The University of Southampton
University of Southampton Institutional Repository

Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking

Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking
Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking
PKC? (protein kinase C? ) is a serine/threonine protein kinase controlled by insulin, various growth factors and phosphoinositide 3-kinase. It has been implicated in controlling glucose transport in response to insulin by the translocation of GLUT4-(glucose transporter 4) containing vesicles to the plasma membrane instimulated cells. How PKC? modulates GLUT4 vesicle trafficking remains unknown. A yeast two-hybrid screen using full-length humanPKC? identified80K-Hprotein as an interactor withPKC? . GST (glutathione S-transferase) pull-down assays with GSTtagged 80K-H constructs confirmed the interaction and showed that the N-terminal portion of 80K-H was not required for the interaction. Immunoprecipitates of endogenous PKC? from Cho cells, 3T3-L1 adipocytes or L6 myotubes contained endogenous 80K-H, demonstrating a physiological interaction. Insulin stimulation enhanced the association 3–5-fold. Immunoprecipitates of endogenous 80K-H contained endogenous munc18c and immunoprecipitates of endogenous munc18c contained endogenous PKC? , with insulin markedly increasing the amount of co-immunoprecipitated protein in each case. These results show that insulin triggers interactions in vivo between PKC? , 80K-H and munc18c. Overexpression of 80K-H constructs mimicked the action of insulin in stimulating both glucose uptake and translocation of Myc-tagged GLUT4 in Cho cells, with the level of effect proportional to the ability of the constructs to associate with munc18c. These results identify 80K-H as a new player involved in GLUT4 vesicle transport and identify a link between a kinase involved in the insulin signalling cascade, PKC?, and a known component of the GLUT4 vesicle trafficking pathway, munc18c. The results suggest amodelwhereby insulin triggers the formation of a PKC?–80K-H–munc18c complex that enhances GLUT4 translocation to the plasma membrane.
glucose uptake, GLUT4, insulin, 80K-H, protein kinase C?, vesicle trafficking
1470-8728
785-793
Hodgkinson, Conrad P.
292cc7c3-b378-4911-8353-9a8c08a1c737
Mander, Ann
2fcb3a33-6bb8-4463-8b06-79cd508e9d80
Sale, Graham J.
81048025-7d8f-4218-969a-bf95ebf50b51
Hodgkinson, Conrad P.
292cc7c3-b378-4911-8353-9a8c08a1c737
Mander, Ann
2fcb3a33-6bb8-4463-8b06-79cd508e9d80
Sale, Graham J.
81048025-7d8f-4218-969a-bf95ebf50b51

Hodgkinson, Conrad P., Mander, Ann and Sale, Graham J. (2005) Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking. Biochemical Journal, 388 (3), 785-793. (doi:10.1042/BJ20041845).

Record type: Article

Abstract

PKC? (protein kinase C? ) is a serine/threonine protein kinase controlled by insulin, various growth factors and phosphoinositide 3-kinase. It has been implicated in controlling glucose transport in response to insulin by the translocation of GLUT4-(glucose transporter 4) containing vesicles to the plasma membrane instimulated cells. How PKC? modulates GLUT4 vesicle trafficking remains unknown. A yeast two-hybrid screen using full-length humanPKC? identified80K-Hprotein as an interactor withPKC? . GST (glutathione S-transferase) pull-down assays with GSTtagged 80K-H constructs confirmed the interaction and showed that the N-terminal portion of 80K-H was not required for the interaction. Immunoprecipitates of endogenous PKC? from Cho cells, 3T3-L1 adipocytes or L6 myotubes contained endogenous 80K-H, demonstrating a physiological interaction. Insulin stimulation enhanced the association 3–5-fold. Immunoprecipitates of endogenous 80K-H contained endogenous munc18c and immunoprecipitates of endogenous munc18c contained endogenous PKC? , with insulin markedly increasing the amount of co-immunoprecipitated protein in each case. These results show that insulin triggers interactions in vivo between PKC? , 80K-H and munc18c. Overexpression of 80K-H constructs mimicked the action of insulin in stimulating both glucose uptake and translocation of Myc-tagged GLUT4 in Cho cells, with the level of effect proportional to the ability of the constructs to associate with munc18c. These results identify 80K-H as a new player involved in GLUT4 vesicle transport and identify a link between a kinase involved in the insulin signalling cascade, PKC?, and a known component of the GLUT4 vesicle trafficking pathway, munc18c. The results suggest amodelwhereby insulin triggers the formation of a PKC?–80K-H–munc18c complex that enhances GLUT4 translocation to the plasma membrane.

PDF
3._Hodgkinson_et_al_(2005).pdf - Other
Restricted to Registered users only
Download (451kB)

More information

Published date: 15 June 2005
Keywords: glucose uptake, GLUT4, insulin, 80K-H, protein kinase C?, vesicle trafficking

Identifiers

Local EPrints ID: 24607
URI: https://eprints.soton.ac.uk/id/eprint/24607
ISSN: 1470-8728
PURE UUID: ba387528-bb7e-4102-87b9-5ba27a8281d4

Catalogue record

Date deposited: 03 Apr 2006
Last modified: 17 Jul 2017 16:13

Export record

Altmetrics

Contributors

Author: Conrad P. Hodgkinson
Author: Ann Mander
Author: Graham J. Sale

University divisions

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of https://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×