Protein expression of the epsilon subspecies of protein kinase C ceases as Swiss 3T6 fibroblasts increase in cell density even though message for the protein is still present

FEBS Letters

Beale, Gary¹  Watson, John¹  Rumsby, Martin¹  Williams, Timothy¹  Littlebury, Paul¹ 

[1] Department of Biology, University of York, York, YO1 5YW, UK

关键词: Protein kinase C subspecies;    Protein kinase C-ϵ;    Protein expression;    mRNA;    3T6 fibroblast;    Passage;    Confluency;    Translational control;    BCA;    biochinchoninic acid;    DAG;    diacylglycerol;    DMEM;    Dulbecco's modified Eagle's medium;    FBS;    foetal bovine serum;    PAGE;    polyacrylamide gel electrophoresis;    PBS;    phosphate-buffered saline;    PKC;    protein kinase C;    PMSF;    phenylmethyl sulphonyl fluoride;    RT-PCR;    reverse transcription–polymerase chain reaction;    SDS;    sodium dodecylsulphate;    TBS;    Tris-buffered saline;    TBST;    Tris-buffered saline/0.2% Tween 20;   

DOI  :  10.1016/S0014-5793(96)01394-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We have noted previously that growth of C6 glioma cells from low cell density to confluency and quiescence in serum is accompanied by changes in protein content of different protein kinase C (PKC) subspecies. Here we show that the same occurs as non-contact-inhibiting Swiss 3T6 fibroblasts grow to high density in the presence of serum. Protein expression of PKC subspecies α and δ increases as the cells increase in density while that of PKC-ζ remains the same. Unusally, protein expression of PKC-ϵ is completely down-regulated as cells grow beyond about 50% confluency and no PKC-ϵ protein can be detected in 3T6 fibroblasts at high density by Western blotting. However, mRNA for PKC-ϵ is expressed at all stages of fibroblast growth as revealed by RT-PCR. When high-density 3T6 fibroblasts are passaged to low density in fresh medium, re-expression of PKC-ϵ protein is observed within 15 min and becomes down-regulated again as cells become more dense. This very rapid synthesis of PKC-ϵ is not blocked by the transcription inhibitor actinomycin D but is inhibited by cycloheximide. PKC-ϵ has some characteristics of a novel ‘early response’ protein whose synthesis in newly passaged 3T6 cells is regulated at the translational level.

【 授权许可】

Unknown