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First published online October 12, 2006
doi: 10.1242/10.1242/jcs.03196

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Scavenging of 14-3-3 proteins reveals their involvement in the cell-surface transport of ATP-sensitive K⁺ channels

Katja Heusser^1,*, Hebao Yuan^1,*, Ioana Neagoe¹, Andrei I. Tarasov², Frances M. Ashcroft² and Blanche Schwappach^1,

¹ Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
² University Laboratory of Physiology, Parks Road, Oxford, OX1 3PT, UK

View larger version (21K): [in a new window]   Fig. 1. Strategy for interfering with 14-3-3-dependent surface expression of membrane proteins. (A) Schematic diagram of the fusion protein employed to sequester 14-3-3 proteins and (below) sequence of the R18 peptide. (B) Endogenous 14-3-3 protein in Xenopus oocytes is associated with the scavenger protein pGpLI-R18. Western blots of 14-3-3 and pGpLI-R18 in cellular extracts (extract) from uninjected or pGpLI-R18-injected oocytes, and in the flow-through (IgG flow-through) and eluate (IgG eluate) after incubation of the extracts with IgG-Sepharose. Gels were probed with an anti-14-3-3 ß antibody detecting all isoforms: the pGpLI-R18 protein is visible because of its protein-G moiety. M indicates molecular mass in kDa. (C) Surface expression of KCNK3, ß2 adrenergic receptor and Kir2.1 measured in the absence (black bars) and presence (grey bars) of the 14-3-3-scavenger pGpLI-R18. Data are normalized to the values measured in the absence of pGpLI-R18. KCNK3PC, n=35 (six batches of oocytes); ß2 adrenergic receptor, n=15 (three batches of oocytes); Kir2.1HA, n=13 (three batches of oocytes). Only for KCNK3 did 14-3-3 sequestration significantly reduce surface expression (P<0.0005).

View larger version (24K): [in a new window]   Fig. 2. Surface expression of KATP channels is impaired by 14-3-3 sequestration. (A) Surface expression in oocytes of KATP channels assembled from independently expressed Kir6.2HA and SUR1 in the absence (black bars) or presence (grey bars) of the 14-3-3-scavenger pGpLI-R18 (n=50, nine batches of oocytes; P<0.0001). (B) Mean steady-state whole-cell currents recorded from control INS-1 cells (n=6) and INS-1 cells expressing pGpLI-R18 (n=6; P<0.05). ATP concentration-inhibition relationships for KATP currents were almost identical for the two groups of cells (data not shown). (C) Coimmunoprecipitation of 14-3-3 proteins and KATP channels. Cells were treated with the crosslinker DSS where indicated. (Top and middle panels) Cell extracts (1-3%) and immunoprecipitates (IP, 40%) obtained with the indicated antibodies were resolved by SDS-PAGE. M indicates molecular mass in kDa. Western blots were probed with antibodies against Kir6.2 to detect Kir6.2 and Kir6.2-11HA (top panel), and against GFP to detect phogrin-EGFP (middle panel). (Bottom panel) Immunoprecipitates (40%) obtained with the indicated antibodies, probed with antibodies to 14-3-3 proteins. Note that the anti-14-3-3 ß antibody recognizes all 14-3-3 isoforms. Monomeric and dimeric species of 14-3-3 proteins are indicated (the dimer was only observed after crosslinking). The presence of an IgG signal in lanes 1 and 2 is because both anti-HA and anti-14-3-3 antibodies were of the same species (mouse). For anti-GFP IP a rabbit antibody was used, hence there is no IgG signal in lanes 3 and 4.

View larger version (31K): [in a new window]   Fig. 3. Role of Arg-based signals in 14-3-3-dependent forward transport. Columns show surface expression of WT and mutant SUR1-Kir6.2-fusion proteins (as indicated) in the absence (black bars) or presence (grey bars) of the 14-3-3-scavenger pGpLI-R18. For each fusion protein, data were obtained from >90 oocytes (from 18 to 22 batches) and were normalized to the values measured in the absence of pGpLI-R18 (see Materials and Methods). Data for SUR1RKR-Kir6.2RKR and SUR1RKR-Kir6.2AAA were significantly different (P<0.0001) in the presence and absence of pGpLI-R18. Western blots of oocyte homogenates for the same constructs probed with an anti-Kir6.2 antibody are shown underneath the bar graph. For each construct, there was no difference in expression when pGpLI-R18 was present. M indicates molecular mass in kDa.

View larger version (39K): [in a new window]   Fig. 4. C-terminal residues other than RKR contribute to 14-3-3 recruitment by the Kir6.2 tail. (A) Sequence of the WT, C10 and C16 Kir6.2 C-terminal tails used in the reporter constructs. (B) Coomassie-Blue-stained gels demonstrating equal loading of the Kir6.2 reporter proteins. The immobilized bait protein was eluted using SDS-PAGE sample buffer, resolved by electrophoresis and stained using Coomassie Blue. M indicates molecular mass in kDa. (C) HeLa cell cytosol (2% input) and eluates after incubation of cytosol with IgG-Sepharose preloaded with tetrameric protein A fusions (pApLI) of the indicated Kir6.2-derived tails (bait proteins, see B). RKR is the full-length WT sequence, KKK and AAA (Michelsen et al., 2006; Yuan et al., 2003; Zerangue et al., 1999) are inactive variants of the RKR motif (KKK preserves the charge), C10 and C16 are the C-terminal deletions (see A), RKR_P-A (KFSISADSLS), RKR_PL-GG (KFSISGDSGS) and RKR_S-A (KFAIAPDALA) are mutant variants of the last ten residues of Kir6.2 (pertinent sequence in parentheses, bold characters indicate mutated residues). (Top panel) Coomassie-Blue-stained gel of the eluates (80%). (Bottom panel) Western blot analysis of the same samples (10%). The membrane was probed with a pan-reacting antibody raised against the ß isoform of 14-3-3 proteins. The lowest panel combines the results for the RKR_S-A variant. The asterisk indicates a band that was not detected by western blotting against 14-3-3. To confirm that the band did not contain 14-3-3, e.g. in a modified form, we analyzed the protein by mass spectrometry. This revealed that the band contains p32/C1QBP, the precursor of a strongly acidic protein destined for the mitochondrial matrix (Dedio et al., 1998; Muta et al., 1997). The results for all constructs were very similar in four independent experiments.

