title: Control of insulin granule dynamics by AMPK dependent KLC1 phosphorylation.
abstract: The movement of insulin granules along microtubules, driven by kinesin-1/Kif5B, is essential for glucose-stimulated insulin secretion from pancreatic β-cells.  5΄AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase, which is activated in β-cells at low glucose concentrations, but inhibited as glucose levels increase.  AMPK activation blocks glucose-stimulated recruitment of secretory granules to the cell surface and insulin secretion, suggesting motor proteins may be targets for this kinase. Whilst both kinesin-1/Kif5B and kinesin light chain-1 (KLC1) contain consensus AMPK phosphorylation sites only a peptide corresponding to Ser520 in mouse KLC1 and purified recombinant GST-KLC1 were phosphorylated by purified AMPK in vitro.  To test the hypothesis that phosphorylation at this site may modulate kinesin-1-mediated granule movement, we developed a novel approach to study the dynamics of the granules within a cell in three-dimensions using Nokigawa spinning disc confocal microscopy.  This cell-wide approach revealed that the number of longer excursions (>10 µm) increased significantly in response to elevated glucose concentration (30 vs 3 mM) in control MIN6 cells.  However, similar changes were seen in cells over-expressing wild-type KLC1, phosphomimetic (S517/520D) or non-phosphorylatable (S517/520A) mutants of KLC1.  Moreover, no evidence for a change in the phosphorylation state of KLC1 at Ser520 after AMPK activation was obtained using an anti-phospho Ser520-specific antibody. Thus, changes in the phosphorylation state of KLC1 at Ser517/520 are unlikely to affect motor function.  In conclusion, we describe a new three-dimensional cell wide approach for the analysis of secretory granule dynamics in living β-cells.
authors.affiliation: Department of Cell Biology, Division of Medicine, Imperial College London, London, UK.
authors.forename: Angela
authors.initials: A
authors.lastname: McDonald
authors.forename: Sarah
authors.initials: S
authors.lastname: Fogarty
authors.forename: Isabelle
authors.initials: I
authors.lastname: Leclerc
authors.forename: Elaine V
authors.initials: EV
authors.lastname: Hill
authors.forename: D Grahame
authors.initials: DG
authors.lastname: Hardie
authors.forename: Guy A
authors.initials: GA
authors.lastname: Rutter
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism* / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D007328:Insulin / Q000378:metabolism*; D016547:Kinesins; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism* / Q000502:physiology; D008954:Models, Biological; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology; D051381:Rats; D055571:Secretory Pathway / Q000502:physiology; D022142:Secretory Vesicles / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D007328:Insulin; C000715690:Klc1 protein, rat; C000715695:Kns2 protein, mouse; D008869:Microtubule-Associated Proteins; D055372:AMP-Activated Protein Kinases; D016547:Kinesins