1. Volume 44, Issue 2, Pages 279-289 (October 2011)
Serine 403 Phosphorylation of p62/SQSTM1 Regulates Selective Autophagic Clearance of Ubiquitinated Proteins 
Gen Matsumoto, Koji Wada, Misako Okuno, Masaru Kurosawa, Nobuyuki Nukina 
Molecular Cell 
Volume 44, Issue 2, Pages (October 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2011 44, 279-289DOI: (10.1016/j.molcel.2011.07.039)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1 p62 Is Phosphorylated during Proteasome Inhibition
(A) Schematic representation showing the functional domains of p62. The amino acid position of phosphorylated serine or threonine residues (letter P in circle) is shown. Bold letters indicate inducible phosphorylation residues.
(B) G-p62 cells on an Atg5-KD background were treated with 2 μM MG132 for the indicated times.
(C and D) G-p62 cells on an Atg5-KD background were treated with or without 0.1 μM Epoxomycin (Epox) (C) or 1 μM okadaic acid (OA) (D) and 1 μM bafilomycin A1 (BafA) (C and D).
(B–D) S403-phosphorylated p62 (S403-P) was detected with polyclonal anti-phospho-p62 (S403) antibody. Total G-p62 was visualized with anti-p62 (monoclonal). The ratio between relative amounts of phosphorylated and total p62, normalized to that at time 24 hr (B), with Epox (C), or BafA (D) treatment is shown.
(E) ATG5 knockout MEF cell was treated with 0.1 μM OA or 0.5 μM BafA for 24 hr as indicated. S403-phos-p62 (S403-P) or endogenous p62 was detected with polyclonal anti-phospho-p62 (S403) or anti-p62 (polyclonal) as shown.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 S403-Phos-p62 Localizes to the p62 Body in Cells and In Vivo
(A) The Atg5-KD G-p62 cells were treated with or without 1 μM okadaic acid (OA) or 1 μM bafilomycin A1 (BafA) for 18 hr as indicated and GFP and S403-phos-p62 (polyclonal anti-phospho-p62 [S403], Alexa633) were visualized by confocal microscopy. The number of S403-phos-p62 positive p62 body par cell in single picture was shown (+OA, p < 0.05; +BafA, p < 0.001). Data are mean ± standard error of the mean (SEM) values of three deferent fields containing more than 10 cells in each field. p values were determined by Student's t test against control. Scale bars represent 10 μm.
(B) Immunohistochemistry of brain sections from control (atg5flox/+), atg5 conditional knockout (atg5flox/flox, nestin-Cre) at 17 weeks of age, DAB staining with an anti-ubiquitin, anti-p62 (p62c), and polyclonal anti-phospho-p62 (S403) antibody as indicated. The scale bar represents 20 μm.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 CK2 Phosphorylates S403 of p62 in Cells
(A) In vitro phosphorylation assay. Purified TF-p62-D69A or TF-p62-D69AS403A was mixed with or without His6-CK2 and/or 1 mM ATP with an ATP regeneration system as indicated. S403-phos-p62 was detected with the monoclonal anti-phospho-p62 (S403) antibody (4F6).
(B and C) G-p62wt cells on the Atg5-KD background were treated with CK2 inhibitors, DMAT, or TBBt, and S403-phos-p62 was accumulated by treatment with 0.1 μM Epox (B) or 1 μM BafA (C) for 18 hr as indicated.
(D) G-p62 cells on the p62-KD background were transiently transfected with RFP-CK2a1 (R-CK2). G-p62 and R-CK2 were visualized by confocal microscopy. The inset shows a magnified image in the square box. Arrows indicate R-CK2 transfected cells and arrowhead indicates non-transfected cells. The scale bar represents 10 μm.
(E) G-p62wt or G-p62-S403A mutant cells on the p62 knockdown background were transiently transfected with R-CK2 for 72 hr and whole-cell lysates were subjected to western blot analysis with monoclonal anti-phospho-p62 (S403) (4F6), anti-p62 (PM045), anti-CK2alpha, and anti-γ-tubulin as indicated.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
S403E Mutation Mimics S403 Phosphorylation and the Mutant Sequestosome Has Distinct Properties
(A) Wild-type or mutant (S403A or S403E) G-p62 cells on a p62-KD background were visualized by confocal microscopy. The GFP and DIC merged image is shown. Scale bars represent 5 μm.
(B) Cells shown in (A) were treated with MG132 (MG) or BafA for 24 hr as indicated. The relative G-p62 protein amount is shown. Error bars represent the SEM (n = 3).