View larger version (34K): [in a new window]   Fig. 5. Reporter proteins distinguish between the effects of 14-3-3 and COPI binding. (A) Schematic representation of the different reporter membrane proteins employed: monomeric CD4, dimeric CD8 and tetrameric CD4pLI. (B-D) Surface expression in COS1 cells of monomeric CD4 (B), dimeric CD8 (C) and tetrameric CD4pLI (D) fused to different C-terminal tails of Kir6.2 (as indicated below). Data are expressed as a fraction of the surface expression observed for the respective AAA construct and represent the mean ± s.e.m. of (B) eight to nine dishes, (C) nine dishes or (D) nine dishes of COS1 cells. (E) Western blots of transfected COS1 cells show similar expression levels for all constructs tested. Detection was performed using antibodies recognizing CD4 or CD8, as indicated. M indicates molecular mass in kDa.

View larger version (45K): [in a new window]   Fig. 6. 14-3-3 binding to the tetrameric CD4pLI reporter proteins correlates with their cell-surface expression. Co-immunoprecipitation of 14-3-3 proteins and indicated CD4pLI-fusion proteins. HEK293T cells transiently transfected with the specified constructs were treated with the crosslinker DSS where indicated. (A) Cell extracts (2% of input) treated with DSS are shown to demonstrate the degree of crosslinking as evident from the ratio between 14-3-3 monomer (lower band) and dimer (upper band). Note that the anti-14-3-3 ß antibody recognizes all 14-3-3 isoforms. M indicates molecular mass in kDa. (B) Anti-CD4 immunoprecipitates (upper panel, 90%, lower panel 10%) were resolved by SDS-PAGE. Western blots were probed with antibodies to 14-3-3 proteins (upper panel) and CD4 (lower panel) to confirm equal precipitation of the membrane proteins in all samples. The position of the weakly cross-reacting IgG heavy chain (IgG hc) is indicated in the upper blot. Note that 14-3-3 co-immunoprecipitation was detected using a maximum-sensitivity substrate for HRP indicating that the 14-3-3-bound population of CD4pLI is small or difficult to solubilize. The relative efficiency of 14-3-3 co-immunoprecipitation with the indicated constructs was, however, identical in three independent experiments. The rabbit anti-14-3-3 ß antibody employed for detection recognizes all 14-3-3 isoforms.

View larger version (28K): [in a new window]   Fig. 7. Impairing 14-3-3 recruitment to the KATP-channel complex results in reduced surface expression but does not alter KATP-channel function. (A) (Top) Surface expression in COS1 cells of SUR1-Kir6.2 proteins containing WT or mutated RKR motifs, and the RKR C10 variant of Kir6.2, as indicated. Data are the mean ± s.e.m. of at least nine dishes of cells in each case. Data are normalized to the values measured for the SUR1RKR-Kir6.2RKR construct. Data for SUR1RKR-Kir6.2RKR C10 were significantly different to SUR1RKR-Kir6.2RKR (P<0.0001) and SUR1AAA-Kir6.2RKR (P<0.001). (Bottom) Western blots of anti-HA immunoprecipitates from cell extracts probed with an anti-HA antibody, showing similar expression levels for the fusion proteins tested. (B) Mean ATP concentration-inhibition relationships for KATP channels. Data were obtained from HEK293 cells expressing recombinant WT channels (triangles), the SUR1-Kir6.2 fusion (circles) or SUR1-Kir6.2 C10 (squares). Black symbols, Mg2+-containing solution. White symbols, Mg2+-free solution. The curves are the best fit of the Hill equation to the mean data, with IC50=20 µM, h=0.8 (, n=6), IC50=389 µM, h=0.8 (, n=6), IC50=370 µM, h=0.7 (, n=6), IC50=80 µM, h=0.9 (, n=6), and IC50=107 µM, h=0.9 (, n=6).

View larger version (15K): [in a new window]   Fig. 8. Model for the sequence of events leading to the functional inactivation of the eight Arg-based signals present in the KATP-channel complex. The shapes illustrating the Kir6.2 tetramer (Antcliff et al., 2005), the heterooctameric KATP-channel complex (Mikhailov et al., 2005), or the 14-3-3 dimer (Petosa et al., 1998; Yaffe et al., 1997) are based on homology models or structural analysis by single-particle electron microscopy or X-ray crystallography. They are shown to scale in order to indicate the relative sizes. As 14-3-3-binding sites other than the one provided by the distal tail of Kir6.2 remain unknown, the position of the 14-3-3 dimer is entirely hypothetical. Filled circles with white Rs represent active Arg-based ER-localization signals, open circles symbolize inactiviation of the signal. (A) The Arg-based signal of SUR1 could be inactivated by direct binding of 14-3-3 to this region of the protein (masking). (B) The Arg-based signal of SUR1 could be indirectly inactivated by 14-3-3 recruitment to a binding site in the vicinity of the distal tail of Kir6.2.

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