Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tnk1 is a nonreceptor tyrosine kinase cloned from CD34+/Lin-/CD38- hematopoietic stem/progenitor cells. The cDNA predicts a 72-kDa protein containing an NH(2)-terminal kinase, a Src Homology 3 (SH3) domain, and a proline-rich (PR) tail. We generated rabbit antiserum to a GST-Tnk1(SH3) fusion protein. Affinity-purified anti-Tnk1 antibodies specifically recognized a 72-kDa protein in Tnk1-transfected COS-1 cells and cells which express Tnk1 mRNA. Western blot analysis indicated that Tnk1 is expressed in fetal blood cells, but not in any other hematopoietic tissues examined. Tnk1 immunoprecipitated from cell lysates possessed kinase activity and was tyrosine phosphorylated. In binding experiments with a panel of GST-fusion constructs, only GST-PLC-gamma1(SH3) interacted with in vitro translated Tnk1. GST-protein precipitations from cell lysates confirmed that GST-PLC-gamma1(SH3) associated with endogenously expressed Tnk1. Conversely, GST-Tnk1(PR) protein constructs complexed with endogenously expressed PLC-gamma1. The association of Tnk1 with PLC-gamma1 suggests a role for Tnk1 in phospholipid signal transduction.
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PMID:Characterization of the tyrosine kinase Tnk1 and its binding with phospholipase C-gamma1. 1087 1

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.
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PMID:Detecting protein-protein interactions using Renilla luciferase fusion proteins. 1244 82

Cyclin-dependent kinase 5 (Cdk5) has been shown to regulate adhesion and migration of lens and corneal epithelial cells. To explore protein-protein interactions that may mediate these functions, we performed yeast two-hybrid screening on an embryonic rat lens library using Cdk5 and its regulators, p35 and p39 as baits. This screen identified an interaction between p39 and non-muscle myosin essential light chain (MLC(17)). GST pull-down experiments demonstrated that p39 binds directly to MLC(17) through a strong binding site in the N-terminal 109 amino acids of p39. Immunoprecipitation of proteins from Cos1 cells co-transfected with GFP-MLC(17) and HA-p39 confirmed that these proteins interact intracellularly. Immunofluorescence microscopy of co-transfected lens epithelial cells showed that GFP-MLC(17) and HA-p39 co-localize along cytoskeletal fibrils. Moreover, endogenous rat lens p39 co-immunoprecipitated with MLC(17) and myosin heavy chain II (MHC II), demonstrating that the interaction is physiological and serves to link p39 to the cytoskeleton.
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PMID:The CDK5 activator, p39, binds specifically to myosin essential light chain. 1727 6