Cysteine‐rich regions of protein kinase Cδ are functionally non‐equivalent

FEBS Letters

Hunn, Martin¹  Quest, Andrew F.G¹ 

[1] Institute of Biochemistry, University of Lausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland

关键词: Protein kinase C isoform;    Protein kinase C delta;    Protein kinase C gamma;    Cysteine-rich region;    Phosphatidylserine;    Phorbol ester;    aPKC;    atypical PKC;    AT;    antisense;    C1;    first conserved region of PKC;    C2;    second conserved region of cPKC;    C6H2;    six conserved cysteines and two conserved histidines of cysteine-rich regions;    cPKC;    calcium-dependent PKC;    Cys1;    first cysteine-rich region of PKC;    Cys2;    second cysteine-rich region of PKC;    DAG;    diacylglycerol;    EGTA;    ethyleneglycol-bis-(2-aminoethyl)-tetraacetic acid;    GST;    glutathione-S-transferase;    GST-PKCδ;    COOH-terminal fusion proteins of GST with regulatory domain elements of PKCδ (see Fig. 1);    HEPES;    N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid];    IPTG;    isopropyl-1-thio-β-d-galactopyranoside;    nPKC;    non-calcium-dependent PKC;    PKC;    protein kinase C;    PDBu;    4β-phorbol-12;    13-dibutyrate;    PMA;    phorbol 12-myristate 13-acetate;    PS;    1;    2-dioleoyl-l-3-phosphatidyl-l-serine;    SDS-PAGE;    sodium dodecyl sulfate-polyacrylamide gel electrophoresis;    SN;    sense;    V1;    first variable region of PKC;    V2;    second variable region of PKC;    ψ;    pseudosubstrate region of PKC;   

DOI  :  10.1016/S0014-5793(96)01395-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Regulatory domain elements of the non-calcium-dependent protein kinase Cδ (nPKCδ), including either or both of the cysteine-rich regions Cys1(δ) and Cys2(δ), were expressed as fusion proteins with glutathione-S-transferase and characterized using liposomal or mixed micellar phorbol ester binding assays. Fusion proteins containing Cys2(δ) bound phorbol-12,13-dibutyrate (PDBu) efficiently in the assay employing phosphatidylserine (PS) vesicles, while no significant binding was seen for proteins containing only Cys1(δ). Likewise, in mixed micellar assays, fusion proteins with Cys2(δ) bound PDBu with high affinity (K _d: 14–37 nM) and to significant stoichiometric levels (0.23–0.66 mol/mol), but no binding could be detected for proteins with Cys1(δ) only. The PS dependence of PDBu binding to Cys2(δ) was highly cooperative with Hill numbers lying in the range of 2.5–5.2. These results demonstrate the presence of striking functional differences between the cysteine-rich regions of nPKCδ and the calcium-dependent isoform, cPKCγ, where both cysteine-rich regions represent functional PDBu binding elements.

【 授权许可】

Unknown