Endoplasmic Reticulum (ER) Stress Enhances Tip60 (A Histone Acetyltransferase) Binding to the Concanavalin A

Herein, we report that the concanavalin A binding of Tip60 (a target of the human immunodeficiency virus type 1-encoded transactivator Tat interacting protein 60 KD; a histone acetyltransferase; HAT) is enhanced as the result of endoplasmic reticulum (ER) stress. The cell expression of Tip60 combined with site-directed mutagenesis analysis was used to identify the glutamine 324 residue as the lecithin binding (Concanavalin A; Con A) site. The Tip60 N324A mutant strain, which seems to be the Con A binding-deficient, was attenuated the protein-protein interactions with FE65 and its protein stability, but its ability of G0-G1 cell cycle arrest was not interrupted. Interestingly, both HAT activity and the nuclear localization of Tip60 N324A mutant were enhanced than those of Tip60 WT. Thus, our results indicate that the Con A binding deficient of Tip60 seems to be one of the most pivotal posttranslational modifications (such as N-glycosylation) for its functional regulation signal, which is generated in response to ER stress

ULK2 Ser 1027 Phosphorylation by PKA Regulates Its Nuclear Localization Occurring through Karyopherin Beta 2 Recognition of a PY-NLS Motif.

Uncoordinated 51-like kinase 2 (ULK2), a member of the serine/threonine kinase family, plays an essential role in the regulation of autophagy in mammalian cells. Given the role of autophagy in normal cellular homeostasis and in multiple diseases, improved mechanistic insight into this process may result in the development of novel therapeutic approaches. Here, we present evidence that ULK2 associates with karyopherin beta 2 (Kapβ2) for its transportation into the nucleus. We identify a potential PY-NLS motif ((774)gpgfgssppGaeaapslRyvPY(795)) in the S/P space domain of ULK2, which is similar to the consensus PY-NLS motif (R/K/H)X(2-5)PY. Using a pull-down approach, we observe that ULK2 interacts physically with Kapβ2 both in vitro and in vivo. Confocal microscopy confirmed the co-localization of ULK2 and Kapβ2. Localization of ULK2 to the nuclear region was disrupted by mutations in the putative Kapβ2-binding motif (P794A). Furthermore, in transient transfection assays, the presence of the Kapβ2 binding site mutant (the cytoplasmic localization form) was associated with a substantial increase in autophagy activity (but a decrease in the in vitro serine-phosphorylation) compared with the wild type ULK2. Mutational analysis showed that the phosphorylation on the Ser1027 residue of ULK2 by Protein Kinase A (PKA) is the regulatory point for its functional dissociation from Atg13 and FIP 200, nuclear localization, and autophagy. Taken together, our observations indicate that Kapβ2 interacts with ULK2 through ULK2's putative PY-NLS motif, and facilitates transport from the cytoplasm to the nucleus, depending on its Ser1027 residue phosphorylation by PKA, thereby reducing autophagic activity

⁷⁷⁴gpgfgssppGaeaapslRyvAY⁷⁹⁵ in ULK2 is required for the interaction between ULK2 and Kapβ2.

<p>(<b>A</b>) Pull-down analysis of Kapβ2 with GST fusion C-terminal ULK2 1–600 or 601–1036 mutants. GST-fusion proteins encompassing the C-terminal of ULK2 were constructed and expressed in <i>E</i>. <i>coli</i>. Approximately 0.1mg of 1–600 or 601–1036 fusion proteins, bound to glutathione-sepharose beads (bottom lane), were incubated with HEK293 cell lysates. (<b>B</b>) Co-immunoprecipitation of ULK2 WT, P794A, or P242A with Kapβ2. HEK293 cells were transiently transfected with the EGFP-ULK2 WT or P794A plasmid. After 48 hours, the cells were lysed, and western blotting carried out with an anti-EGFP antibody and protein A agarose beads. Western blotting assays were performed with rabbit anti-ULK2 or mouse anti-Kapβ2 antibodies. The large size (~110kDa) of Kapβ2 is shown here. To monitor the amount of total protein in the cell lysate, western blotting was also performed with an anti-actin antibody. The negative control was untransfected HEK293 cell lysate. (C) Interaction between ULK2 and WT, P794A, or P242A <i>in vitro</i>. HEK293 cells were transiently transfected with the EGFP-ULK2 WT, P794A, or 242A plasmid. After 48 hours, the cells were lysed, and pull-down assays were conducted with GST-Kapβ2 beads. Western blotting was performed with rabbit anti-ULK2 or mouse anti-Kapβ2 antibodies. The large size (~110 kDa) of Kapβ2 is shown. GST-beads were used as the negative control.</p

Autophagic ability of WT and mutant ULK2.

