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7598
HTScan® PKCι Kinase Assay Kit
Cellular Assay Kits
Assay Kit

HTScan® PKCι Kinase Assay Kit #7598

Citations (0)
HTScan® PKCι Kinase Assay Kit: Image 1

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

HTScan® PKCι Kinase Assay Kit: Image 2

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

HTScan® PKCι Kinase Assay Kit: Image 3

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

HTScan® PKCι Kinase Assay Kit: Image 4

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

HTScan® PKCι Kinase Assay Kit: Image 5

Figure 1. PKCiota kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 5 mM MgCl2, 1.32 mM CaCl2, 1 mM EDTA, 1.25 mM EGTA, 0.25 µg/50 µl phosphatidylserine, 50 ng/50 µl 1.2 Dioleyl-glycerol, 1.2 mM DTT, ATP: variable , 2.5 µg/50 µl PEG20,000, Substrate: PKC(19-31) (RFARKGSLRQKNV), 0.5µg/50 µl and Recombinant PKCiota: 50 ng/50 µl.

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Product Description

The kit provides a means of performing kinase activity assays with recombinant human PKCiota kinase. It includes active PKCiota 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-PKCiota Kinase: 97 kDa.

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 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).

PKCiota has been suggested to mediate signaling pathways supporting cell proliferation, differentiation, and survival in response to growth factors and nutrients. PKCiota also plays a critical role in cell-cell adhesion, cell polarization process, and tight junction formation in epithelial cells (10, 11).

  1. Nishizuka, Y. (1984) Nature 308, 693-8.
  2. Keranen, L.M. et al. (1995) Curr Biol 5, 1394-403.
  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. Suzuki, A. et al. (2003) J Biochem (Tokyo) 133, 9-16.
  9. Mackay, H.J. and Twelves, C.J. (2003) Endocr. Relat. Cancer 10, 389-96.

Pathways & Proteins

Explore pathways + proteins related to this product.

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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.