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 Ser517/Ser520 in glucose-stimulated insulin granule movement.
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 mum) 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.
McDonald
Angela
A
Division of Medicine, Department of Cell Biology, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK.
Fogarty
Sarah
S
Leclerc
Isabelle
I
Hill
Elaine V
EV
Hardie
D Grahame
DG
Rutter
Guy A
GA
D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000818:Animals; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D016547:Kinesins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*
D007328:Insulin; C000715688:KLC1 protein, human; D008869:Microtubule-Associated Proteins; D020033:Protein Isoforms; D012694:Serine; D055372:AMP-Activated Protein Kinases; D016547:Kinesins; D005947:Glucose