
Author(s):  
Haiwen Zhang ◽  
Hao Feng ◽  
Junwen Zhang ◽  
Rongchao Ge ◽  
Liyuan Zhang ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Regulatory Mechanisms ◽  
K Channel ◽  
Interacting Proteins ◽  
The Novel ◽  
Voltage Gated ◽  
Primary Core ◽  
High Salt Stress ◽  
Hordeum Brevisubulatum

Abstract K+/Na+ homeostasis is the primary core response for plant to tolerate salinity. Halophytes have evolved novel regulatory mechanisms to maintain a suitable K+/Na+ ratio during long-term adaptation. The wild halophyte Hordeum brevisubulatum can adopt efficient strategies to achieve synergistic levels of K+ and Na+ under high salt stress. However, little is known about its molecular mechanism. Our previous study indicated that HbCIPK2 contributed to prevention of Na+ accumulation and K+ reduction. Here, we further identified the HbCIPK2-interacting proteins including upstream Ca2+ sensors, HbCBL1, HbCBL4, and HbCBL10, and downstream phosphorylated targets, the voltage-gated K+ channel HbVGKC1 and SOS1-like transporter HbSOS1L. HbCBL1 combined with HbCIPK2 could activate HbVGKC1 to absorb K+, while the HbCBL4/10–HbCIPK2 complex modulated HbSOS1L to exclude Na+. This discovery suggested that crosstalk between the sodium response and the potassium uptake signaling pathways indeed exists for HbCIPK2 as the signal hub, and paved the way for understanding the novel mechanism of K+/Na+ homeostasis which has evolved in the halophytic grass.

scholarly journals Single-Nucleotide Mutations in FMR1 Reveal Novel Functions and Regulatory Mechanisms of the Fragile X Syndrome Protein FMRP

2015 ◽  
Vol 9s2 ◽  
pp. JEN.S25524 ◽  
Author(s):  
Joshua A. Suhl ◽  
Stephen T. Warren
Keyword(s):  
Intellectual Disability ◽  
Fragile X Syndrome ◽  
Fragile X ◽  
Point Mutations ◽  
Regulatory Mechanisms ◽  
Cgg Repeat ◽  
Novel Variants ◽  
Sequencing Studies ◽  
The Cost ◽  
Syndrome Protein

Fragile X syndrome is a monogenic disorder and a common cause of intellectual disability. Despite nearly 25 years of research on FMR1, the gene underlying the syndrome, very few pathological mutations other than the typical CGG-repeat expansion have been reported. This is in contrast to other X-linked, monogenic, intellectual disability disorders, such as Rett syndrome, where many point mutations have been validated as causative of the disorder. As technology has improved and significantly driven down the cost of sequencing, allowing for whole genes to be sequenced with relative ease, in-depth sequencing studies on FMR1 have recently been performed. These studies have led to the identification of novel variants in FMR1, where some of which have been functionally evaluated and are likely pathogenic. In this review, we discuss recently identified FMR1 variants, the ways these novel variants cause dysfunction, and how they reveal new regulatory mechanisms and functionalities of the gene.

scholarly journals MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach

PLoS ONE ◽  
2015 ◽  
Vol 10 (10) ◽  
pp. e0140872 ◽  
Author(s):  
Kalyan C. Tirupula ◽  
Dongmei Zhang ◽  
Appledene Osbourne ◽  
Arunachal Chatterjee ◽  
Russ Desnoyer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Interacting Proteins ◽  
Proteomic Approach

scholarly journals Ubiquitylation of Cdk9 by Skp2 Facilitates Optimal Tat Transactivation

2005 ◽  
Vol 79 (17) ◽  
pp. 11135-11141 ◽  
Author(s):  
Matjaz Barboric ◽  
Fan Zhang ◽  
Mojca Besenicar ◽  
Ana Plemenitas ◽  
B. Matija Peterlin
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Cyclin Dependent Kinase ◽  
Primary Mouse ◽  
Immunodeficiency Virus ◽  
F Box Protein ◽  
Hiv 1

