In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase Cμ

FEBS Letters

Pfizenmaier, Klaus¹  Link, Gisela¹  Herget, Thomas²  Dieterich, Sabine¹  Böttinger, Heiner¹  Johannes, Franz-Josef¹ 

[1] Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany;Institute of Physiological Chemistry, Duesbergweg 6, 55099 Mainz, Germany

关键词: Protein kinase Cμ;    Phorbol ester binding;    Baculo expression;    Activation condition;    MARCKS phosphorylation;    PKC;    protein kinase C;    PDBu;    phorbol 12;    13-dibutyrate;    PtdIns-4;    5-P2;    l-α-phosphatidyl-d-myo-inositol-4;    5-bisphosphate;    PMSF;    phenylmethylsulfonylfluoride;    PS;    l-α-phosphatidyl-l-serine;    DAG;    1;    2-dioctanoyl-sn-glycerol;    MARCKS;    myristoylated alanine-rich C-kinase substrate;    l-PC;    l-α-lysophosphatidylcholine;    PA;    l-α-dipalmitoyl phosphatidic acid;    AA;    arachidonic acid;    Cer;    C16-ceramide;    HEPES;    N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid;   

DOI  :  10.1016/0014-5793(96)00116-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

To study enzymatic activity and activation conditions of the recently identified novel protein kinase C μ (PKCμ) subtype, epitope tagged PKCμ was propagated in the baculovirus expression system and was purified to homogeneity. PKCμ displays high affinity phorbol ester binding (K _d = 7 nM) resulting in enhanced phosphatidylserine-dependent kinase activity. From various lipid second messengers known to activate PKCs only diacylglycerol and PtdIns-4,5-P₂, were found to promote PKCμ kinase activity. Two peptides derived from the glycogen synthase, GS-peptide and syntide 2, were found to be phosphorylated efficiently in vitro. MARCKS (myristoylated alanine-rich C-kinase substrate) served as an in vitro substrate for PKCμ too. However, in contrast to other PKCs, a peptide derived from the MARCKS phosphorylation domain is phosphorylated only at serine 156, and not at serines 152 and 163, implicating a differential regulation by PKCμ.

【 授权许可】

Unknown