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doi: 10.1242/10.1242/jcs.00069

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Binding of protein kinase B to the plakin family member periplakin

A. Pieter J. van den Heuvel1, Alida M. M. de Vries-Smits1, Pascale C. van Weeren, Pascale F. Dijkers1,*, Kim M. T. de Bruyn1, Jürgen A. Riedl1 and Boudewijn M. T. Burgering1,{ddagger}

1 Laboratory of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, Stratenum, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands
* Present address: Department of Biochemistry and Biophysics, UCSF, San Francisco, CA 94143-0448, USA



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  		Fig. 1. PKB binds periplakin. (A) The yeast two-hybrid fragments of PKB (fish) and periplakin (bait). Periplakin consists of six {alpha}-helical subdomains (aa 16-920), a rod domain (aa 945-1554) and a linker domain (L; aa 1556-1756). (B) GST pull-down assay with GST-c-ppl. A14 cells were transiently transfected with either wild-type or kinase-dead HA-tagged PKB. Lysates were incubated for 1 hour with GST-c-ppl. Samples were analysed for the presence of PKB by western blotting using the anti-HA antibody. A direct immunoprecipitation of HA-PKB using anti-HA was used as a control. The experiment was also performed with either GST or GST-c-ppl (right panel). (C) COS-7 cells were transiently co-transfected with myc-tagged c-ppl and either HA-tagged PKB or p70S6K. Co-immunoprecipitations were performed with anti-HA and anti-myc antibodies and immunocomplexes were resolved with SDS-PAGE and immunoblotted with anti-myc and anti-HA antibodies, respectively.

 


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  		Fig. 2. Co-immunoprecipitation of endogenous PKB and periplakin. (A) Characterization of generated anti-periplakin antibodies. Purified GST and GST-c-ppl were separated by SDS-PAGE and immunoblotted with anti-GST or anti-GST-c-ppl (5117). (B) Cell lysates of COS-7 cells transfected with myc-c-ppl were either immunoprecipitated with anti-GST-c-ppl (5117 and 5118), anti-periplakin peptide serum (7445 and 7446; left panel), or anti-myc (9E10; two right panels). Immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-myc (9E10; right panel), anti-periplakin peptide serum (7445) and anti-GST-c-ppl (5117; two left panels). (C) MCF-7 cells were lysed and periplakin was immunoprecipitated with anti-GST-c-ppl or anti-peptide serum. (NI) is an immunoprecipitation with non-immune serum as a control. Immunoprecipitates were separated by SDS-PAGE and immunoblotted for the presence of periplakin with 5117. (D) Epithelial cells express periplakin. Various cell lines were lysed and analyzed by western blotting using the anti-periplakin 5117 antibody. (E) Periplakin interacts with PKB endogenously. Periplakin and PKB were immunoprecipitated from MCF-7 cells using the anti-periplakin 5117 antibody and anti-PKB antibody, respectively, and the immune complex was resolved by SDS-PAGE and immunoblotted with the anti-periplakin 5117 antibody. Non-immune anti-serum was used as a negative control. (F) c-ppl expression does not affect insulin-induced PKB activation. A14 cells were transfected with HA-PKB either in the presence or absence of myc-c-ppl. After 36 hours cells were either treated with insulin for 7 minutes or left untreated. HA-PKB was immunoprecipitated and analysed for kinase activity as described (Burgering and Coffer, 1995Go).

 


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  		Fig. 3. Determination of the binding site of PKB to periplakin. (A) COS-7 cells were transiently transfected with myc-tagged c-ppl in combination with HA-tagged PKB constructs as indicated. HA-PKB was immunoprecipitated with anti-HA and analysed by western blotting for myc-c-ppl binding by immunoblotting with anti-myc (9E10) (upper panel). Expression of HA-PKB constructs and myc-c-ppl was checked by immunoblotting with anti-HA and anti-myc, respectively. (B) The outcome of the interaction studies by co-immunoprecipitation and yeast two-hybrid analysis. nd, not done. (C) Primary structure of the PH domain of PKB. The positions of the ß-sheets and {alpha}-helix are indicated by arrows on top and were determined by sequence alignment with other PH domains (see also Ferguson et al., 2000Go). The nine amino acid deletion that results in loss of periplakin binding is indicated by a black line below the sequence. The conserved W99 residue is depicted in bold and indicated by an arrow.

 


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  		Fig. 4. Periplakin binds to the intermediate filament vimentin. (A) Periplakin interacts with vimentin. COS-7 cells were transiently transfected with myc-tagged c-ppl and co-immunoprecipitations were performed in PKB buffer using anti-myc and anti-vimentin antibodies. Samples were analysed by western blotting using either the anti-myc (anti-myc 9E10) or anti-vimentin (anti-vimentin, Oncogene Science) antibody. (B) Periplakin colocalises with vimentin. Rat-1 cells were transiently transfected with myc-tagged c-ppl and immunostained for myc-c-ppl and endogenous vimentin. Bars, 10 µm. (C) Myc-c-ppl is present in vimentin fractions. A14 cells were transiently transfected with myc-tagged c-ppl and fractionated using a KI-protocol. The fractions were immunostained for vimentin, actin and myc-c-ppl. Fraction 1, total; 2, cytosolic and membrane; 3, actin; and 4, vimentin. (D) Comparison of C-terminal deletion mutants and their binding to PKB and vimentin. The C-terminal part of periplakin is shown. Binding was determined by co-immunoprecipitation and yeast two-hybrid analysis.

 


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  		Fig. 5. Periplakin colocalises with mitochondira. (A) Confocal immunostaining for periplakin. MCF-7 cells were pre-incubated with Mitotracker and co-immunostained for periplakin using the anti-periplakin 5117 antibody (upper panel). Similar staining was observed with the other antibodies (5118, 7445 and 7446) and with affinity-purified 5117. No staining was observed using pre-immune serum from the rabbits (5117 and 5118) used for immunization with GST-c-ppl. MCF-7 cells were transiently transfected with HA-Ppl and immunostained for HA-Ppl using the HA antibody and for cytochrome c (lower panel). Bars, 10 µm. (B/C) Periplakin is found in mitochondrial fractions. (B) COS-7, MCF-7/FR and Rat-1 cells were fractionated using a Percoll gradient and the fractions were analysed by western blotting using anti-periplakin 5117, anti-PKB, anti-cox4 and anti-MAPK antibodies. (C) MCF-7/FR and Rat-1 cells were fractionated using the mitochondria affinity purification protocol. Fractions were analysed by immunoblotting, using the same antibodies as in B.

 


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  		Fig. 6. Periplakin expression modulates PKB-dependent FOXO4 regulation. (A) 293T cells were transfected with a TK-renilla-luciferase (internal control) and 6xDBE luciferase reporter construct (Medema et al., 2000Go), in the absence or presence of FOXO4. Different amounts (µg) of full-length periplakin, myc-c-ppl or HA-PKB were cotransfected with FOXO4. 36 hours after transfection lysates were prepared and luciferase counts were measured and normalized against renilla luciferase counts. 6xDBE activity in the absence of FOXO4 was set at 1. Results represent the averages of three independent experiments. (B) Periplakin expression reduces PKB-dependent FOXO4 phosphorylation. 293T cells were transfected with the 0.5 µg full-length periplakin or 1.0 µg c-ppl and 1 µg HA-FOXO4. HA-FOXO4 was immunoprecipitated and the level of PKB-dependent phosphorylation was determined by immunoblotting using a phospho-specific antibody against pSer193.

 




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