ABSTRACT By recruiting the positive transcriptional elongation factor b (P-TEFb) to paused RNA polymerase II, the transactivator Tat stimulates transcriptional elongation of the human immunodeficiency virus type 1 (HIV-1) genome. We found that cyclin-dependent kinase 9 (Cdk9), the catalytic subunit of P-TEFb, is ubiquitylated in vivo. This ubiquitylation depended on the Skp1/Cul1/F-box protein E3 ubiquitin ligase Skp2. Likewise, Tat required Skp2 since its transactivation of the HIV-1 long terminal repeat decreased in primary mouse embryonic fibroblasts, which lacked Skp2. The ubiquitylation of Cdk9 by Skp2 facilitated the formation of the ternary complex between P-TEFb, Tat, and transactivation response element. Thus, our findings underscore the requirement of ubiquitylation for the coactivator function in regulating HIV-1 transcriptional elongation.

scholarly journals Identification of Different Proteins Binding to Na, K-ATPase α1 in Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome Cell Model by Proteomic Analysis

2021 ◽  
Author(s):  
Xu-Peng Wen ◽  
Yue-Zhong Zhang ◽  
He Huang ◽  
Tao-Hua Liu ◽  
Qi-Quan Wan
Keyword(s):  
Acute Respiratory Distress Syndrome ◽  
Tight Junction ◽  
Respiratory Distress Syndrome ◽  
Respiratory Distress ◽  
A549 Cell ◽  
Distress Syndrome ◽  
Cell Model ◽  
Interacting Proteins ◽  
Proteomic Approach ◽  
And Control

Abstract Acute respiratory distress syndrome (ARDS) is characterized by refractory hypoxemia caused by accumulation of pulmonary fluid, which is related to inflammatory cell infiltration, impaired tight junction of pulmonary epithelium and impaired Na, K-ATPase function, especially Na, K-ATPase α1 subunit. Up until now, the pathogenic mechanism at the level of protein during lipopolysaccharide- (LPS-) induced ARDS remains unclear. Using an unbiased, discovery and quantitative proteomic approach, the discovery of differentially expressed proteins binding to Na, K-ATPase α1 between LPS-induced A549 cell and control-A549 group is of particular interest for the current study. These proteins may help the clinical diagnosis and facilitate the personalized treatment of ARDS. We screened these Na, K-ATPase α1 interacting proteins, carried out the related Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and found evident phenomena of ubiquitination and deubiquitination, as well as the pathways related to autophagy. We also chose some of the differentiated expressing proteins with significant performance for further verification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among proteins with rich abundance, there were several intriguing ones, including the deubiquitinase (OTUB1), the tight junction protein zonula occludens-1 (ZO-1), the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complexes (CUL4B) and the autophagy-related protein sequestosome-1 (SQSTM1). Protein-protein interaction network showed that there were 244 significantly enriched co-expression among 60 proteins in the group control-A549. while the group LPS-A549 showed 43 significant enriched interactions among 29 proteins. In conclusion, our quantitative discovery-based proteomic approach identified commonalities, and revealed targets related to the occurrence and development of ARDS, being the first study to investigate significant differences in Na, K-ATPase α1 interacting proteins between LPS-induced ARDS cell model and control-A549 cell.

scholarly journals Npac Is a Co-factor of Histone H3K36me3 and Regulates Transcriptional Elongation in Mouse ES Cells

2020 ◽  
Author(s):  
Sue Yu ◽  
Jia Li ◽  
Guanxu Ji ◽  
Zhen Long Ng ◽  
Jiamin Siew ◽  
...  
Keyword(s):  
Es Cells ◽  
Chromatin Modification ◽  
Embryonic Stem ◽  
Transcriptional Elongation ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Mouse Es Cells ◽  
Reprogramming Efficiency ◽  
Pluripotency Maintenance ◽  
Pluripotency Genes

AbstractChromatin modification contributes to pluripotency maintenance in embryonic stem cells (ESCs). However, the related mechanisms remain obscure. Here, we show that Npac, a “reader” of histone H3 lysine 36 trimethylation (H3K36me3), is required to maintain mouse ESC pluripotency since knockdown of Npac causes mouse ESC differentiation. Depletion of Npac in mouse embryonic fibroblasts (MEFs) inhibits reprogramming efficiency. Furthermore, our Npac ChIP-seq results reveal that Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes in mESCs. Interestingly, we find that Npac interacts with p-TEFb, RNA Pol II Ser2 and Ser5. Depletion of Npac disrupts transcriptional elongation of pluripotency genes Nanog and Rif1. Taken together, we propose that Npac is essential for transcriptional elongation of pluripotency genes by recruiting of p-TEFb and interacting with RNA Pol II Ser2 and Ser5.

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