Abstract
The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-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 beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-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 beta-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 beta (PP2Bbeta) 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 beta-granules from the storage pool to replenish the readily releasable pool of beta-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 beta-granule transport in beta-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.
Highlights
The specific biochemical steps required for glucoseregulated insulin exocytosis from -cells are not well defined
We present evidence that kinesin heavy chains in -granules are phosphorylated under basal condition in -cells by casein kinase-2 (CK2), and dephosphorylated by phosphoprotein-2B (PP2B, known as calcineurin) in a Ca2ϩdependent manner under conditions that stimulate insulin secretion
No incorporation was observed when purified kinesin alone was incubated with ATP. These results strongly suggest that phosphorylation of kinesin heavy chain in -granule was the result of a direct action of CK2 on kinesin
Summary
The specific biochemical steps required for glucoseregulated insulin exocytosis from -cells are not well defined. We present evidence that kinesin heavy chains in -granules are phosphorylated under basal condition in -cells by casein kinase-2 (CK2), and dephosphorylated by phosphoprotein-2B (PP2B, known as calcineurin) in a Ca2ϩdependent manner under conditions that stimulate insulin secretion.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have