作者: Carter, Gay ; Patel, Achintya ; Kong, Xiaoyuan ; Harding, Linette ; Sparks, Robert P. ; Dobbins, Tradd ; Cooper, Denise R. ; Patel, Niketa A. ; Patel, Rehka

AbstractExcessive dietary intake of fat results in its storage in white adipose tissue (WAT). Energy expenditure through lipid oxidation occurs in brown adipose tissue (BAT). Certain WAT depots can undergo a change termed beiging where markers that BAT express are induced. Little is known about signalling pathways inducing beiging. Here, inhibition of a signalling pathway regulating alternative pre‐mRNA splicing is involved in adipocyte beiging. Clk1/2/4 kinases regulate splicing by phosphorylating factors that process pre‐mRNA. Clk1 inhibition by TG003 results in beige‐like adipocytes highly expressing PGC1α and UCP1. SiRNA for Clk1, 2 and 4, demonstrated that Clk1 depletion increased UCP1 and PGC1α expression, whereas Clk2/4 siRNA did not. TG003‐treated adipocytes contained fewer lipid droplets, are smaller, and contain more mitochondria, resulting in proton leak increases. Additionally, inhibition of PKCβII activity, a splice variant regulated by Clk1, increased beiging. PGC1α is a substrate for both Clk1 and PKCβII kinases, and we surmised that inhibition of PGC1α phosphorylation resulted in beiging of adipocytes. We show that TG003 binds Clk1 more than Clk2/4 through direct binding, and PGC1α binds to Clk1 at a site close to TG003. Furthermore, we show that TG003 is highly specific for Clk1 across hundreds of kinases in our activity screen. Hence, Clk1 inhibition becomes a target for induction of beige adipocytes.

2020-09-01·Gastric Cancer1区 · 医学

Phosphoproteomic analysis identifies CLK1 as a novel therapeutic target in gastric cancer

1区 · 医学

Article

作者: Pinto, Sneha M ; Thiyagarajan, Saravanan ; Subbannayya, Tejaswini ; Radhakrishnan, Padhma ; Radhakrishna, Vinod D ; Syed, Nazia ; Rajagopalan, Pavithra ; Kumar, Rekha V ; Chatterjee, Aditi ; Pandey, Akhilesh ; Muthusamy, Oliyarasi ; Gopisetty, Gopal ; Majumder, Pradip ; Navani, Sanjay ; Mohan, Sonali V ; Rajappa, Manoj ; Sathe, Gajanan ; Rajkumar, Thangarajan ; Babu, Niraj ; Gowda, Harsha ; Advani, Jayshree ; Biswas, Manjusha

BACKGROUNDPhosphorylation is an important regulatory mechanism of protein activity in cells. Studies in various cancers have reported perturbations in kinases resulting in aberrant phosphorylation of oncoproteins and tumor suppressor proteins.METHODSIn this study, we carried out quantitative phosphoproteomic analysis of gastric cancer tissues and corresponding xenograft samples. Using these data, we employed bioinformatics analysis to identify aberrant signaling pathways. We further performed molecular inhibition and silencing of the upstream regulatory kinase in gastric cancer cell lines and validated its effect on cellular phenotype. Through an ex vivo technology utilizing patient tumor and blood sample, we sought to understand the therapeutic potential of the kinase by recreating the tumor microenvironment.RESULTSUsing mass spectrometry-based high-throughput analysis, we identified 1,344 phosphosites and 848 phosphoproteins, including differential phosphorylation of 177 proteins (fold change cut-off ≥ 1.5). Our data showed that a subset of differentially phosphorylated proteins belonged to splicing machinery. Pathway analysis highlighted Cdc2-like kinase (CLK1) as upstream kinase. Inhibition of CLK1 using TG003 and CLK1 siRNA resulted in a decreased cell viability, proliferation, invasion and migration as well as modulation in the phosphorylation of SRSF2. Ex vivo experiments which utilizes patient's own tumor and blood to recreate the tumor microenvironment validated the use of CLK1 as a potential target for gastric cancer treatment.CONCLUSIONSOur data indicates that CLK1 plays a crucial role in the regulation of splicing process in gastric cancer and that CLK1 can act as a novel therapeutic target in gastric cancer.

2019-05-01·Journal of Human Genetics3区 · 生物学

Verification and rectification of cell type-specific splicing of a Seckel syndrome-associated ATR mutation using iPS cell model

3区 · 生物学

Article

作者: Hagiwara, Masatoshi ; Takahashi, Jun ; Awaya, Tomonari ; Samata, Bumpei ; Suzuki, Naoya M ; Saito, Megumu K ; Ichisima, Jose ; Nakahata, Tatsutoshi

AbstractSeckel syndrome (SS) is a rare spectrum of congenital severe microcephaly and dwarfism. One SS-causative gene is Ataxia Telangiectasia and Rad3-Related Protein (ATR), and ATR (c.2101 A>G) mutation causes skipping of exon 9, resulting in a hypomorphic ATR defect. This mutation is considered the cause of an impaired response to DNA replication stress, the main function of ATR, contributing to the pathogenesis of microcephaly. However, the precise behavior and impact of this splicing defect in human neural progenitor cells (NPCs) is unclear. To address this, we established induced pluripotent stem cells (iPSCs) from fibroblasts carrying the ATR mutation and an isogenic ATR-corrected counterpart iPSC clone. SS-patient-derived iPSCs (SS-iPSCs) exhibited cell type-specific splicing; exon 9 was dominantly skipped in fibroblasts and iPSC-derived NPCs, but it was included in undifferentiated iPSCs and definitive endodermal cells. SS-iPSC-derived NPCs (SS-NPCs) showed distinct expression profiles from ATR non-mutated NPCs with negative enrichment of neuronal genesis-related gene sets. In SS-NPCs, abnormal mitotic spindles occurred more frequently than in gene-corrected counterparts, and the alignment of NPCs in the surface of the neurospheres was perturbed. Finally, we tested several splicing-modifying compounds and found that TG003, a CLK1 inhibitor, could pharmacologically rescue the exon 9 skipping in SS-NPCs. Treatment with TG003 restored the ATR kinase activity in SS-NPCs and decreased the frequency of abnormal mitotic events. In conclusion, our iPSC model revealed a novel effect of the ATR mutation in mitotic processes of NPCs and NPC-specific missplicing, accompanied by the recovery of neuronal defects using a splicing rectifier.

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