Fig 1.
Interaction between the putative PY-NLS motif of ULK2 and Kapβ2 and the subcellular localization of ULK2 and Kapβ2.
(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 [1,9]. 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 [9,28]. (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)X2–5PY well [1,11,14–16]. (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 Materials and methods). 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).
Fig 2.
774gpgfgssppGaeaapslRyvAY795 in ULK2 is required for the interaction between ULK2 and Kapβ2.
(A) 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 E. coli. 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) 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 in vitro. 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.
Fig 3.
Subcellular localization of exogenous ULK2 PY mutants.
A) Confocal fluorescence micrographs of EGFP-ULK2 WT, P794A, and P242A in HEK293 cells. All EGFP constructs fluoresced green, while Kapβ2 fluoresced red. Transfected EGFP-ULK2 WT merged with Kapβ2 is predominantly in the nucleus (A). Yellow color resulting from merging of the red and green indicates co-localization of both proteins. This is found mainly in the nucleus, similar to the results obtained for endogenous ULK2 shown in Fig 1C. The transfected EGFP-ULK2 PY-NLS mutant (P794A, which is not detected in the nucleus) is not co-localized with Kapβ2 in the cytoplasm either (B). A greater number of puncta are shown (B). Subcellular localization of the ULK2 P242A mutant is also visualized in (C) This mutant is localized with Kapβ2 mainly in the nucleus (yellow color). The cell’s nuclear region is visualized with Hoescht staining (blue). Representative images based on five replicate experiments for each construct are shown. Fluorescence images of (A-C) 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 Fig 3D shows the mean ± SEM (error bars; n ≥ 10) from a single assay representative of three separate experiments (* P < 0.05; ** P < 0.01).
Fig 4.
Autophagic ability of WT and mutant ULK2.
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 (A) or its mutants P794A (B), P242A (C) 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 (D), P794A (E), and P242A (F) 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 Fig 3. To visualize co-localization of ULK2 WT (G), P794A (H), and P242A (I) 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 (G-I) 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 Materials and Methods section).
Fig 5.
Comparison of the serine phosphorylation status of ULK2 WT and its mutants.
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) [1,3,5].
Fig 6.
Subcellular localization and autophagic ability of ULK2 Ser1027 mutants.
To visualize co-localization of EGFP-ULK2 WT, S1027A, and S1027D (green) with endogenous human Atg13 (A-C) or FIP200 (E-G) 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 Materials and Methods 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 (H-J). 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 (K-M). Fluorescence images of Fig 6A–6C 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 Fig 6D 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) (N-P). Confocal fluorescence micrographs were taken in a similar manner to that described in the legend to Fig 3. PCC between ULK2 and Kapβ2 was measured using quantitative confocal microscopy (n = 5). Each PCC is indicated on the left side of Fig 6N–6P. The nuclear (N) or cytoplasmic (C) fluorescence intensity (FI) profile (each 43μm distance) of ULK2 is shown on the right side of Fig 6N–6P. The bar indicates the nuclear region. Fluorescence images of Fig 6N–6P 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 Fig 6Q shows the percentage of endogenous ULK2 localization in the nucleus, depending on H89 or FSK treatment.
Fig 7.
Both PY-NLS and Ser1027 residue phosphorylation in ULK2 confer a unique role in autophagy.
P794A (774gpgfgssppGaeaapslRyvPY795 changed to 774gpGfgssppgaeaapslRyvAY795) or P242A (220qdlrmfyeKnRnotslmpSipRetsPY243 changed to 220qdlrmfyekKnRslmpSipRetsAY243) mutants showed an increased autophagic activity, but less serine phosphorylation. Membrane attachment and interaction with Atg13-focal adhesion kinase family-interacting protein 200 (FIP200) appears to mask the ULK2 PY NLS motif (Left side). The protein complex is likely dissociated by phosphorylation by the protein kinase (such as AMPK, mTOR1, PKA, or ULK2), consequently making the PY-NLS motif in ULK2 accessible by Kapβ2 (Right side), even though the phosphorylation sites on Atg13-FIP200 by the protein kinases are not completely characterized. Due to the fact that the PY-NLS mutant of ULK2 and WT ULK1 (“X” indicates blockade of Kapβ2 binding) are not transported to the nucleus, these two seem to be more active in autophagy than WT ULK2. The ULK2 S1027A mutant (dephosphorylated analogy form) can easily bind Atg13-FIP200 to promote cell autophagy and apoptosis. These protein associations result in its PY-NLS motif hiding through steric inhibition, resulting in the blockade of its nuclear localization by Kapβ2 (Left side). Meanwhile, the PY-NLS motif of the ULK2 S1027D mutant (PKA phosphorylated analogy form), which is free from Atg13-FIP200 association, is exposed to Kapβ2, and the mutant protein can be imported into the nuclei (Right side). The freed ULK2 S1027D mutant can induce neither cell autophagy nor apoptosis. Thus, the phosphorylation on the Ser1027 residue (RRlSA) of ULK2 by PKA seems to be a major regulatory event in its autophagic functions (see the main body text for more detail).
Table 1.
Comparison of the effect of ULK2 WT and its mutants on cell survival.