Cell Signaling Technology

Product Pathways - Tyrosine Kinase/ Adaptors

Met Kinase #7760

Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.

Description

Purified recombinant human Met Kinase (Lys956-Ser1390), supplied as a GST fusion protein.

Source / Purification

The GST-Met fusion protein was produced using a baculovirus expression system with a construct expressing human Met (Lys956-Ser1390) (GenBank accession No. X54559) with an amino-terminal CST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

Gel Staining

Gel Staining

Figure 1. The purity of the GST-Met fusion protein was analyzed using SDS/PAGE followed by anti-Met Western blot (A) or Silver stain (B).

Kinase Assay - Radiometric

Kinase Assay - Radiometric

Figure 2. Met kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20,000, Substrate: PolyAEKY, 1.5 µg/50 µl and 100 ng/50 µl Recombinant Met.

Kinase Assay - DELFIA

Kinase Assay - DELFIA

Figure 3. Dose dependence curve of Met kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1315) by Met kinase. In a 50 µl reaction, increasing amounts of Met and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)


Quality Control

The theoretical molecular weight of the GST-Met fusion protein is 79 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. Met kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure Met activity using HTScan™ Met Kinase Assay Kit #7440 [Fig.3].

Background

Met, a high affinity tyrosine kinase receptor for hepatocyte growth factor (HGF, also known as scatter factor), is a disulfide-linked heterodimer made of 45 kDa α- and 145 kDa β-subunits (1,2). The α-subunit and the amino-terminal region of the β-subunit form the extracellular domain. The remainder of the β-chain spans the plasma membrane and contains a cytoplasmic region with tyrosine kinase activity. Interaction of Met with HGF results in autophosphorylation at multiple tyrosines, which recruit several downstream signaling components, including Gab1, c-Cbl and PI3 kinase (3). These fundamental events are important for all of the biological functions involving Met kinase activity. Addition of a phosphate at cytoplasmic Tyr1003 is essential for ubiquitination and Met protein degradation (4). Phosphorylation of Tyr1234/1235 in the Met kinase domain is critical to kinase activation. Phosphorylation of Tyr1349 in the Met cytoplasmic domain provides a direct binding site for Gab1 (5). Altered Met levels and/or tyrosine kinase activities are found in several types of tumors, including renal, colon and breast. Thus, Met is an attractive cancer therapeutic and diagnostic target (6).

  1. Weidner, K.M. et al. (1993) J. Cell Biol. 121, 145-154.
  2. Park, M. et al. (1986) Cell 45, 895-904.
  3. Bardelli, A. et al. (1997) Oncogene 15, 3103-3111.
  4. Taher, T.E. et al. (2002) J. Immunol. 169, 3793-3800.
  5. Schaeper, U. et al. (2000) J. Cell Biol. 149, 1419-1432.
  6. Traxler, P. et al. (2001) Med. Res. Rev. 21, 499-512.

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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.

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