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

Publication ,  Journal Article
Hodgkinson, CP; Mander, A; Sale, GJ
Published in: Biochem J
June 15, 2005

PKCzeta (protein kinase Czeta) 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 in stimulated cells. How PKCzeta modulates GLUT4 vesicle trafficking remains unknown. A yeast two-hybrid screen using full-length human PKCzeta identified 80K-H protein as an interactor with PKCzeta. GST (glutathione S-transferase) pull-down assays with GST-tagged 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 PKCzeta 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 PKCzeta, with insulin markedly increasing the amount of co-immunoprecipitated protein in each case. These results show that insulin triggers interactions in vivo between PKCzeta, 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, PKCzeta, and a known component of the GLUT4 vesicle trafficking pathway, munc18c. The results suggest a model whereby insulin triggers the formation of a PKCzeta-80K-H-munc18c complex that enhances GLUT4 translocation to the plasma membrane.

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Published In

Biochem J

DOI

EISSN

1470-8728

Publication Date

June 15, 2005

Volume

388

Issue

Pt 3

Start / End Page

785 / 793

Location

England

Related Subject Headings

  • Two-Hybrid System Techniques
  • Saccharomyces cerevisiae
  • Protein Kinase C
  • Protein Binding
  • Phosphorylation
  • Phosphoproteins
  • Munc18 Proteins
  • Multiprotein Complexes
  • Mice
  • Intracellular Signaling Peptides and Proteins
 

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Hodgkinson, C. P., Mander, A., & Sale, G. J. (2005). Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking. Biochem J, 388(Pt 3), 785–793. https://doi.org/10.1042/BJ20041845
Hodgkinson, Conrad P., Ann Mander, and Graham J. Sale. “Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking.Biochem J 388, no. Pt 3 (June 15, 2005): 785–93. https://doi.org/10.1042/BJ20041845.
Hodgkinson CP, Mander A, Sale GJ. Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking. Biochem J. 2005 Jun 15;388(Pt 3):785–93.
Hodgkinson, Conrad P., et al. “Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking.Biochem J, vol. 388, no. Pt 3, June 2005, pp. 785–93. Pubmed, doi:10.1042/BJ20041845.
Hodgkinson CP, Mander A, Sale GJ. Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking. Biochem J. 2005 Jun 15;388(Pt 3):785–793.

Published In

Biochem J

DOI

EISSN

1470-8728

Publication Date

June 15, 2005

Volume

388

Issue

Pt 3

Start / End Page

785 / 793

Location

England

Related Subject Headings

  • Two-Hybrid System Techniques
  • Saccharomyces cerevisiae
  • Protein Kinase C
  • Protein Binding
  • Phosphorylation
  • Phosphoproteins
  • Munc18 Proteins
  • Multiprotein Complexes
  • Mice
  • Intracellular Signaling Peptides and Proteins