Exchange protein directly activated by cAMP (EPAC) interacts with the light chain (LC) 2 of MAP1A

Maria M. Magiera, Mona Gupta, Catherine J. Rundell, Nilima Satish, Isabelle Ernens, S. J. Yarwood

    Research output: Contribution to journalArticle

    27 Citations (Scopus)

    Abstract

    Using EPAC1 (exchange protein directly activated by cAMP 1) as bait in two-hybrid screens of foetal and adult human brain libraries, we identified the LC2 (light chain 2) of MAP1A (microtubule-associated protein 1A) as a protein capable of interaction with EPAC1. We applied an immunoprecipitation assay to demonstrate protein interaction between EPAC1 and LC2 in co-transfected human embryonic kidney 293 cells. EPAC2 also co-immunoprecipitated with LC2 from extracts of rat cerebellum. Immunolocalization in co-transfected human embryonic kidney 293 cells revealed that EPAC1 co-localizes with LC2 throughout the cell body. We found that endogenous EPAC2 is also immunolocalized with LC2 in PC12 cells. Immunolocalization of EPAC1 in transfected COS1 cells showed that EPAC1 is associated with the perinuclear region surrounding the nucleus and filamentous structures throughout the cell. Removal of the cAMP-binding domain of EPAC1 (DeltacAMP-EPAC1) appeared to disrupt targeting of EPAC1 in cells resulting in a more dispersed staining pattern. Using two-hybrid assay, we tested the ability of LC2 to interact with DeltacAMP-EPAC1 and DeltaDEP-EPAC1, which lacks a DEP domain (dishevelled, Egl-10 and pleckstrin homology domain). We found that deletion of the cAMP-binding domain inhibited interaction between EPAC1 and LC2 in a two-hybrid assay, but removal of the DEP domain had little effect. LC2 was found to interact with a glutathione-S-transferase-fusion protein of the cAMP-binding domain of EPAC1 in a pull-down assay, but not the DEP, REM (Ras exchange motif) or CAT (catalytic) domains. Together with our two-hybrid results, this suggests that the cAMP-binding domain of EPAC1 mediates interaction with LC2.

    Original languageEnglish
    Pages (from-to)803-810
    Number of pages8
    JournalBiochemical Journal
    Volume382
    Issue number3
    DOIs
    Publication statusPublished - 15 Sep 2004

    Keywords

    • Adult
    • Animals
    • Binding Sites
    • Brain Chemistry
    • Cell Line
    • Cercopithecus aethiops
    • Cyclic AMP
    • Fetus
    • Guanine Nucleotide Exchange Factors
    • Humans
    • Immunohistochemistry
    • Immunoprecipitation
    • Microtubule-Associated Proteins
    • Peptide Fragments
    • Protein Binding
    • Rats
    • Recombinant Fusion Proteins
    • Subcellular Fractions

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