QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 16 November 2004
doi: 10.1242/jcs.01517

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Berwick, D. C. Articles by Tavaré, J. M. Search for Related Content PubMed PubMed Citation Articles by Berwick, D. C. Articles by Tavaré, J. M.

Protein kinase B phosphorylation of PIKfyve regulates the trafficking of GLUT4 vesicles

Daniel C. Berwick^1,*,, Ghislaine C. Dell^1,, Gavin I. Welsh^1,, Kate J. Heesom¹, Ingeborg Hers¹, Laura M. Fletcher¹, Frank T. Cooke² and Jeremy M. Tavaré^1,

¹ Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK
² Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, W1E 6BT, UK

View larger version (17K): [in a new window]   Fig. 1. Protein kinase B phosphorylates recombinant PIKfyve on serine318 and stimulates its 5-kinase activity. (A) Recombinant GST-PIKfyve fusion protein was incubated in the absence or presence of recombinant active PKB as indicated and with [-32P]ATP. The upper panel shows an autoradiogram of the resulting gel. The lower panel shows a western blot of the same samples probed with anti-pS318 antibody. Data are representative of three separate experiments. (B) Recombinant GST-PIKfyve was incubated in the absence or presence of recombinant active [-32P]ATP and PKB, as indicated. The activity of PIKfyve was determined using PtdIns(3)P as substrate according to Materials and Methods. The phosphorylated lipids were then separated by thin layer chromatography. The upper panel shows an autoradiogram of an experiment performed in triplicate. The lower panel shows data from three independent experiments. *P<0.05 compared to activity in the control.

View larger version (32K): [in a new window]   Fig. 2. Insulin stimulates PIKfyve serine318 phosphorylation in intact cells. (A) Cells transiently transfected with wild-type GFP-PIKfyve (WT), the GFP PIKfyve[S318A] mutant or vector alone (V) were pre-incubated in the absence (C) or presence of wortmannin (W) for 30 minutes and then in the absence or presence of insulin (I) for a further 15 minutes. Cell lysates were then subjected to western blotting with either the anti-pS318 or anti-PIKfyve antibodies as indicated. Note that the anti-pS318 antibody crossreacts with a phosphoprotein that migrates just below PIKfyve and exhibits an increase in phosphorylation in the presence of wortmannin. The identity of this phosphoprotein is unknown but it is not endogenous PIKfyve as demonstrated by the lack of reactivity with an anti-PIKfyve antibody (lower panel). (B) Wild-type GFP-PIKfyve or a GFP-PIKfyve[R192A] mutant were transiently transfected into CHO.T cells. 24 hours later the cells were examined by confocal microscopy. Typical images of the distribution of these proteins are shown. Bar, 10 µm. (C) Cells were transfected with wild-type GFP-PIKfyve or the GFP-PIKfyve[R192A] mutant and treated as described in panel A for western blotting with either the anti-pS318 or anti-PIKfyve antibodies. (D) Data from at least three experiments performed according to panel C were quantified by densitometric scanning of the autoradiographs. The data are corrected for the amount of GFP-PIKfyve loaded onto the gels and are expressed relative to the appropriate control.

View larger version (24K): [in a new window]   Fig. 3. PIKfyve serine318 phosphorylation is stimulated by constitutively active PKB and PtdIns 3-kinase mutants. Cells were transiently transfected with wild-type GFP-PIKfyve and vector alone, constitutively active PI3-kinase catalytic subunit (p110*) or constitutively active PKB (Myr-PKB). 48 hours later the cells were serum starved and then incubated in the absence or presence of insulin for 15 minutes, as indicated. Cell lysates were then subjected to western blotting with either the anti-pS318 or anti-PIKfyve antibodies as shown. The data are representative of three separate experiments.

View larger version (123K): [in a new window]   Fig. 4. IRAP and PIKfyve colocalise on highly dynamic vesicles. (A) A 3T3-L1 adipocyte, under basal conditions, coexpressing GFP-PIKfyve (left panel) and IRAP-mRFP1 (middle panel) was imaged by confocal microscopy. The right hand panel shows an overlay of the two images. Boxes B and C indicate two different GFP-PIKfyve and IRAP-mRFP1 coexpressing vesicles that are magnified and indicated with arrows in panels B and C, respectively. In Box D, another GFP-PIKfyve and IRAP-mRFP1 coexpressing vesicle enters the perinuclear region and exhibits behaviour suggestive of fusion with other membranous structures. For optimal viewing of this vesicle see the animation provided as supplementary material. (B,C) Sequential images of GFP-PIKfyve (upper panels) and IRAP-mRFP1 (lower panels) expression, 1 second apart were collected over two different time periods. This time-lapse sequence is best viewed in the accompanying supplementary material. Note that the IRAP-mRFP1 fluorescence has been enhanced to improve the resolution on peripheral vesicular structures; this results in the overexposure of fluorescence in the perinuclear region. Bars, 10 µm.

View larger version (41K): [in a new window]   Fig. 5. The PIKfyve[S318A] mutant enhances insulin-stimulated IRAP translocation. (A) 3T3-L1 adipocytes stably expressing IRAP with an exofacial (luminal) HA tag, were microinjected with plasmids encoding GFP, wild-type GFP-PIKfyve (WT) or the PIKfyve[S318A] mutant (S318A). The cells were then treated in the absence (Con) or presence of insulin (Ins) as indicated. Cells were fixed, but not permeabilised, and stained with an anti-HA antibody that only detects surface-exposed IRAP. GFP-expressing cells exhibiting a clear surface-associated staining (i.e. a plasma membrane ring of fluorescence) were counted as having undergone IRAP translocation. In the absence of insulin, no cells were detected that exhibited surface staining. Quantitative data are provided for either two (control) or four (insulin) independent experiments. (B) Images of individual insulin-stimulated cells from one representative experiment are shown. HA-stained surface IRAP is shown in red, with the corresponding wild-type (WT, upper panel) or [S318A] mutant GFP-PIKfyve (lower panel) expression in the same cell in green. In this experiment 13 cells out of 33 wild-type GFP-PIKfyve expressing cells exhibited the translocation phenotype (39.3%). In cells expressing PIKfyve[S318A] this became 12 out of 19 (63%). Bar, 10 µm.