Bioinformatic Prediction and Confirmation of beta-Adducin as a Novel Substrate of Glycogen Synthase Kinase 3

Hovik Farghaian, Ann M. Turnley, Calum Sutherland, Adam R. Cole

    Research output: Contribution to journalArticlepeer-review

    21 Citations (Scopus)

    Abstract

    It is important to identify the true substrates of protein kinases because this illuminates the primary function of any kinase. Here, we used bioinformatics and biochemical validation to identify novel brain substrates of the Ser/Thr kinase glycogen synthase kinase 3 (GSK3). Briefly, sequence databases were searched for proteins containing a conserved GSK3 phosphorylation consensus sequence ((S/T)PXX(S/T)P or (S/T)PXXX(S/T)P), as well as other criteria of interest (e.g. brain proteins). Importantly, candidates were highlighted if they had previously been reported to be phosphorylated at these sites by large-scale phosphoproteomic studies. These criteria identified the brain-enriched cytoskeleton-associated protein beta-adducin as a likely substrate of GSK3. To confirm this experimentally, it was cloned and subjected to a combination of cell culture and in vitro kinase assays that demonstrated direct phosphorylation by GSK3 in vitro and in cells. Phosphosites were mapped to three separate regions near the C terminus and confirmed using phosphospecific antibodies. Prior priming phosphorylation by Cdk5 enhanced phosphorylation by GSK3. Expression of wild type, but not non-phosphorylatable (GSK3 insensitive), beta-adducin increased axon and dendrite elongation in primary cortical neurons. Therefore, phosphorylation of beta-adducin by GSK3 promotes efficient neurite outgrowth in neurons.

    Original languageEnglish
    Pages (from-to)25274-25283
    Number of pages10
    JournalJournal of Biological Chemistry
    Volume286
    Issue number28
    DOIs
    Publication statusPublished - 15 Jul 2011

    Keywords

    • RESPONSE MEDIATOR PROTEIN-2
    • RABBIT SKELETAL-MUSCLE
    • ALZHEIMERS-DISEASE
    • PHOSPHOPROTEOMIC ANALYSIS
    • DIFFERENTIAL REGULATION
    • IN-VIVO
    • SYNAPTIC PLASTICITY
    • MOUSE-BRAIN
    • A-BETA
    • PHOSPHORYLATION

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