PKC-θ激酶体外活性试验

Xudong  Wang; Yingqiu Li

doi:10.21769/BioProtoc.1980

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Peer-reviewed

PKC-θ in vitro Kinase Activity Assay

PKC-θ激酶体外活性试验

Xudong Wang

YL Yingqiu Li email

发布: 2016年10月20日第6卷第20期 DOI: 10.21769/BioProtoc.1980 浏览次数: 9357

评审: Ivan ZanoniMichael EnosMareta Ruseva

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Cover of Nature Immunology, featuring study using the protocol.

Oct 2015

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Abstract

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.

Keywords: In vitro kinase assay (体外激酶测定)

PKC-θ (PKC)

Radiolabeled ATP (放射性标记的ATP)

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.

Materials and Reagents

Jurkat E6.1 cells or HEK293T cells
Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010023 )
RPMI-1640 medium (GE Healthcare, HyClone^TM, catalog number: SH30027.01 )
Heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270-106 )
Anti-PKC-θ (Santa Cruz Biotechnology, catalog number: sc-1875 )
Anti-Myc (9E10) (Santa Cruz Biotechnology, catalog number: sc-40 )
Protein G sepharose (GE Healthcare, catalog number: 17-0618-01 )
Phosphatidylserine from PepTag^® Non-Radioactive PKC assay kit (Promega, catalog number: V5330 )
Tris (Sangon Biotech, catalog number: A600194 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
Ethylenediaminetetraacetate (EDTA) (Sangon Biotech, catalog number: A610185 )
Nonidet P40 (Sangon Biotech, catalog number: A600385 )
Aprotinin (EMD Millipore, catalog number: 616370 )
Leupeptin (Calbiochem, catalog number: 108976 )
Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
Sodium pyrophosphate tetrabasic decahydrate (NaPPi) (Sigma-Aldrich, catalog number: S6422 )
Sodium orthovanadate (Na₃VO₄) (Sigma-Aldrich, catalog number: 567540 )
HEPES (Thermo Fisher Scientific, Gibco^TM, catalog number: 11344041 )
Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M4880 )
EGTA (Sigma-Aldrich, catalog number: E3889 )
ATP (Sigma-Aldrich, catalog number: A2383 )
[γ-³²P]ATP (PerkinElmer, catalog number: NEG002Z001MC )
Myelin basic protein (MBP) (Sigma-Aldrich, catalog number: M1891 )
PMA (Sigma-Aldrich, catalog number: P1585 )
Bromophenol blue (Bio-Rad Laboratories, catalog number: 1610404 )
DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
Glycerol (Sangon Biotech, catalog number: A100854 )
ATP-competitive PKC-θ inhibitor rottlerin (EMD Millipore, catalog number: 557370 )
DMSO
Lysis buffer (see Recipes)
PKC-θ kinase buffer (see Recipes)
Reaction buffer (see Recipes)
5x SDS-PAGE loading buffer (see Recipes)