Product Pathways - HTScan Kinase Assay Kits
HTScan® PKCα Kinase Assay Kit #7579
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 |
|---|---|
| ATP (10 mM) # 9804 | 1 milliliters |
| Phospho-PKA Substrate (RRXS/T) (100G7E) Rabbit mAb # 9624 | 30 microliters |
| Kinase Buffer (10X) # 9802 | 15 milliliters |
| CREB (Ser133) Biotinylated Peptide # 1331 | 1250 microliters |
| PKCα Kinase # 7578 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human PKCalpha kinase. It includes active PKCalpha 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-CREB (Ser133): 2,326 Daltons. GST-PKCalpha Kinase: 110 kDa.
Peptide Core Sequence
RRPS*YRK
Kinase Assay - Radiometric
Figure 1. PKCalpha 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, 1 µM ATP, 2.5 µg/50 µl PEG20,000, Substrate: Histone H1, 5 µg/50 µl and Recombinant PKCalpha: 25 ng/50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of PKCalpha kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKCalpha kinase. In a 50 µl reaction, increasing amounts of PKCalpha and 1.5 µM substrate peptide were used per reaction at room temperature for 15 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 5. Staurosporine inhibition of PKCalpha kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKCalpha substrate peptide (#1331) by PKCalpha kinase. In a 50 µl reaction, 10 ng PKCalpha, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 15 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 4. Peptide concentration dependence of PKCalpha kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKCalpha kinase. In a 50 µl reaction, 10 ng of PKCalpha and increasing concentrations of substrate peptide were used per reaction at room temperature for 15 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 2. Time course of PKCalpha kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKCalpha substrate peptide (#1331) by PKCalpha kinase. In a 50 µl reaction, 10 ng PKCalpha and 1.5 uM 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 full length human PKCα (Met1-Val672) (GenBank Accession No. NM_002737) 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-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified PKCα kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the PKCα kinase 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 PKCα activity using the PKCα substrate peptide provided in this kit. PKCα sensitivity to the inhibitor staurosporine was measured using the PKCα substrate peptide provided in this kit [Fig.5].
Background
Activation of protein kinase C (PKC) is one of the earliest events in a cascade that controls a variety of cellular responses, including secretion, gene expression, proliferation and muscle contraction (1,2). PKC isoforms belong to three groups based on calcium dependency and activators. Classical PKCs are calcium-dependent via their C2 domains and are activated by phosphatidylserine (PS), diacylglycerol (DAG) and phorbol esters (TPA, PMA) through their cysteine-rich C1 domains. Both novel and atypical PKCs are calcium-independent, but only novel PKCs are activated by PS, DAG and phorbol esters (3-5). Members of these three PKC groups contain a pseudo-substrate or autoinhibitory domain that binds to substrate-binding site in the catalytic domain to prevent activation in the absence of cofactors or activators.Control of PKC activity is regulated through three distinct phosphorylation events. Phosphorylation of Thr500 in the activation loop, the autophosphorylation site at Thr641 and at carboxy-terminal hydrophobic site Ser660 occurs in vivo (2). Atypical PKC isoforms lack hydrophobic region phosphorylation, which correlates with the presence of glutamic acid rather than the serine or threonine residues found in more typical PKC isoforms. Either the enzyme PDK1 or a close relative is responsible for PKC activation.A recent addition to the PKC superfamily is PKCμ (PKD), which is regulated by DAG and TPA through its C1 domain. PKD is distinguished by the presence of a PH domain and by its unique substrate recognition and Golgi localization (6). PKC-related kinases (PRK) lack the C1 domain and do not respond to DAG or phorbol esters. Phosphatidylinositol lipids activate PRKs and small Rho-family GTPases bind to the homology region 1 (HR1) to regulate PRK kinase activity (7).
PKCalpha consists of 672 amino acids and is expressed ubiquitously. It performs important roles in the control of major cellular functions such as cell proliferation, apoptosis, differentiation and motility. PKCalpha is also associated with the malignant phenotype of several tumors, such as gliomas and breast cancers (10,11).
- Nishizuka, Y. (1984) Nature 308, 693-698.
- Keranen, L.M. et al. (1995) Curr. Biol. 5, 1394-1403.
- Mellor, H. and Parker, P.J. (1998) Biochem J. 332 (Pt 2), 281-292.
- Ron, D. and Kazanietz, M.G. (1999) FASEB J. 13, 1658-1676.
- Moscat, J. and Diaz-Meco, M.T. (2000) EMBO Rep. 1, 399-403.
- Baron, C.L. and Malhotra, V. (2002) Science 295, 325-328.
- Flynn, P. et al. (2000) J. Biol. Chem. 275, 11064-11070.
- Nakashima, S. (2002) J. Biochem. (Tokyo) 132, 669-75.
- Mackay, H.J. and Twelves, C.J. (2003) Endocr. Relat. Cancer 10, 389-96.
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
- 7578 PKCα Kinase
- 9624 Phospho-PKA Substrate (RRXS/T) (100G7E) Rabbit mAb
- 1331 CREB (Ser133) Biotinylated Peptide
- 9953 Staurosporine
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.
