Protease inhibitors prevent the protein kinase A-dependent loss of Rap1 GTPase from the particulate fraction of COS1 cells

Catherine J. Rundell, Claire E. Repellin, S. J. Yarwood

    Research output: Contribution to journalArticle

    6 Citations (Scopus)

    Abstract

    Immunoblotting with a monoclonal Rap1 antibody, we found that elevation of cyclic AMP, with forskolin and IBMX or CPT-cAMP, led to a rapid reduction in the levels of Rap1 protein associated with particulate, nuclear/perinuclear fractions from PC12 and COS1 cells. In contrast, cytoplasmic levels of Rap1 remained constant following cyclic AMP stimulation. To gain independent confirmation that cyclic AMP promoted loss of Rap1 in nuclear/perinuclear fractions we used a polyclonal Rap1 antibody, which gave similar results to the monoclonal antibody. This demonstrated that the loss in Rap1 immunoreactivity was not due to phosphorylation-dependent changes that alter immunorecognition. The reduction in Rap1 levels was blocked by PKA inhibitors and by a Rap1 serine to alanine PKA-phosphorylation site mutant (S180A). Peptide inhibitors of the proteasome, cathespin, and calpain II also inhibited the decrease in Rap1 levels, indicating that proteolytic degradation may contribute to maintaining Rap1 levels in the nuclear/perinuclear fraction of cells.

    Original languageEnglish
    Pages (from-to)1077-1081
    Number of pages5
    JournalBiochemical and Biophysical Research Communications
    Volume315
    Issue number4
    DOIs
    Publication statusPublished - 19 Mar 2004

    Keywords

    • 1-Methyl-3-isobutylxanthine
    • Animals
    • Antibodies
    • COS Cells
    • Cell Fractionation
    • Colforsin
    • Cyclic AMP
    • Cyclic AMP-Dependent Protein Kinases
    • Cytosol
    • Enzyme Activation
    • Gene Expression
    • PC12 Cells
    • Protease Inhibitors
    • Rats
    • Transfection
    • rap1 GTP-Binding Proteins

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