Ca2+-dependent Dephosphorylation of Kinesin Heavy Chain on β-Granules in Pancreatic β-Cells

The specific biochemical steps required for glucose-regulated insulin exocytosis from β-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+] i and biphasic release of insulin from both a readily releasable and a storage pool of β-granules. The effect of elevated [Ca2+] i on phosphorylation of isolated β-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+] i . One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa β-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+] i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B β (PP2Bβ) as [Ca2+] i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of β-granules from the storage pool to replenish the readily releasable pool of β-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and β-granule transport in β-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+] i .