Molecular mechanisms regulating exocytosis : studies of insulin secretion and neurotransmitter release

In order to achieve normal physiology the secretion of hormones and neurotransmitters needs to be firmly controlled. The basic molecular machinery mediating regulated exocytosis of hormones and neurotransmitters is in principal revealed, but knowledge about how the individual components are spatially and temporarily coordinated is limited. This thesis was primarily aimed to clarify molecular mechanisms that modulate insulin secretion, with the emphasis on protein phosphorylation, but it also aimed to compare the regulation of related secretory machineries responsible for secretory granule and synaptic vesicle exocytosis.

Cyclin-dependent kinase 5 (Cdk5) associated with either p35 or p39 forms a multifunctional kinase that is primarily expressed in neurons. Cdk5 was identified in pancreatic ?-cells where it functions as a positive regulator of insulin secretion. Although ?-cells expressed both of the identified Cdk5 regulatory subunits, only Cdk5, activated by p39, enhanced insulin secretion. Co-expression of Cdk5, p35 or p39 with munc18-1, mutated in potential phosphorylation sites, revealed that Cdk5/p39 activity facilitated secretion by phosphorylating the syntaxin 1 interacting protein munc18-1.

The role of Cdk5 activity in spontaneous neurotransmitter release and in functional synapse formation was examined using the neuroblastoma/glioma cell line NG108-15, which when co-cultured with myotubes form cholinergic synapses. NG108-15 cells endogenously expressed Cdk5 and both of its activators. In contrast to the ?-cell, which predominantly expressed the p39 activator, the NG108-15 cell mainly expressed the p35 protein. NG108-15 cells overexpressing a dominant negative mutant of Cdk5 showed a reduced mEPP frequency and had less ability to form functional synaptic-like structures with muscle cells as compared to non-transfected cells.

Overexpression of either Cdk5/p35 or Cdk5/p39 enhanced both the mEPP frequency and functional synapse formation to a similar extent, indicating that Cdk5 activity facilitated spontaneous neurotransmitter release as well as functional synapse formation in NG108-15 cells. Protein phosphatase 1 (PP1) is regarded as an important regulator of insulin exocytosis, but regulation of its activity in ?-cells is unknown. RT-PCR, Western blotting and immunohistochemistry revealed expression of the endogenous PP1 inhibitors DARPP-32 and inhibitor-1 in ?-cells, suggesting a potential role for DARPP-32 and inhibitor-1 in the regulation of PP1 activity in signal transduction and insulin exocytosis.

List of scientific papers

I. Lilja L, Yang SN, Webb DL, Juntti-Berggren L, Berggren PO, Bark C (2001). Cyclin-dependent kinase 5 promotes insulin exocytosis. J Biol Chem. 276(36): 34199-205.
https://doi.org/10.1074/jbc.M103776200

II. Lilja L, Johansson JU, Gromada J, Mandic SA, Fried G, Berggren PO, Bark C (2004). Cyclin-dependent kinase 5 associated with p39 promotes Munc18-1 phosphorylation and Ca(2+)-dependent exocytosis. J Biol Chem. 279(28): 29534-41.
https://doi.org/10.1074/jbc.M312711200

III. Johansson JU, Lilja L, Chen XL, Higashida H, Meister B, Noda M, Zhong ZG, Yokoyama S, Berggren PO, Bark C (2005). Cyclin-dependent kinase 5 activators p35 and p39 facilitate formation of functional synapses. [Submitted]

IV. Lilja L, Meister B, Berggren PO, Bark C (2005). DARPP-32 and inhibitor-1 are expressed in pancreatic beta-cells. [Submitted]