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. 2009 Sep;20(18):4010-20.
doi: 10.1091/mbc.e09-02-0173. Epub 2009 Jul 22.

Par-4 is an essential downstream target of DAP-like kinase (Dlk) in Dlk/Par-4-mediated apoptosis

Meike Boosen  1 , Susanne Vetterkind, Jan Kubicek, Karl-Heinz Scheidtmann, Susanne Illenberger, Ute Preuss
Affiliations

Par-4 is an essential downstream target of DAP-like kinase (Dlk) in Dlk/Par-4-mediated apoptosis

Meike Boosen et al. Mol Biol Cell. 2009 Sep.

Abstract

Prostate apoptosis response-4 (Par-4) was initially identified as a gene product up-regulated in prostate cancer cells undergoing apoptosis. In rat fibroblasts, coexpression of Par-4 and its interaction partner DAP-like kinase (Dlk, which is also known as zipper-interacting protein kinase [ZIPK]) induces relocation of the kinase from the nucleus to the actin filament system, followed by extensive myosin light chain (MLC) phosphorylation and induction of apoptosis. Our analyses show that the synergistic proapoptotic effect of Dlk/Par-4 complexes is abrogated when either Dlk/Par-4 interaction or Dlk kinase activity is impaired. In vitro phosphorylation assays employing Dlk and Par-4 phosphorylation mutants carrying alanine substitutions for residues S154, T155, S220, or S249, respectively, identified T155 as the major Par-4 phosphorylation site of Dlk. Coexpression experiments in REF52.2 cells revealed that phosphorylation of Par-4 at T155 by Dlk was essential for apoptosis induction in vivo. In the presence of the Par-4 T155A mutant Dlk was partially recruited to actin filaments but resided mainly in the nucleus. Consequently, apoptosis was not induced in Dlk/Par-4 T155A-expressing cells. In vivo phosphorylation of Par-4 at T155 was demonstrated with a phospho-specific Par-4 antibody. Our results demonstrate that Dlk-mediated phosphorylation of Par-4 at T155 is a crucial event in Dlk/Par-4-induced apoptosis.

