Product Pathways - Ca / cAMP / Lipid Signaling

PKCε Kinase #7492

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 PKCepsilon (Met1-Pro737) 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 PKCepsilon (Met1-Pro737) (GenBank Accession No. NM_005400) 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-PKCepsilon fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.

Kinase Assay - Radiometric

Figure 2. PKCepsilon 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, 40 ng/μl BSA, 50 μg/ml phosphatidylserine, 5 μg/ml diacylglycerol, 0.1 mM sodium orthovanadate, 0.1 mM dithioreitol, 0.1 mM CaCl2, 50 μM ATP, Substrate: PKCepsilon substrate peptide 300 ng/μL and recombinant PKCepsilon: variable.

Quality Control

The theoretical molecular weight of the GST-PKCepsilon fusion protein is 110 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. PKCepsilon kinase activity was determined using a radiometric assay [Fig.2].

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).

PKCepsilon regulates various physiological functions and is involved in the activation of nervous, endocrine, exocrine, inflammatory and immune systems. Disruption of PKCepsilon regulation has been related to diseases including cardiac ischemia, Alzheimer’s, malignant tumors and diabetes (10).

Application References

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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.