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7594
HTScan® PKCη Kinase Assay Kit

HTScan® PKCη Kinase Assay Kit #7594

This product is discontinued

Kinase Assay - Radiometric Image 1

Figure 1. PKCeta 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, variable and Recombinant PKCeta, variable.

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Kinase Assay - DELFIA Image 2

Figure 3. Dose dependence curve of PKCeta kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKCeta kinase. In a 50 µl reaction, increasing amounts of PKCeta and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

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Kinase Assay - DELFIA Image 3

Figure 5. Staurosporine inhibition of PKCeta kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKCeta substrate peptide (#1331) by PKCeta kinase. In a 50 µl reaction, 10 ng PKCeta, 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.)

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Kinase Assay - DELFIA Image 4

Figure 4. Peptide concentration dependence of PKCeta kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKCeta kinase. In a 10 µl reaction, 50 ng of PKCeta and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

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Kinase Assay - DELFIA Image 5

Figure 2. Time course of PKCeta kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKCeta substrate peptide (#1331) by PKCeta kinase. In a 50 µl reaction, 10 ng PKCeta and 1.5 uM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

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Product Includes Quantity (with Count) Solution Color
ATP (10 mM) 9804 1 x 1 ml
Kinase Buffer (10X) 9802 1 x 15 ml

The kit provides a means of performing kinase activity assays with recombinant murine PKCeta kinase. It includes active PKCeta 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 Formula:

Biotin-peptide: 2,326 Daltons. GST-PKCeta Kinase: 110 kDa.

Peptide Core Sequence:

RRPS*YRK

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 sites 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 occurs in vivo at Thr500 in the activation loop, at Thr641 through autophosphorylation, and at the carboxy-terminal hydrophobic site Ser660 (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. 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).

PKCeta is predominantly expressed in squamous epithelial tissue. Overexpression of PKCeta induces G1 arrest and differentiation in keratinocytes. PKCeta knockout mice show a much higher sensitivity to carcinogenesis, suggesting PKCeta behaves as a tumor suppressor in epithelial tissues. In addition to epithelial cells, PKCeta expression and activation also plays an important role in early B cell development (10,11).

  1. Nishizuka, Y. (1984) Nature 308, 693-698.
  2. Keranen, L.M. (1995) Curr. Biol. 5, 1394-1403.
  3. Mellor, H. and Parker, P.J. (1998) Biochem J 332 ( Pt 2), 281-92.
  4. Ron, D. and Kazanietz, M.G. (1999) FASEB J 13, 1658-76.
  5. Moscat, J. and Diaz-Meco, M.T. (2000) EMBO Rep 1, 399-403.
  6. Baron, C.L. and Malhotra, V. (2002) Science 295, 325-8.
  7. Flynn, P. et al. (2000) J Biol Chem 275, 11064-70.
  8. Kashiwagi, M. et al. (2002) J. Biochem. (Tokyo) 132, 853-7.
  9. Mackay, H.J. and Twelves, C.J. (2003) Endocr. Relat. Cancer 10, 389-96.
Entrez-Gene Id
5583
Swiss-Prot Acc.
P24723
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DELFIA is a registered trademark of PerkinElmer, Inc.

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