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.
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