Identification of MELK-interacting proteins in HEK293T cells using IP-MS
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Epithelial Cell, Cell Culture
SUBMITTER: 
Kuai Yu
LAB HEAD: Kuai Yu
PROVIDER: PXD057095 | Pride | 2026-04-01
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| R20220801404_L_1.msf | Msf | |||
| R20220801404_L_1.raw | Raw | |||
| R20220801404_L_2.raw | Raw | |||
| R20220801404_M_1.raw | Raw | |||
| R20220801404_M_2.raw | Raw |
Items per page: 5 1 - 5 of 6 |
Similar Datasets
Project description:To investigate whether MELK phosphorylates Cofilin-1 at residue Threonine 70 (T70), we performed a phosphoproteomic validation using immunoprecipitation coupled with mass spectrometry (IP-LC-MS/MS). HEK293T cells were divided into three experimental groups: (1) MOCK control (Empty Vector), (2) MELK overexpression, and (3) MELK overexpression treated with the inhibitor OTSSP167. Endogenous Cofilin-1 was enriched by immunoprecipitation. The study aimed to identify and quantify the phosphorylation status of Cofilin-1, specifically focusing on the T70 site, and to assess the impact of MELK kinase activity and its inhibition by OTSSP167 on this phosphorylation event.
2026-04-01 | PXD074164 | Pride
Project description:The key objectives of this study were to evaluate the selectivity profiles of three MELK inhibitors, 8a, HTH, and OTS, using a cell-based assay, in order to identify a highly selective inhibitor to subsequently investigate MELK function. To this end, we utilized a chemical proteomics approach called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS) to characterize the selectivity of these MELK inhibitors in TNBC cells.
2020-01-09 | PXD016022 | Pride
Project description:Maternal Embryonic Leucine Zipper Kinase (MELK), a Ser/Thr protein kinase, is highly over expressed in stem and cancer cells. The oncogenic role of MELK is attributed to its capacity to disable critical cell cycle checkpoints and to enhance replication. Most functional studies have relied on the use of siRNA/shRNA-mediated gene silencing, but this is often compromised by off target effects. Here we present the cellular validation of a novel, potent and selective small molecule MELK inhibitor, MELK-T1, which has enabled us to explore the biological function of MELK. Strikingly, the binding of MELK-T1 to endogenous MELK triggers a rapid and proteasome dependent degradation of the MELK protein. Treatment of MCF-7 breast adenocarcinoma cells with MELK-T1 leads to an accumulation of stalled replication forks and double strand breaks, followed by a replicative senescence phenotype. This phenotype correlates with a rapid and long-lasting ATM activation and phosphorylation of CHK2. Furthermore, MELK-T1 induces strong phosphorylation of p53 and prolonged up-regulation of p21. Our data generated with MELK-T1 indicate that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA damage tolerance. Thus, targeting MELK by combined inhibition of its catalytic function and inhibitor-induced degradation might sensitize tumors to DNA-damaging agents or radiation therapy, by lowering the DNA damage threshold.
2015-12-11 | GSE62477 | GEO
Project description:Melanoma accounts for over 80% of skin cancer-related deaths and current therapies provide only short-term benefit to patients. Here, we show in melanoma cells that maternal embryonic leucine zipper kinase (MELK) is transcriptionally upregulated by the MAP kinase pathway via transcription factor E2F1. MELK knockdown or pharmacological inhibition blocked melanoma growth and enhanced the effectiveness of BRAFV600E inhibitor against melanoma cells. To identify mediators of MELK function, we performed stable isotope labeling with amino acids in cell culture (SILAC) and identified 469 proteins that had downregulated phosphorylation after MELK inhibition. Remarkably, 139 of these proteins were previously reported as substrates of BRAF or MEK, demonstrating that MELK is an important downstream mediator of the MAPK pathway. Furthermore, we show that MELK promotes melanoma growth by activating NF-B pathway activity via Sequestosome 1 (SQSTM1/p62). Collectively, these results underpin an important role for MELK in melanoma growth, downstream of the MAPK pathway.
2017-12-07 | PXD007872 | Pride
Project description:Maternal embryonic leucine zipper kinase (MELK) has been implicated in cell-cycle regulation, yet its transcriptional programs in megakaryocytic cells remain incompletely defined. To profile MELK-dependent gene expression changes, we performed bulk RNA-seq on the human megakaryoblastic cell line MEG-01 transduced with a control pLKO.1 vector or a MELK-targeting shRNA (sh2-MELK), with three independent biological replicates per condition. Paired-end sequencing reads (raw FASTQ files) and processed gene-level expression quantifications are provided to support differential expression and pathway analyses of MELK knockdown in megakaryocytic cells.