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Figures

Figure 1.
Figure 1.
Dlk/Par-4 complex formation and catalytic activity of Dlk are essential for Dlk/Par-4–mediated apoptosis. REF52.2 cells were transiently transfected either with Par-4 wt-GFP/FLAG-Dlk wt (a–d), Par-4 L3-GFP/FLAG-Dlk wt (e–h) and Par-4 wt-GFP/FLAG-Dlk K42A (i–l). Twenty-four hours after transfection, the cells were fixed with formaldehyde and stained for indirect immunofluorescence microscopy with the monoclonal anti-FLAG M2 antibody and with Cy3-labeled secondary antibodies (b, f, and j). Nuclei were visualized with DAPI to analyze induction of apoptosis (c, g, and k). Scale bar, 10 μm.
Figure 2.
Figure 2.
(A) Schematic representation of potential Dlk phosphorylation sites in the Par-4 protein. Full-length rat Par-4 (Par-4 wt, 332 amino acids) contains two nuclear localization signals (NLS1 and NLS2), and a leucine zipper motive (LZ) located within the death domain (DD) at the carboxy-terminus (shaded box). The putative Dlk phosphorylation sites in the rat Par-4 protein are indicated. (B) Sequence comparison of known Dlk substrates. The positions of the required arginine residue at the −3 position preceding the phospho-acceptor site are highlighted in all substrates, suggesting a minimal Dlk consensus motif of R-X-X-(S/T).
Figure 3.
Figure 3.
Time-dependent in vitro phosphorylation of recombinant rat Par-4 by Dlk and PKA. Purified recombinant Par-4, 0.5–1 μg, was phosphorylated by Dlk (A and C) or with the catalytic subunit of PKA (B and D) in the presence of [γ-32P]ATP. Phosphorylation was monitored over a period of 30 min. (A and B) Autoradiography of a 10% SDS-PAGE. Note that phosphate incorporation constantly increases over time (lanes 1–5). For Dlk (A) phosphorylation of Par-4 was also analyzed for the kinase-dead mutant Dlk K42A in comparison to wild-type protein (lanes 6–8). (C and D) Phosphoproteins were blotted onto nitrocellulose membrane, isolated, and subjected to acid hydrolysis. Phosphoamino acids were separated by electrophoresis on thin-layer cellulose plates and visualized by autoradiography. Note that Dlk phosphorylated Par-4 at threonine and serine residues, whereas PKA phosphorylated Par-4 only at serine residues.
Figure 4.
Figure 4.
Coexpression of Par-4 phospho-mutants and Dlk in rat fibroblasts. (A) REF52.2 cells were cotransfected with FLAG-Dlk and either Par-4 wt-GFP (a–d), Par-4 S154A-GFP (e–h), Par-4 T155A-GFP (i–l), Par-4 S220A-GFP (m–p), and Par-4 S249A-GFP (q–t). At 24 h after transfection, the cells were fixed with formaldehyde and stained for indirect immunofluorescence microscopy with the monoclonal anti-FLAG M2 antibody and anti-mouse IgG-Cy3 (b, f, j, n, and r) and with DAPI to analyze induction of apoptosis (c, g, k, o, and s). Note that after coexpression of Dlk and Par-4 phospho-mutant T155A the kinase was only partially recruited to actin filaments and was mainly localized in the nucleus of the cotransfected cells. Scale bar, 10 μm. (B) Quantitative analysis of apoptosis induction in REF52.2. FLAG-Dlk and the C-terminally GFP-tagged Par-4 constructs Par-4 wt, S154A, T155A, S220A, and S249A were coexpressed in REF52.2 cells. Twenty-four hours after transfection, the cells were fixed with formaldehyde and stained with the anti-FLAG M2 antibody and with DAPI to visualize nuclei. The percentage of cells that showed apoptotic morphology (i.e., fragmented nuclei, condensed chromatin and membrane blebbing) was determined among the cotransfected cells by fluorescence microscopy, counting 100–200 positive cells in each experiment. The graph represents the mean value from three independent experiments; error bars, SD (*** p < 0.001).
Figure 5.
Figure 5.
In vitro phosphorylation of Par-4 wt and different Par-4 phospho-mutants by Dlk. Purified recombinant Par-4 wt (2 μg, lane 1), Par-4 S154A (lane 2), Par-4 T155A (lane 3), and Par-4 S154A/T155A (lane 4) were phosphorylated by recombinant Dlk in the presence of [γ-32P]ATP, analyzed on 10% SDS-PAGE, and visualized by autoradiography (top). Equal protein loading of all Par-4 constructs was demonstrated by Coomassie staining of the same gel (bottom). For better depiction of differences in signal intensities a shorter exposure time was chosen for autoradiography than in Figure 3. Hence autophosphorylation of Dlk is not visible.
Figure 6.
Figure 6.
The Par-4(P)T155 antibody recognizes Par-4 when phosphorylated by Dlk at T155. (A) REF52.2 cells were cotransfected with YFP-Dlk and either Par-4 wt-CFP (a–d), Par-4 S154A-CFP (e–h), or Par-4 T155A-CFP (i–l). As a control, REF52.2 cells were also transfected with Par-4 wt-CFP and the kinase-inactive mutant Dlk K42A (m–p). Twenty-four hours after transfection, the cells were fixed with formaldehyde and stained with the rabbit polyclonal Par-4(P)T155 antibody and anti-rabbit IgG-Cy3 (c, g, k, and o). The phospho-specific Par-4(P)T155 antibody failed to stain cells either coexpressing YFP-Dlk/Par-4 T155A-CFP or Par-4 wt-CFP/YFP-Dlk K42A, suggesting phosphorylation at T155 by Dlk. At higher magnification (q–t) of the insets marked in (a–d) the colocalization of Dlk (r) and phosphorylated Par-4 (s) becomes more evident. Note that both analyses show a slightly punctate staining pattern that matches in the merged image (t). Scale bar, 10 μm. (B) Immunoprecipitation of Par-4 with the phospho-specific antibody Par-4(P)T155. REF52.2 cells were transfected either with GFP-vector, Par-4 wt-GFP, Par-4 S154A-GFP, or Par-4 T155A-GFP alone (lanes 1, 2, 4, and 6, respectively) or cotransfected with the same Par-4 constructs and FLAG-Dlk (lanes 3, 5, and 7). Twenty-four hours after transfection, cell extracts were prepared and subjected to immunoprecipitation with the rabbit polyclonal Par-4(P)T155 antibody. The proteins were separated by SDS-PAGE and analyzed by Western blotting with the monoclonal anti-GFP antibody. Note that in conjunction with Dlk only Par-4 wt-GFP (lane 3) and Par-4 S154A-GFP (lane 5) were precipitated with the anti-Par-4(P)T155 antibody but not Par-4 T155A-GFP (lane 7). The input (bottom) represents 20 μg of whole-cell lysate, which was simultaneously subjected to SDS-PAGE and Western blot analysis with the anti-GFP antibody.
Figure 7.
Figure 7.
Introduction of acidic amino acids cannot mimic phosphorylation of Par-4 at T155. REF52.2 cells were cotransfected either with FLAG-Dlk and the GFP-tagged Par-4 phospho-mutant T155D (a–d) or with FLAG-Dlk and the GFP-tagged phospho-mutant T155E (e–h). Twenty-four hours after transfection, the cells were fixed and stained for indirect fluorescence microscopy with the monoclonal anti-FLAG M2 antibody and with Cy3-labeled secondary antibodies (b and f). Nuclei were visualized with DAPI to analyze induction of apoptosis (c and g). Scale bar, 10 μm.
Figure 8.
Figure 8.
Phosphorylation of endogenous Par-4 protein in REF52.2 cells. (A) Immunoprecipitation of endogenous Par-4 with the phospho-specific antibody Par-4(P)T155 (top). Cells were left untreated (lane 2), serum-starved for 16 h (SST, lane 3), stimulated with 10 μM LPA lysophosphatidic acid for 15 min after serum starvation (LPA, lane 4), or treated with 10 μM forskolin for 15 min (FSK, lane 5). As control for antibody specificity the IP on untreated cells was also performed with preimmune serum (lane 1). Immunoprecipitates were analyzed by Western blot analysis with the mouse monoclonal anti-Par-4 antibody. The input (bottom) represents 20 μg of whole-cell lysate. (B) Densitometric analysis of four independent experiments. Par-4 bands after immunoprecipitation with the Par-4(P)T155 antibody were normalized to Par-4 bands in the respective input samples, and the phosphorylation level in unstimulated cells was defined as 100% (* p < 0.05).
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