Cell-wide analysis of secretory granule dynamics in three dimensions in living pancreatic beta-cells: evidence against a role for AMPK-dependent phosphorylation of KLC1 at Ser(517)/Ser(520) in glucose-stimulated insulin granule movement

Angela McDonald, Sarah Fogarty, Isabelle Leclerc, Elaine V. Hill, D. Grahame Hardie, Guy A. Rutter

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    Abstract

    Glucose-stimulated insulin secretion from pancreatic beta-cells requires the kinesin-1/Kif5B-mediated transport of insulin granules along microtubules. 5'-AMPK (5'-AMP-activated protein kinase) is a heterotrimeric serine/threonine kinase which is activated in beta-cells at low glucose concentrations, but inhibited as glucose levels increase. Active AMPK blocks glucose-stimulated insulin secretion and the recruitment of insulin granules to the cell surface, suggesting motor proteins may be targets for this kinase. While both kinesin-1/Kif5B and KLC1 (kinesin light chain-1) contain consensus AMPK phosphorylation sites (Thr(693) and Ser(520), respectively) only recombinant GST (glutathione transferase)-KLC1 was phosphorylated by purified AMPK in vitro. To test the hypothesis that phosphorylation at this site may modulate kinesin-1-mediated granule movement, we developed an approach to study the dynamics of all the resolvable granules within a cell in three dimensions. This cell-wide approach revealed that the number of longer excursions (>10 mu m) increased significantly in response to elevated glucose concentration (30 versus 3 mM) in control MIN6 beta-cells. However, similar changes were seen in cells overexpressing wild-type KLC1, phosphomimetic (S517D/S520D) or non-phosphorylatable (S517A/S520A) mutants of KLC1 Thus, changes in the phosphorylation state of KLC1 at Ser(517)/Ser(520) seem unlikely to affect motor function.

    Original languageEnglish
    Pages (from-to)205-208
    Number of pages4
    JournalBiochemical Society Transactions
    Volume38
    DOIs
    Publication statusPublished - Feb 2010

    Keywords

    • AMP-activated protein kinase (AMPK)
    • four-dimensional confocal imaging
    • insulin secretion
    • kinesin
    • protein neuropeptide
    • ACTIVATED PROTEIN-KINASE
    • KINESIN LIGHT-CHAIN
    • HEAVY-CHAIN
    • CONVENTIONAL KINESIN
    • GENE-EXPRESSION
    • EXOCYTOSIS
    • RELEASE
    • IDENTIFICATION
    • MITOCHONDRIA
    • REGULATOR

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