Product Pathways - Kinases
MYLK2 Kinase #7498
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 full length human MYLK2 (Met1-Val596) 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 full length human MYLK2 (Met1-Val596) (GenBank Accession No. NM_033118.2) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using GSH-agarose.
Gel Staining
Figure 1. The purity of the GST-MYLK2 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. MYLK2 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, 0.4 mM CaCl2, 3 μM Na-orthovanadate, 1.2 mM DTT, 0.04 mg/ml calmodulin, 2.5 μg/50 μl PEG20,000, ATP variable, Substrate: GSK3 derived peptide (R11-SGRARTSSFAEPGGK) 200 ng/μL, and recombinant MYLK2: 200 ng/50 μl.
Quality Control
The theoretical molecular weight of the GST-MYLK2 fusion protein is 95 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. MYLK2 kinase activity was determined using a radiometric assay [Fig.2].
Background
Myosin light chain kinases (MYLKs) are Ca2+/calmodulin-dependent protein kinases that regulate myosin motor activities through phosphorylation of myosin regulatory light chain (RLC). This phosphorylation increases the actin-activated myosin ATPase activity and is thought to play major roles in a number of biological processes, including cell spreading and migration, transepithelial permeability and muscle contraction (1). In vertebrates there are 2 genes for MYLK. The skeletal MYLK (MYLK2) is expressed predominantly in skeletal muscles and regulates tension potentiation due to repetitive stimulation in fast-twitch skeletal muscles (2). The smooth muscle MYLK is more ubiquitously expressed with the greatest expression observed in smooth muscle tissue (3). Skeletal MYLK can phosphorylate myosin light chains isolated from skeletal or smooth muscle. In contrast, smooth muscle MYLK specifically phosphorylates myosin light chains isolated from smooth muscle only (4).
- Kamm, K.E. and Stull, J.T. (2001) J. Biol. Chem. 276, 4527-4530.
- Zhi, G. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 17519-17524.
- Lazar, V. and Garcia, J.G. (1999) Genomics 57, 256-267.
- Herring, B.P. et al. (1992) J. Biol. Chem. 267, 25945-25950.
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.
