Product Pathways - Neuroscience
TrkA Kinase #7422
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Description
Purified recombinant human TRKA (Asn440-Gly796) kinase, supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing a fragment of human TrkA (Asn440-Gly796) (GenBank Accession No. NM_002529) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-TrkA fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. TrkA kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 5 mM MnCl2, 0.05 mM DTT, 50 ng/μl BSA, 50 μM ATP, Substrate: Poly (EY. 4:1) 800 ng/μL and recombinant TrkA: variable.
Quality Control
The theoretical molecular weight of the GST-TrkA fusion protein is 66 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. TrkA kinase activity was determined using a radiometric assay [Fig.2].
Background
The family of Trk receptor tyrosine kinases consists of TrkA, TrkB and TrkC. While the sequence of these family members is highly conserved, they are activated by different neurotrophins: TrkA by NGF, TrkB by BDNF or NT4, and TrkC by NT3. TrkA regulates proliferation and is important for development and maturation of the nervous system (1). Phosphorylation at Tyr490 is required for Shc association and activation of the Ras-MAP kinase cascade. Residues Tyr674/675 lie within the catalytic domain, and phosphorylation at this site reflects TrkA kinase activity (2-6). Point mutations, deletions and chromosomal rearrangements (chimeras) cause ligand-independent receptor dimerization and activation of TrkA. Many malignancies including breast, colon, prostate and thyroid carcinomas and acute myeloid leukemia have activated TrkA. Expression of TrkA in neuroblastomas is a good prognostic marker because it signals growth arrest and differentiation of cells originating from the neural crest (1).
- Pierotti, M.A. and Greco, A. (2006) Cancer Lett. 232, 90-98.
- Segal, R.A. and Greenberg, M.E. (1996) Annu. Rev. Neurosci. 19, 463-489.
- Stephens, R.M. et al. (1994) Neuron 12, 691-705.
- Obermeier, A. et al. (1993) EMBO J. 12, 933-941.
- Obermeier, A. et al. (1994) EMBO J. 13, 1585-1590.
- Yao, R. and Cooper, G.M. (1995) Science 267, 2003-2006.
Application References
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Companion Products
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
