Cell Signaling Technology

Product Pathways - HTScan Kinase Assay Kits

HTScan® MAPKAPK-5 Kinase Assay Kit #7660

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.

Kit Includes Quantity
Phospho-Tyrosine Hydroxylase (Ser40) Antibody # 2791 30 microliters
Kinase Buffer (10X) # 9802 15 milliliters
ATP (10 mM) # 9804 1 milliliters
Tyrosine Hydroxylase (Ser40) Biotinylated Peptide # 1132 1.25 milliliters
MAPKAPK-5 Kinase # 7659 2 x 5 micrograms

Description

The kit provides a means of performing kinase activity assays with recombinant human MAPKAPK-5 kinase. It includes active MAPKAPK-5 kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-serine/threonine antibody for detection of the phosphorylated form of the substrate peptide.

Molecular Weights

Peptide substrate, Biotin- Tyrosine Hydroxylase (Ser40): 2,326 Daltons. GST-MAPKAPK-5 Kinase: 84 kDa.

Peptide Core Sequence

RRQS*LIE

Kinase Assay - Radiometric

Kinase Assay - Radiometric

Figure 1. MAPKAPK-5 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: Tetra (LRRWSLG), 3 µg/50 µl and recombinant MAPKAPK-5: 200 ng/50 µl.

DELFIA

DELFIA

Figure 5. Staurosporine inhibition of MAPKAPK-5 kinase activity: DELFIA® data generated using Phospho-Tyrosine Hydroxylase (Ser40) Antibody #2791 to detect phosphorylation of MAPKAPK-5 substrate peptide #1132 by MAPKAPK-5 kinase. In a 50 µl reaction, 100 ng MAPKAPK-5, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

DELFIA

DELFIA

Figure 3. Dose dependence curve of MAPKAPK-5 kinase activity: DELFIA® data generated using Phospho-Tyrosine Hydroxylase (Ser40) Antibody #2791 to detect phosphorylation of substrate peptide #1132 by MAPKAPK-5 kinase. In a 50 µl reaction, increasing amounts of MAPKAPK-5 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)


DELFIA

DELFIA

Figure 4. Peptide concentration dependence of MAPKAPK-5 kinase activity: DELFIA® data generated using Phospho-Tyrosine Hydroxylase (Ser40) Antibody #2791 to detect phosphorylation of substrate peptide #1132 by MAPKAPK-5 kinase. In a 50 µl reaction, 100 ng of MAPKAPK-5 and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

DELFIA

DELFIA

Figure 2. Time course of MAPKAPK-5 kinase activity: DELFIA® data generated using Phospho-Tyrosine Hydroxylase (Ser40) Antibody #2791 to detect phosphorylation of MAPKAPK-5 substrate peptide #1132 by MAPKAPK-5 kinase. In a 50 µl reaction, 100 ng MAPKAPK-5 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

Source / Purification

The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing human MAPKAPK-5 (Met1-Gln473) (GenBank Accession No. NM_139078) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

Quality Control

The substrate peptide was selected using our Serine/Threonine Kinase Substrate Screening Kit #7400. Phospho-Tyrosine Hydroxylase (Ser40) Antibody #2791 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified MAPKAPK-5 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain. MAPKAPK-5 kinase specific activity was determined using a radiometric assay [Fig.1]. Time course [Fig.2], kinase dose-dependency [Fig.3] and substrate dose-dependency [Fig.4] assays were performed to verify MAPKAPK-5 activity using the MAPKAPK-5 substrate peptide provided in this kit. MAPKAPK-5 sensitivity to the inhibitor staurosporine was measured using the MAPKAPK-5 substrate peptide provided in this kit [Fig.5].

Background

MAPKAPK5 belongs to the mitogen-activated protein kinase (MAPK) activated protein kinases (MK) subfamily that includes MAPKAPK2/MK2 and MK3/3pK. The MK subfamily is part of a family of protein kinase subfamilies downstream of MAPK pathways and includes ribosomal S6 kinase (RSKs), mitogen and stress activated kinases (MSKs) and MAPK-interacting kinases (MNKs). All MKs are activated by MAPK pathways and mediate important processes such as gene expression, cell-cycle progression and have been implicated in inflammation and cancer (1,2). MAPKAPK5 shows binding to and activation by the p38 kinase and extracellular-regulated kinases (ERK) (3,4). Recently, MAPKAPK5 was shown to be activated by ERK3 and act as a chaperone to ERK3 (5,6). While overexpressed MAPKAPK5 shares similar substrates with MAPKAPK2, such as HSP27 and glycogen synthase, recent work with MAPKAPK5 knock-out mice indicate distinct substrates and functional properties (7).

  1. Roux, P.P. and Blenis, J. (2004) Microbio.l Mol. Biol. Rev. 68, 320-344.
  2. Gaestel, M. (2006) Nat. Rev. Mol. Cell Biol. 7, 120-130.
  3. New, L. et al. (1998) EMBO J. 17, 3372-3384.
  4. Ni, H. et al. (1998) Biochem. Biophys. Res. Commun. 243, 492-496.
  5. Schumacher, S. et al. (2004) EMBO J. 23, 4770-4779.
  6. Seternes, O.M. et al. (2004) EMBO J. 23, 4780-4791.
  7. Shi, Y. et al. (2003) Mol. Cell Biol. 23, 7732-7741.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

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.

Products