<p>EGFP ULK2 WT or its mutants (P242A, P794A) were transfected in HEK293 cells as described in the Materials and Methods section. Accumulation of microtubule-associated protein light chain 3-II (LC3-II) in cells is indicated with arrows. The same number of HEK293 cells were transfected with ULK2 WT (<b>A</b>) or its mutants P794A (<b>B</b>), P242A (<b>C</b>) for 48 hours, then LC3-II appearance was analyzed by western blotting at different time points (0, 2, 4, 6 hours) after starvation induction. The amount of ULK2 protein was monitored using an ULK2 antibody (lower lane). To visualize co-localization of ULK2 WT (<b>D</b>), P794A (<b>E</b>), and P242A (<b>F</b>) with endogenous LC3-II in HEK293 cells, cells were stained with an LC3-II antibody 12 hours after starvation induction. Confocal fluorescence micrographs were taken in a similar manner to that described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#pone.0127784.g003" target="_blank">Fig 3</a>. To visualize co-localization of ULK2 WT (<b>G</b>), P794A (<b>H</b>), and P242A (<b>I</b>) with endogenous human WD-repeat protein interacting with phosphoinositides (WIPI; another autophagic marker protein) in HEK293 cells, cells were stained with a WIPI antibody 12 hours after starvation induction. Confocal fluorescence micrographs (<b>G-I</b>) were taken in a similar manner to that described in the legend to D-F. The antibody against LC3-II or WIPI was used according to the manufacturer’s recommendations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#sec002" target="_blank">Materials and Methods</a> section).</p

Comparison of the serine phosphorylation status of ULK2 WT and its mutants.

<p>EGFP ULK2 WT and its mutants (P242A and P794A) were purified with an EGFP antibody, as described in the Materials and Methods section. Phosphorylation of ULK2 was detected with IB using anti-phosphor Ser (upper lane) or ULK2 antibodies. The amount of ULK2 protein in the experiment was monitored using a ULK2 antibody (lower lane) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#pone.0127784.ref001" target="_blank">1</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#pone.0127784.ref003" target="_blank">3</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#pone.0127784.ref005" target="_blank">5</a>].</p

Interaction between the putative PY-NLS motif of ULK2 and Kapβ2 and the subcellular localization of ULK2 and Kapβ2.

<p>(A) ULK2 (Gene ID; KIAA0623) contains two putative-conserved Kapβ2 binding motifs. (²²⁰qdlrmfyeKnRslmpSipRetsPY²⁴³) in its protein kinase domain and (⁷⁷⁴gpGfgssppgaeaapslRyvPY⁷⁹⁵) within the serine/proline (S/P)-rich space domain. Two point mutations (P242A and P794A) were prepared to define the binding motif. Mutated sequences are indicated with arrows. P794A mutant (⁷⁷⁴gpGfgssppgaeaapslRyvPY⁷⁹⁵ changed to ⁷⁷⁴gpGfgssppgaeaapslRyvAY⁷⁹⁵) or P242A mutant (²²⁰qdlrmfyeKnRnotslmpSipRetsPY²⁴³ changed to ²²⁰qdlrmfyekKnRslmpSipRetsAY²⁴³) were constructed. Putative PKA-phosphorylation sites (Ser468 and Ser1027) are also indicated with arrows [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#pone.0127784.ref001" target="_blank">1</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#pone.0127784.ref009" target="_blank">9</a>]. Regions of the protein in the C-terminal domain (CTD) that are involved in membrane attachment and interaction with Atg13-focal adhesion kinase family-interacting protein 200 (FIP200) are indicated [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#pone.0127784.ref009" target="_blank">9</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#pone.0127784.ref028" target="_blank">28</a>]. (B) Two putative ULK2 PY NLS motifs were aligned with the defined PY NLS motif. Both motifs (⁷⁷⁴gpGfgssppgaeaapslRyvPY⁷⁹⁵ and ²²⁰qdlrmfyeKnRslmpSipRetsPY²⁴³) matched the consensus PY-NLS motif of (ΦA/G/SΦΦ—R/K/H)<i>X</i>_2–5PY well [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#pone.0127784.ref001" target="_blank">1</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#pone.0127784.ref011" target="_blank">11</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#pone.0127784.ref014" target="_blank">14</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#pone.0127784.ref016" target="_blank">16</a>]. (C) Following immunoprecipitation (IP) with an anti-ULK2 antibody, immunoblotting (IB) was performed using an antibody against Kapβ2 (left). Conversely, anti-Kapβ2 immunoprecipitated complexes were subjected to immunoblotting using an anti-ULK2 antibody (right). Co-immunoprecipitation of Kapβ2 with ULK2 confirms the presence of a ULK2-Kapβ2 complex in the cell. As a control for immunoprecipitation, an unrelated antibody against EGFP was used. For the control of immunoblotting, an antibody against actin was used (bottom). (D) Confocal fluorescence micrographs showing endogenous ULK2 and Kapβ2 in HEK293 cells. These proteins were visualized by immunofluorescence in fixed and permeabilized cells using monoclonal or polyclonal antibodies against human Kapβ2 or ULK2, and Alexa Fluor 568-conjugated donkey anti-rabbit IgG or Alexa Fluor 488-conjugated mouse anti-rabbit IgG. The yellow pattern resulting from the merging of red and green colors indicates co-localization of the proteins in the cytoplasm (white color in the nucleus). The cell’s nuclear region was visualized using Hoescht staining (blue color). To determine the co-localization of ULK2 and Kapβ2, the enlarged co-localization image of the specific merged region is shown (white color). PCC between ULK2 and Kapβ2 was measured by quantitative confocal microscopy. (E) Confocal fluorescence micrographs showing localization of endogenous ULK1 (green color; not containing a PY-NLS motif), which is in the cytosol but not in the nucleus in HEK293 cells, for comparison. The cell’s nuclear region was visualized using Hoescht staining (blue color). The image of Kapβ2 is not present. Fluorescence images were analyzed to calculate the nuclear-to-total fluorescence ratio (Fn/t; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#sec002" target="_blank">Materials and methods</a>). The graph shows the percentage of ULK1 and ULK2 localization in the nucleus; the mean ± SEM (error bars; n ≥ 10) from a single assay representative of three separate experiments (* P<0.05; ** P<0.01; Student’s t-test is used throughout).</p

