Protein kinase C-θ (PKC-θ), a member of the Ca2+-independent PKC subfamily of kinases, serves as a regulator of T cell activation by mediating the T cell antigen receptor (TCR)- and coreceptor CD28-induced activation of the transcription factors NF-κB and AP-1 and, to a lesser extent, NFAT, and, subsequently, interleukin 2 (IL-2) production and T cell proliferation. In T cells, TCR and CD28 stimulation-induced activation of PKC-θ is the integrated result of diacylglycerol-mediated membrane recruitment, GLK-mediated phosphorylation at activation loop, CD28, Lck, and sumoylation-mediated central immunological synapse localization (Wang et al., 2015; Monks et al., 1997; Kong et al., 2011; Isakov and Altman, 2012; Chuang et al., 2011). Phosphatidylserine (PtdSer) and the phorbol ester Phorbol 12-myristate 13-acetate (PMA, a surrogate of diacylglycerol [DAG]) are the cofactors for the Ca2+-independent PKC subfamily that bind to PKC directly and activate it by changing its conformation (Nishizuka, 1995). A protocol to analyze the PKC-θ kinase activity in vitro is described here. Myelin basic protein is used as the substrate and its phosphorylation is detected by the incorporation of radioactive phosphate into the substrate, which is analyzed by a laser scanner.
[Background] Specified by their divergent regulatory domains, the PKC family can be divided into distinct subgroups: the conventional PKCs (cPKCs, comprising PKC-α, β and γ), the novel PKCs (nPKCs, including PKC-δ, ε, θ and η) and the atypical PKCs (aPKCs, PKCι and ζ). The cPKCs are activated by a combination of diacylglycerol, phospholipid and Ca2+. The nPKCs are activated by diacylglycerol and phorbol esters but do not require Ca2+. In contrast, the aPKCs do not depend on Ca2+ or diacylglycerol for activation (Rosse et al., 2010). Here we used anti-Myc or anti-PKC-θ to isolate PKC-θ from the cell lysates, then performed the radiolabeled ATP based kinase assay in the Reaction buffer containing PMA, PtdSer and EGTA (a selective chelator for Ca2+). The assay protocol described here is quick, sensitive and specific, provides a direct measurement of PKC-θ activity. This protocol could be modified to analyze the activity of other nPKC isoforms.
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