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Protein kinase-? interacts with munc18c: role in GLUT4 trafficking

By C.P. Hodgkinson, A. Mander and G.J. Sale


Aims/hypothesis: Insulin-stimulated glucose transport requires a signalling cascade through kinases protein kinase (PK) C?/? and PKB that leads to movement of GLUT4 vesicles to the plasma membrane. The aim of this study was to identify missing links between the upstream insulin-regulated kinases and the GLUT4 vesicle trafficking system.<br/>Materials and methods: A yeast twohybrid screen was conducted, using as bait full-length mouse munc18c, a protein known to be part of the GLUT4 vesicle trafficking machinery.<br/>Results: The yeast twohybrid screen identified PKC? as a novel interactor with munc18c. Glutathione S transferase (GST) pull-downs with GST-tagged munc18c constructs confirmed the interaction, mapped a key region of munc18c that binds PKC? to residues 295–338 and showed that the N-terminal region of PKC? was required for the interaction. Endogenous munc18c was shown to associate with endogenous PKC? in vivo in various cell types. Importantly, insulin stimulation increased the association by approximately three-fold. Moreover, disruption of PKC? binding to munc18c by deletion of residues 295–338 of munc18c or deletion of the N-terminal region of PKC? markedly inhibited the ability of insulin to stimulate glucose uptake or GLUT4 translocation.<br/>Conclusions/interpretation: We have identified a physiological interaction between munc18c and PKC? that is insulin-regulated. This establishes a link between a kinase (PKC?) involved in the insulin signalling cascade and a known component of the GLUT4 vesicle trafficking pathway (munc18c). The results indicate that PKC? regulates munc18c and suggest a model whereby insulin triggers the docking of PKC? to munc18c, resulting in enhanced GLUT4 translocation to the plasma membrane

Topics: RB, QH301
Year: 2005
OAI identifier: oai:eprints.soton.ac.uk:24606
Provided by: e-Prints Soton
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