Comparison of the effect of ULK2 WT and its mutants on cell survival.

<p>Comparison of the effect of ULK2 WT and its mutants on cell survival.</p

Subcellular localization and autophagic ability of ULK2 Ser1027 mutants.

<p>To visualize co-localization of EGFP-ULK2 WT, S1027A, and S1027D (green) with endogenous human Atg13 <b>(A-C)</b> or FIP200 <b>(E-G)</b> in HEK293 cells, cells were stained with an Atg13 or FIP200 antibody (red color) 12 hours after starvation induction. The antibodies against Atg13 and FIP200 were used according to the manufacturer’s recommendations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#sec002" target="_blank">Materials and Methods</a> section). To compare co-localization of ULK2 WT, S1027A, and S1027D with endogenous LC3-II (as a marker of autophagy) in HEK293 cells, cells were stained with an LC3-II antibody (red color) 12 hours after starvation induction <b>(H-J)</b>. To further examine co-localization of ULK2 WT, S1027A, and S1027D with endogenous Kapβ2 in HEK293 cells, cells were stained with a Kapβ2 antibody (red color) 12 hours after starvation induction <b>(K-M)</b>. Fluorescence images of Fig <b>6A–6C</b> were analyzed to calculate the nuclear-to-total fluorescence ratio (Fn/t). Histograms show the mean of three experiments, bars indicate SD (error bars; n ≥ 10) from a single assay of three separate experiments (* P < 0.05). The graph on the <b>Fig 6D</b> shows the percentage of ULK2 WT, S1027A, or S1027D mutant localization in the nucleus. The endogenous ULK2 in HEK293 cells was visualized with a PKA inhibitor (H89 5μM for 12 hours) and activator (FSK 30μM for 12 hours). ULK2 was stained green and kapβ2 was stained red. The cell’s nuclear region was visualized with Hoescht staining (blue color) <b>(N-P)</b>. Confocal fluorescence micrographs were taken in a similar manner to that described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127784#pone.0127784.g003" target="_blank">Fig 3</a>. PCC between ULK2 and Kapβ2 was measured using quantitative confocal microscopy (n = 5). Each PCC is indicated on the left side of <b>Fig 6N–6P</b>. The nuclear (N) or cytoplasmic (C) fluorescence intensity (FI) profile (each 43μm distance) of ULK2 is shown on the right side of <b>Fig 6N–6P</b>. The bar indicates the nuclear region. Fluorescence images of <b>Fig 6N–6P</b> were analyzed to calculate the nuclear-to-total fluorescence ratio (Fn/t). Histograms indicate SD (error bars; n ≥ 10) from a single assay of three separate experiments (* P < 0.05; ** P < 0.01). The graph on the <b>Fig 6Q</b> shows the percentage of endogenous ULK2 localization in the nucleus, depending on H89 or FSK treatment.</p