Figure 1. PKCtheta 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 PEG 20.000, Substrate: Histone H2B, 2 μg/50 μl and recombinant PKCtheta: 200 ng/50 µl.
The kit provides a means of performing kinase activity assays with recombinant human PKCtheta kinase. It includes active PKCtheta 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.
Peptide substrate, Biotin-peptide: 2,326 Daltons. GST-PKCtheta Kinase domain: 110 kDa.
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).
PKCtheta is expressed in a relatively selective manner in T lymphocytes and is essential for the activation of mature T cells. PKCtheta is also expressed in muscles cell and may play a role in regulation of insulin sensitivity (10).
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