(C and D) G-p62wt (circle), G-p62-S403A (triangle), and G-p62-S403E (square) cells on an Atg5-KD background were subjected to FRAP analysis as indicated. The arrow in (C) indicates the photobleached sequestosome. The fluorescence recovery in a sequestosome was measured and the relative fluorescence intensity (RFI) was determined at each time point and represented as the mean ± SEM (n ≥ 10 sequestosomes).
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Increased Ubiquitin Binding Property in S403E Mutant Results in the Efficient Autophagic Degradation of Ub Proteins
(A) R-ubiquitin and wild-type or mutant (S403A or S403E) G-p62 double-stable cells were visualized by confocal microscopy as indicated. The ratio of R-Ub accumulated G-p62 bodies out of total G-p62 was shown. Data are mean ± SEM values of 12 cells in each group (p < 0.01 against wild-type for each mutant). Scale bars represent 5 μm.
(B) G-p62wt, G-p62-S403A, and G-p62-S403E cells on an Atg5-KD background were immunoprecipitated with anti-GFP antibody. The ratio between coimmunoprecipitated polyUb proteins and G-p62 is shown. Data are mean ± SEM values of three independent results. ∗ and ∗∗ represent p < 0.05 and p < 0.01, respectively.
(C) Purified TF-p62 with the indicated mutations was mixed with differently branched in vitro polymerized ubiquitin chain (poly-UB) and TF-p62-polyubiquitin complex was purified with IMAC. The fraction eluted by 0.5 M imidazole was analyzed by western blot as indicated.
(D) G-p62wt, G-p62-S403A, or G-p62-S403E cells on the mp62-KD background were treated with 0.1 μM epoxomycin for 12 hr as indicated. An additional 12 hr treatment was performed with epoxomycin with or without 2 μM BafA, followed by western blot analysis using the indicated antibody.
(E) The ratio is shown of polyubiquitinated proteins in samples described in (D) with or without BafA treatment. Data are mean ± SEM values of three independent results.
(F) S403-phosphorylated G-p62 was immunopurified from OA-treated G-p62wt cells on an Atg5-KD background and dephosphorylated with alkaline-phosphatase followed by incubation with the indicated blanched purified poly-UB chain. The G-p62-polyUB complex was immunoprecipitated with anti-GFP.
(G) Immunopurified refolded GFP-p62 was incubated with K63-linked poly-UB chain followed by the treatment with alkaline-phosphatase. S403-phos-p62 protected from phosphatase was detected. The ratio between S403-phos-p62 and total p62 was shown.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
CK2 Overexpression Prevents the Large Inclusion Formation of Mutant Huntingtin Protein in a p62-Dependent Manner
(A) Doxycycline (dox)-inducible httQ150-YFP cell line was transfected with RFP or RFP-CK2 (R-CK2) and differentiated with dibutyl-cAMP. httQ150-YFP was induced for 3 days with dox. Cells expressing R-CK2 with or without inclusions were counted in several fields (n = 10). The average number of transfected cells with inclusions in the total cells with RFP fluorescence is shown. Each field contains at least ten cells. Error bars represent the SEM (n = 10).
(B) Total cell lysate (20 μg) of R-CK2 transfected cells shown in (A) was subjected to the filter trap assay and aggregates were detected with anti-GFP antibody. Error bars represent the SEM (n = 4).
(C) HttQ150-YFP cells were transiently transfected with RFP or RFP-miR-p62, differentiated as in (A), and induced with dox for 3 days with or without 10 nM OA, as indicated. Total cell lysates (20 μg) were subjected to the filter trap assay and aggregates were detected by anti-GFP antibody. The amount of filter-trapped httQ150-YFP was measured and the relative amounts of aggregates are depicted. Error bars represent the SEM (n = 4). p value of Student's t test is shown.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
A Regulatory Mechanism for p62-Mediated Selective Autophagy of Ubiquitinated Protein
Schematic representation of the proposed mechanism of p62-mediated selective autophagy. In normal conditions, p62 is in equilibrium between S403-phosphorylated by CK2 and dephosphorylated states. Once proteasome is inhibited or overloaded, polyubiquitinated proteins (polyUb proteins) are accumulated, and S403-phos-p62 binds with the accumulated Ub proteins. The S403-phos-p62 binding with Ub proteins becomes dephosphorylation resistant. Then, the S403-phosphorylation balance shifts to the increased S403-phos-p62 state. The S403-phos-p62 with Ub proteins forms sequestosomes. The isolation membrane is recruited to the sequestosome by LC3-p62 interaction and the autophagosome grows on the surface of sequestosome. The autophagosomes holding p62 eventually fuse with lysosomes. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions