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First published online April 28, 2005
doi: 10.1242/10.1242/jcs.02321

Journal of Cell Science 118, 1923-1934 (2005)
Published by The Company of Biologists 2005
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14-3-3 and calmodulin control subcellular distribution of Kir/Gem and its regulation of cell shape and calcium channel activity

Pascal Béguin1,*,{ddagger}, Ramasubbu N. Mahalakshmi1,*, Kazuaki Nagashima2, Damian H. K. Cher1, Akira Takahashi2, Yuichiro Yamada2, Yutaka Seino2 and Walter Hunziker1,{ddagger}

1 Epithelial Cell Biology Laboratory, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Republic of Singapore
2 Department of Diabetes and Clinical Nutrition, Graduate School of Medicine Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan



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  		Fig. 1. Association of 14-3-3 and Kir/Gem. (A) 14-3-3 binding to Kir/Gem. (a) The Ras-like core domain (white bar), N- and C-terminal extensions (black bar) and the location of the binding sites for 14-3-3, CaM (white circles) and GTP (black circle) are shown. Mutations that affect the different binding sites are indicated. Kir/Gem-R-18 is a chimera in which the C-terminus (including S288A) was substituted by a stretch of 18 amino acids (R-18) that mediates constitutive 14-3-3 binding. (b) Cells were cotransfected with cDNAs for wt or mutated Myc-Kir/Gem proteins and GST–14-3-3 or GST–14-3-3 K49E {zeta} isoform. GST–14-3-3 proteins were precipitated and associated Kir/Gem detected by western blot using a Myc antibody. (c) Cells were cotransfected with cDNAs for wt or mutated Myc-Kir/Gem proteins and GST–14-3-3 or GST–14-3-3 K49E. Kir/Gem was immunoprecipitated and associated GST–14-3-3 and endogenous 14-3-3 was detected by western blot using a 14-3-3 antibody. The IgG heavy chain, migrating just below the GST–14-3-3 in (c), is marked by an asterisk. (d and e) Cell lysates were blotted with Myc (d) or GST (e) antibodies to monitor the expression level of Myc-Kir/Gem or GST–14-3-3; st, proteins markers of known molecular mass. (B) Association of Kir/Gem with 14-3-3 isoforms. (a-c, lanes 1-7) Cells were cotransfected with cDNAs for the different GST–14-3-3 isoforms and wt or mutated Myc-Kir/Gem. GST–14-3-3 proteins were precipitated and associated Kir/Gem was detected by western blot using a Myc antibody. (a-c, lanes 8-14) Cell lysates were blotted with Myc antibodies to monitor Kir/Gem expression levels. (d) One example of cell lysates blotted with a GST antibody to monitor expression levels of the GST–14-3-3 isoforms.

 


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  		Fig. 2. 14-3-3 dimerization is required for efficient binding to Kir/Gem. (A) Coprecipitation. (a) Cells were cotransfected with cDNAs for wt Myc-Kir/Gem or the R18 chimera and GST–14-3-3 or a dimerization defective mutant (dim). GST–14-3-3 proteins were precipitated and associated Kir/Gem was detected by Western blot using a Myc antibody. (b and c) Cell lysates were blotted with Myc (b) or GST (c) antibodies to monitor Kir/Gem protein and GST–14-3-3 expression levels. (B) Coimmunoprecipitation. Myc-Kir/Gem was immunoprecipitated from cells coexpressing GST–14-3-3 or a GST–14-3-3 dimerization mutant. Overexpressed and endogenous 14-3-3 associated with Myc-Kir/Gem were revealed by western blot analysis using a 14-3-3 antibody. The asterisk indicates the band for IgG heavy chain. (C) Pull-down experiment. (a) Wt and mutated immobilized recombinant GST–14-3-3 were tested for interaction with wt or mutated Myc-Kir/Gem present in homogenates of transfected cells. Kir/Gem proteins were revealed by western blot using Myc antibody. (b) Cell lysates were blotted with a Myc antibody to monitor Myc-Kir/Gem protein expression levels.

 



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  		Fig. 3. 14-3-3 and CaM regulate the subcellular distribution of Kir/Gem and Kir/Gem-mediated changes in cell morphology. (A and B) COS-1 cells were transfected with cDNAs for wt or mutated Kir/Gem, either alone, or together with GST–14-3-3, GST–14-3-3 K49E or GST–14-3-3 dimerization mutant. Cells were processed for immunofluorescence microscopy using Myc and GST antibodies to label Kir/Gem (red) and GST–14-3-3 (green), respectively. Areas of colocalization are in yellow in the merged image. Nuclei were stained with Hoechst 2022 (A and B, panel b). (C) Quantification of the 14-3-3-mediated cytosolic relocalization of Kir/Gem. 150-200 transfected cells were randomly selected and analyzed in 3-5 independent experiments. The fraction of cells showing efficient (black bars), partial (gray bar) or no (white bars) nuclear clearance is plotted. (D) Quantification of the 14-3-3-mediated cytosolic relocalization of Kir/Gem. Transfected cells were subjected to subcellular fractionation to obtain nuclear (N) and cytoplasmic (C) fractions. Kir/Gem in the different fractions was detected by SDS-PAGE and western blot analysis (Fig. S1 in supplementary material). The bands were scanned and the fraction of total Kir/Gem recovered in the nuclear and cytoplasmic fraction of three independent experiments is plotted. (E) Quantification of the Kir/Gem-induced morphological changes. 150-200 transfected cells were randomly selected and analyzed in 3-5 independent experiments. The fraction of cells showing one or more dendrite-like extensions, defined as thin protrusions of at least half a cell diameter, is plotted.

 


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  		Fig. 4. Binding of 14-3-3 and CaM to Kir/Gem. (A) 14-3-3 binding. Cells were cotransfected with cDNAs for wt or mutated Myc-Kir/Gem and GST–14-3-3, GST–14-3-3 K49E or GST–14-3-3 dimerization mutant. (a) GST–14-3-3 proteins were precipitated and associated Myc-Kir/Gem was detected by western blot using Myc antibody. (b and c) Cell lysates were blotted with Myc (b) or GST (c) antibodies to monitor Kir/Gem and GST–14-3-3 expression levels. (B) CaM binding. (a) Cells were transfected with cDNAs for wt or mutated Myc-Kir/Gem. Cell homogenates were incubated with CaM beads and bound Myc-Kir/Gem detected by western blot using a Myc antibody. (b) Cell lysates were blotted with Myc antibodies to monitor Kir/Gem expression levels.

 


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  		Fig. 5. Binding of CaM and 14-3-3 to Kir/Gem is mutually exclusive (A) and Kir/Gem in COS-1 cells is in a constitutively active, GTP-bound form (B and C). (A) Binding of 14-3-3/Kir/Gem complexes to CaM. (a) Cells were cotransfected with cDNAs for wt or mutated Myc-Kir/Gem and Flag–14-3-3. Flag–14-3-3 was first immunoprecipitated and Flag–14-3-3 (and associated Kir/Gem, if any) was eluted from the beads (under conditions that do not disrupt the 14-3-3/Kir/Gem complex; see Fig. S2a in supplementary material) and incubated with CaM beads. Flag–14-3-3 and Myc-Kir/Gem proteins associated with the CaM beads were detected by western blot by probing with both Flag and Myc antibodies. (b) The supernatant after the CaM pull-down was blotted with both Flag and Myc antibodies to detect unbound Flag–14-3-3 and Myc-Kir/Gem. (c and d) Cell lysates were blotted with Myc (c) and Flag (d) antibodies to monitor Flag–14-3-3 and Myc-Kir/Gem expression levels. (B) Cavß3 binds to the GTP but not GDP form of Kir/Gem. Homogenates from cells expressing Flag-Cavß3 were incubated with recombinant GST-Kir/Gem preloaded with either GDP-ßS (lane 2) or GTP-{gamma}S (lane 3) and bound Flag-Cavß3 was detected by western blot using a Flag antibody. Recombinant GST served as a control (lane 1). Cell lysates were blotted with a Flag antibody to monitor Flag-Cavß3 protein expression levels (lane 4). (C) Binding of cellular Kir/Gem to Cavß3. Cells were transfected with cDNAs for wt or mutated Myc-Kir/Gem. Cell homogenates were incubated with immobilized recombinant GST-Cavß3 and associated Kir/Gem was detected by western blot using a Myc antibody. During the pull-down experiment, homogenates were preincubated either in the absence or in the presence of exogenous nucleotides. Recombinant GST served as a control (lane 1). Cell lysates were blotted with a Myc antibody to monitor Kir/Gem expression levels (lanes 11-13).

 


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  		Fig. 6. Cavß3 and 14-3-3 compete for binding to Kir/Gem (A) and CaM displaces Cavß3 from Kir/Gem (B). (A) Binding of 14-3-3, Cavß3 and Kir/Gem. Cells were cotransfected with cDNAs for wt or mutated Myc-Kir/Gem and GST–14-3-3, either with or without Flag-Cavß3. (a) GST–14-3-3 was precipitated and associated Myc-Kir/Gem and Flag-Cavß3 was detected by western blot by sequentially probing with Myc and Flag antibodies, respectively. (b) Flag-Cavß3 subunits were immunoprecipitated and associated Myc-Kir/Gem and GST–14-3-3 were detected by western blot by sequentially probing with Myc and GST antibodies, respectively. (c-e) Cell lysates were blotted with Myc (c), Flag (d) or GST (e) antibodies to monitor expression levels of Myc-Kir/Gem, Flag Cavß3 and GST–14-3-3, respectively. (B) CaM binding. (a) Cells were cotransfected with cDNAs for wt or mutated Myc-Kir/Gem and Flag-Cavß3. Flag-Cavß3 was immunoprecipitated. Flag-Cavß3 (and associated Kir/Gem, if any) was eluted from the beads (under conditions that do not disrupt the Flag-Cavß3/Kir/Gem complex; see Fig. S2b in supplementary material) and incubated with CaM beads. Flag-Cavß3 and Myc-Kir/Gem associated with the CaM beads were detected by western blot by probing with both Flag and Myc antibodies. (b) The supernatant after the CaM pull-down was blotted with both Flag and Myc antibodies to detect unbound Flag-Cavß3 and Myc-Kir/Gem. (c and d) Cell lysates were blotted with Myc (c) and Flag (d) antibodies to monitor Flag-Cavß3 and Myc-Kir/Gem protein expression levels.

 


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  		Fig. 7. Kir/Gem downregulates Ca2+ channel activity. (A) PC12 cells were cotransfected with a GFP plasmid and cDNAs for wt or mutated Kir/Gem, either with or without 14-3-3 or 14-3-3 K49E. GFP-positive cells were selected for electrophysiology and the average of the maximal current detected at +20 mV for endogenous Ca2+ channels was measured. Between 9 and 16 independent experiments were carried out for each condition. (B) An example of the I-V relationship of Ca2+ channels in PC-12. A, Ampere; F, Faraday. Cells transfected only with the GFP cDNA served as a control.

 


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  		Fig. 8. Working model for the regulatory role of 14-3-3 and CaM on Kir/Gem localization and function. Since CaM and 14-3-3 compete for Kir/Gem binding, two pathways can be considered. Activation of CaM by intracellular Ca2+ results in association of CaM with Kir/Gem and retains it in the cytoplasm. Dissociation of CaM allows Kir/Gem to bind the Ca2+ channel ß-subunit, interfering with plasma membrane expression of the {alpha}1-subunit. Alternatively, in the absence of CaM activation, 14-3-3 can relocalize phosphorylated Kir/Gem from a submembranous location to the cytoplasm to block the formation of dendrite-like extensions. 14-3-3 can also relocalize Kir/Gem from the nucleus to the cytoplasm. 14-3-3 bound to Kir/Gem may be exchanged for the ß-subunit. Upon activation of CaM by Ca2+, Kir/Gem dissociates from the ß-subunit. Kir/Gem is in its active, GTP-bound form when located in the nucleus or bound to the ß-subunit; however, the activation state when associated with CaM or 14-3-3 remains to be determined.

 

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© The Company of Biologists Ltd 2005