2026-04-02 | GSE322507 | GEO
Project description:In order to address the putative role of MELK and UBE2C in prostate cancer development and progression, we performed functional analysis upon siRNA-based knockdown, and searched for downstream genes and processes by microarray experiments. RNAi-based inhibition of MELK and UBE2C was efficient in PC3 prostate cancer cells and decreased transcriptional level down to about 30% remaining expression level. Illumina microarray experiments were done upon siRNA based knockdown 48h after transfection of PC3 cells in triplicates.
2012-10-19 | E-GEOD-32873 | biostudies-arrayexpress
Project description:Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Recent studies including our own identified that the mitotic kinase, maternal embryonic leucine-zipper kinase (MELK), is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we provide evidence that the kinase activity of MELK is essential for the action of MELK in GSCs and vital for GBM growth. We utilized in silico structure-based analysis for protein-compound interaction to predict that a recently identified small molecule, Compound 1 (C1), binds to the kinase-active site of MELK protein and eliminates MELK kinase activity in nanomolar concentrations. When treated with C1, GSCs undergo mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed using MELK shRNA-mediated knockdown. C1 treatment strongly induces tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuates growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors.
2013-08-28 | GSE50227 | GEO
Project description:Oncogenic EZH2 is overexpressed and extensively involved in the pathophysiology of different cancers including extranodal natural killer/T-cell lymphoma (NKTL). However, the mechanisms regarding EZH2 upregulation is poorly understood, and it still remains untargetable in NKTL. In this study, we examine EZH2 protein turnover in NKTL and identify MELK kinase as a regulator of EZH2 ubiquitination and turnover. Using quantitative mass spectrometry (MS) analysis, we observed a MELK-mediated increase of EZH2 S220 phosphorylation along with a concomitant loss of EZH2 K222 ubiquitination, suggesting a phosphorylation-dependent regulation of EZH2 ubiquitination. MELK inhibition through both chemical and genetic means led to ubiquitination and destabilization of EZH2 protein. Importantly, we determine that MELK is upregulated in NKTL, and its expression correlates with EZH2 protein expression as determined by tissue microarray derived from NKTL patients. Interestingly, FOXM1, which connected MELK to EZH2 signaling in glioma, was not involved in mediating EZH2 ubiquitination. Furthermore, we identify USP36 as the deubiquitinating enzyme which deubiquitinates EZH2 at K222. These findings uncover an important role of MELK and USP36 in mediating EZH2 stability in NKTL. Moreover, MELK overexpression led to decreased sensitivity to Bortezomib treatment in NKTL based on deprivation of EZH2 ubiquitination. Therefore, modulation of EZH2 ubiquitination status by targeting MELK may be a new therapeutic strategy for NKTL patients with poor Bortezomib response.
2019-08-26 | PXD015008 | Pride
Project description:Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Recent studies including our own identified that the mitotic kinase, maternal embryonic leucine-zipper kinase (MELK), is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we provide evidence that the kinase activity of MELK is essential for the action of MELK in GSCs and vital for GBM growth. We utilized in silico structure-based analysis for protein-compound interaction to predict that a recently identified small molecule, Compound 1 (C1), binds to the kinase-active site of MELK protein and eliminates MELK kinase activity in nanomolar concentrations. When treated with C1, GSCs undergo mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed using MELK shRNA-mediated knockdown. C1 treatment strongly induces tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuates growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors. Microarray-based expression analysis of glioma stem cells treated with MELK-signaling inhibitors
2013-08-28 | E-GEOD-50227 | biostudies-arrayexpress
Project description:Triple-negative breast cancer (TNBC) has high relapse and metastasis rates and a high proportion of cancer stem-like cells (CSCs), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, is known to promote CSC maintenance and malignant transformation. Our study showed that MELK knockdown using siRNA or MELK inhibition using the MELK inhibitor MELK-In-17 significantly reduced invasiveness, reversed epithelial-to-mesenchymal transition (EMT), and reduced CSC self-renewal and maintenance in TNBC cells. Nude mice injected with CRISPR MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and improved overall survival compared with mice injected with control cells. Furthermore, MELK-In-17 suppressed 4T1 tumor growth in syngeneic BALB/c mice. Our findings indicate that MELK supports metastasis by promoting EMT and the CSC phenotype in TNBC. In our microarray analysis, we identified potential downstream targets of MELK, including STAT5 and NF-kB target genes, as well as genes involved in tumor progression and metastasis (i.e., EMT, angiogenesis, hypoxia, and apical junction). EMT was the most strongly enriched hallmark among genes highly expressed in Cas9-p15 control cells, further confirming that EMT is a major factor contributing to MELK-induced metastasis in TNBC. We also identified a direct physical interaction partner (PRKAB2) of MELK and a set of intermediate proteins (CDC25B, EZH2, FOXM1, JUN, MAP3K5, PRKAB1, PRKAB2, and SMAD2), suggesting that these proteins are key components of MELK-induced signal transduction.
2023-05-01 | GSE227774 | GEO