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:Protein Ser/Thr kinase CK2 is involved in a myriad of cellular processes including cell growth and proliferation by phosphorylating hundreds of substrates, yet the regulation process of CK2 function is poorly understood. The CK2 catalytic subunit, CK2α, is phosphorylated at Thr344 and phosphorylation on the C-terminal tail of CK2α is required for interaction with Pin1 protein. The substrate selectivity for protein kinase CK2α was examined by performing kinase assays on protein microarrays spotted with 17,000 human proteins. Semisynthetic CK2α proteins were prepared to contain an unmodified C-terminal tail or phospho-Thr (pThr) at T344. These semisynthetic proteins were used to determine if the phosphorylation-dependent interaction of CK2α with Pin1 can modulate the substrate selectivity for CK2. The different semisynthetic CK2α proteins (unmodified and pThr344) were tested alone and in the presence of the recombinant Pin1 protein. Pin1 has been shown to interaction with CK2α only when CK2α is phoshorylated on its C-terminal site (including Thr344). In the study presented here, kinase assays were performed using two different semisynthetic CK2α proteins: unmodified C-terminal tail and phospho-Thr (pThr) at 344. The semisynthetic proteins were each tested alone and in the presence of the recombinant Pin1 protein. There were four different kinase conditions and each condition was performed in duplicate.
Project description:To identify the key phosphorylation residues of SlLYK4, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS)in vitro kinase assays with liquid of GST-SlLYK4 and MBP-SlLYK13CD or MBP (mock) after in vitro kinase assays. And then SlLYK4-FLAG was transiently expressed in tomato hairy roots and samples were collected post EPS or chitin treatment, followed by immunoprecipitation-mass spectrometry analysis.
Project description:To identify the specific site of STX17 phosphorylated by ULK, in vitro kinase assays were performed with ULK1 immunoprecipitated from 293T cells and STX17 purified from E. coli. Purified GST-STX17 proteins were used for kinase assay as substrates to determine phosphorylation sites.
Project description:To identify putative phosphorylation sites on TCF3 (E47), TCF4, and IRF4, in vitro kinase assays were performed using recombinant MKK6E (a p38α activator) and p38α as kinases, and TCF3(E47), TCF4, or IRF4 proteins as substrates, the phosphorylation sites were analyzed by mass spectrometry (MS).
Project description:Protein Ser/Thr kinase CK2 is involved in a myriad of cellular processes including cell growth and proliferation by phosphorylating hundreds of substrates, yet the regulation process of CK2 function is poorly understood. The CK2 catalytic subunit, CK2α, is modified by O-GlcNAc on Ser347 proximal to a Cdk1 phosphorylation site at Thr344 on the same protein. The substrate selectivity for protein kinase CK2 was examined by performing kinase assays on protein microarrays spotted with 17,000 human proteins. Semisynthetic CK2α proteins were prepared to contain an unmodified C-terminal tail, S-GlcNAc-Serine at S347, or Pfa (non-hyrdolyzeable phosphomimic) at T344. These semisynthetic proteins were used to determine if the posttranslational modifications on CK2 alpha modulate the substrate selectivity for this pleiotropic kinase. The different semisynthetic CK2α proteins were tested alone and in the presence of the regulatory subunit CK2β since it is known that the CK2α subunit is active both in its isolated state and in the heterotetrameric state formed in the presence of the regulatory beta subunit. The CK2β subunit has been shown to modulate CK2 activity with some substrates and not others. In the study presented here, kinase assays were performed using three different semisynthetic CK2 alpha proteins: unmodified C-terminal tail; S-GlcNAc-Ser at 347; and Pfa (phosphomimic) at 344. The semisynthetic proteins were each tested alone and in the presence of the regualatory CK2 beta subunit. There were six different kinase conditions and each condition was performed in duplicate and one no kinase control was performed to eliminate autophorylated proteins.
Project description:Protein Ser/Thr kinase CK2 is involved in a myriad of cellular processes including cell growth and proliferation by phosphorylating hundreds of substrates, yet the regulation process of CK2 function is poorly understood. The CK2 catalytic subunit, CK2α, is phosphorylated at Thr344 and phosphorylation on the C-terminal tail of CK2α is required for interaction with Pin1 protein. The substrate selectivity for protein kinase CK2α was examined by performing kinase assays on protein microarrays spotted with 17,000 human proteins. Semisynthetic CK2α proteins were prepared to contain an unmodified C-terminal tail or phospho-Thr (pThr) at T344. These semisynthetic proteins were used to determine if the phosphorylation-dependent interaction of CK2α with Pin1 can modulate the substrate selectivity for CK2. The different semisynthetic CK2α proteins (unmodified and pThr344) were tested alone and in the presence of the recombinant Pin1 protein. Pin1 has been shown to interaction with CK2α only when CK2α is phoshorylated on its C-terminal site (including Thr344).
Project description:The αNAC (alpha chain of the Nascent polypeptide-Associated Complex) transcriptional coregulator is developmentally expressed in osteoblasts and regulates osteoblast differentiation in vitro and in vivo. αNAC can activate or repress gene transcription, a function that is dynamically regulated by post-translational modification. Phosphorylation of residue Ser132 stimulates the sumoylation of αNAC on Lys127 to repress gene expression. Using in vitro kinase assays, we show that Ser132 phosphorylation is mediated by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Pharmacological inhibition of DNA-PKcs kinase activity or gene silencing of Prkdc (encoding DNA-PKcs) in murine osteoblastic MC3T3-E1 cells and human adipose-derived mesenchymal stromal cells markedly enhanced osteogenesis and the expression of osteoblast differentiation marker genes. ChIP-seq identified Ezh2 as a target of the αNAC/DNA-PKcs signaling pathway. Mechanistically, inhibition of DNA-PKcs repressed Ezh2 expression, induced cell cycle block, and increased osteogenesis by significantly enhancing the bone morphogenetic protein 2 (BMP-2) response in osteoblasts and other mesenchymal cells. Importantly, in vivo inhibition of the kinase enhanced bone biomechanical properties, and bones from osteoblast-specific conditional Prkdc-knockout mice exhibited increased stiffness. In conclusion, DNA-PKcs is a negative regulator of osteoblast differentiation, and therefore DNA-PKcs inhibitors may have therapeutic potential for bone regeneration and metabolic bone diseases.
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.