Project description:Here we describe a bead-based method capable of profiling tyrosine kinase phosphorylations in a multiplexed, high-throughput and low-cost manner. This approach allows for the discovery of tyrosine kinase-activating events, even when the DNA sequence is wild-type. In an effort to pilot the establishment of a tyrosine kinase activation catalog, we profiled tyrosine phosphorylation levels of 62 tyrosine kinases in 130 human cancer lines, and followed-up on the frequent SRC phosphorylation in glioblastoma. Keywords: quantitative measurements of tyrosine phosphorylation levels on tyrosine kinases Total protein lysates were collected from 130 cancer cell lines. Tyrosine phosphorylation levels on 62 tyrosine kinases were measured with the bead assay.
Project description:Here we describe a bead-based method capable of profiling tyrosine kinase phosphorylations in a multiplexed, high-throughput and low-cost manner. This approach allows for the discovery of tyrosine kinase-activating events, even when the DNA sequence is wild-type. In an effort to pilot the establishment of a tyrosine kinase activation catalog, we profiled tyrosine phosphorylation levels of 62 tyrosine kinases in 130 human cancer lines, and followed-up on the frequent SRC phosphorylation in glioblastoma. Keywords: quantitative measurements of tyrosine phosphorylation levels on tyrosine kinases
Project description:Supporting MSMS dataset for phosphorylation mapping reported in the paper
"Structural basis of human 5,10-methylenetetrahydrofolate reductase (MTHFR) regulation by phosphorylation and S-adenosylmethionine inhibition."
Froese et al.
Project description:Numerous leucine-rich repeat kinase 2 mutations identified throughout the protein are associated with Parkinson disease, however the activating G2019S kinase domain mutation is currently regarded as the most common cause of familial and sporadic forms of this disorder. Despite studies demonstrating the prominent role that its kinase activity plays in the pathobiology of leucine-rich repeat kinase 2, few substrates have been identified and only a subset of these have been linked to disease. Therefore, we utilized protein microarrays to screen over 9,000 human proteins in an unbiased radiometric assay for potential targets of the kinase.
Project description:Numerous leucine-rich repeat kinase 2 mutations identified throughout the protein are associated with Parkinson disease, however the activating G2019S kinase domain mutation is currently regarded as the most common cause of familial and sporadic forms of this disorder. Despite studies demonstrating the prominent role that its kinase activity plays in the pathobiology of leucine-rich repeat kinase 2, few substrates have been identified and only a subset of these have been linked to disease. Therefore, we utilized protein microarrays to screen over 9,000 human proteins in an unbiased radiometric assay for potential targets of the kinase. ProtoArrayM-bM-^DM-" Human Protein Microarrays v5.0 (Invitrogen, Carlsbad, CA, USA) were used following the manufactureM-bM-^@M-^Ys protocol (ProtoArray Kinase Substrate Identification Kit). Briefly, slides were equilibrated at 4C for 15 min before blocking in 1% BSA in PBS for 1 h at 4oC with gentle shaking. Recombinant G2019S or D1994A glutathione-S-transferase (GST)-LRRK2 (970-2527) (Invitrogen) was diluted to 50nM in 20mM Tris (pH 7.5), 10mM MgCl2, 1mM EGTA, 1mM Na3VO4, 5mM beta-glycerophosphate, 2mM DTT, 0.02% polysorbate 20, and 10 mCi /mL of [gamma- 33P]ATP (33 nM final concentration) in a total volume of 120uL. Slides were overlayed with buffer alone, or buffer containing G2019S or D1994A LRRK2, then covered with a coverslip and placed in a 50 mL conical tube for 1 h at 30oC. Afterwards, slides were washed with 0.5% SDS buffer and water followed by centrifugation. Dried slides were exposed to a PhosphorImager plate (Amersham Biosciences, Piscataway, NJ, USA), and scanned on a Storm 840 (Molecular Dynamics, Inc., Sunnyvale, CA, USA) at 50 microns.
Project description:We performed phosphorylation site mapping on of budding yeast Atg13 applying a quantitative mass spectrometry-based proteomics approach based on stable isotope labeling with amino acids in cell culture (SILAC). We determined changes in the Atg13 phosphorylation pattern in response of inhibition of kinase Atg1 and starvation conditions (using rapamycin), respectively. To do so, Atg13 was tandem purified via a histidine-biotin (HB) tag from cells growing at the respective experimental condition. Different stimulus-dependent PTM profiles of Atg13 have been identified.
Project description:We report a synthetic chromatin kinase, by fusing nuclease-null CRISPR/Cas9 to a hyperactive, truncated variant of the human MSK1 histone kinase (dCas9-dMSK1). To investigate the role of MSK1 on transcriptional regulation, we explore the different transcriptome between wildtype and MSK1 knockout HEK 293T cell lines by RNA-seq. To test the efficacy of dCas9-dMSK1 at thousands of human promoters in high-throughput and to evaluate if histone phosphorylation could uncover novel mediators of pathological gene expression, we performed a CRISPRa screening assay in A375 cells using the genome wild gRNA library. The enriched gRNAs were measured by Miseq.
Project description:Protein expression profile was analyzed by antibody array for cell cycle control phosphorylation with 238 antibodies with bladder cancer cell line, TCCSUP, and KSHV-infected TCCSUP cells. Protein was extracted from uninfected bladder cancer cell line, TCCSUP, and KSHV-infected TCCSUP cells, and they were analyzed by antibody array for cell cycle control phosphorylation.
Project description:Examine protein phosphorylation status during peripheral nerve regeneration when using autologous nerve graft or tissue engineered nerve graft to bridge nerve gap.