Product Pathways - Lymphocyte Signaling
Hck Kinase #7448
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Description
Purified recombinant human Hck (Asn230-Arg497) 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 human Hck (Asn230-Arg497) (GenBank Accession No. NM_002110) 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-Hck fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. Hck kinase activity was measured in a radiometric assay using the following reaction conditions: 4 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: Poly(Glu-Tyr), 400 ng/μL and recombinant Hck: variable.
Quality Control
The theoretical molecular weight of the GST-Hck fusion protein is 56 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. Hck kinase activity was determined using a radiometric assay [Fig.2].
Background
Hck (hemopoietic cell kinase) is a protein tyrosine kinase of the Src family prominently expressed in the lymphoid and myeloid lineages of hemopoiesis (1). It participates in transducing a variety of extracellular signals, which ultimately affect cellular processes including proliferation, differentiation and migration.The well-defined modular structure of Hck comprises a relatively divergent, NH2-terminal "unique" domain, which is subject to post-translational lipid modifications thereby targeting Hck to the plasma membrane. Src homology 3 (SH3) and 2 (SH2) domains, and a tyrosine kinase catalytic domain follow the "unique" domain. The catalytic activity of Hck is regulated, both positively and negatively, by tyrosine phosphorylation of highly conserved tyrosine (Y) residues. Phosphorylation of a single conserved Tyr499 residue in the COOH terminus of Hck by the protein kinase Csk renders Hck inactive as a result of an intramolecular interaction between the phosphorylated tyrosine (pY) residue and its own SH2 domain. Disruption of this interaction, either as a result of dephosphorylation, or substitution of the COOH-terminal regulatory Y residue with phenylalanine (F; e.g., HckY499F), or COOH-terminal truncation mutations as observed in the virally transduced v-Src oncoprotein, results in constitutive activation of Hck. In contrast to phosphorylation of the COOH-terminal regulatory tyrosine residue, autophosphorylation of a tyrosine residue (Tyr388) within the kinase domain of Hck acts to positively regulate its catalytic activity. Thus, activation of Hck requires both disruption of the COOH-terminal regulatory tyrosine-SH2 domain interaction and autophosphorylation of the regulatory tyrosine residue within the kinase domain ( 2, 3). The dysfunction or dysregulation of Hck may contribute to the pathogenesis of some human leukemias (4).
- Quintrell, N. et al. (1987) Mol. Cell. Biol. 7, 2267-2275.
- Ziegler, C.A. et al. (1989) Mol. Cell. Biol. 9, 2724-2727.
- Kefalas, P. et al. (1995) Int. J. Biochem. Cell. Biol. 27, 551-563.
- Hu, Y. et al. (2004) Nat. Genet. 36, 453-461.
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.
