Degradation of AF1Q by chaperone-mediated autophagy.

E XP ER I ME NTAL C E LL RE S E ARCH

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Research Article

Degradation of AF1Q by chaperone-mediated autophagy Peng Lia, Min Jia, Fei Lua, Jingru Zhanga, Huanjie Lib, Taixing Cuib, Xing Li Wangb, Dongqi Tangb,c,n, Chunyan Jia,nn a

Department of Hematology, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012, P.R. China b Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012, P.R. China c Center for Stem Cell & Regenerative Medicine, The Second Hospital of Shandong University, Jinan 250033, P.R. China

article information

abstract

Article Chronology:

AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker

Received 28 November 2013

for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult

Received in revised form

myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differ-

8 May 2014

entiation and development but its regulatory mechanism has not been defined clearly. In the

Accepted 20 May 2014

present study, we used pharmacological and genetic approaches to influence chaperonemediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological

Keywords: AF1Q Chaperone-mediated autophagy Protein degradation macroautophagy

inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA. & 2014 Elsevier Inc. All rights reserved.

Introduction The AF1Q gene, located on chromosome 1q21, is initially identified as a mixed lineage leukemia gene fusion partner in two infant

patients diagnosed with acute myelomonocytic leukemia, which carried a t(1; 11) (q21; q23) translocation [1]. AF1Q encodes a small protein of 9 k Da, which is not similar to any other known proteins, and no functional domains have been described for this

Abbreviations: 3-MA, 3-methyladenine; 6-AN, 6-aminonicotinamide; AML, acute myeloid leukemia; CMA, chaperone-mediated autophagy; CQ, chloroquine; HBSS, Hanks balanced salt solution; HSPA8, heat shock 70 kDa protein 8; LAMP-2 A, lysosomeassociated membrane protein 2A n Corresponding author at: Center for Stem Cell & Regenerative Medicine, The Second Hospital of Shandong University, 274 Beiyuan Street, Jinan 250033, P.R. China. Tel.: þ86 531 85875489. nn Corresponding author. Fax: þ86 531 86927544. E-mail addresses: [email protected] (D. Tang), [email protected] (C. Ji).

http://dx.doi.org/10.1016/j.yexcr.2014.05.013 0014-4827/& 2014 Elsevier Inc. All rights reserved.

Please cite this article as: P. Li, et al., Degradation of AF1Q by chaperone-mediated autophagy, Exp Cell Res (2014), http://dx.doi.org/ 10.1016/j.yexcr.2014.05.013

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small protein [1]. AF1Q is expressed in various tissues, such as colon and small intestine [1]. In hematopoietic tissues, its expression is highly regulated in normal lineage committed hematopoietic progenitor cells and thymus [1]. AF1q cooperates with the Notch signaling pathway to foster the emergence of bone marrow prothymocytes and drive subsequent intrathymic specification toward the T-cell lineage [2]. It is confirmed that elevated AF1Q expression is a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome [3–5]. The biological function of AF1Q has not been completely characterized, but increasing evidence has shown a potentially protooncogenic function of this protein in solid tumors such as thyroid oncocytic tumors, breast cancer and testicular germ cell tumors [6–9]. In addition to the proposed proto-oncogenic role of AF1Q, this protein also participates in the regulation of apoptosis in tumor cells. In human squamous carcinoma cells, AF1Q activates BAD-mediated apoptotic pathway via NF-kB [10,11]. In ovarian cancer cells, AF1Q mediates basal and 4-HPR-induced apoptosis [12]. It has been shown that AF1Q expression can be directly regulated by microRNA-29b [13]; the catabolism of AF1Q protein is ubiquitinmediated degradation by the proteasome in the centrosomal area [2]. However, mechanisms underlying the regulation of AF1Q and its degradation pathway are not totally understood. The ubiquitinproteasome system and the autophagy-lysosome system compare the two major intracellular proteolytic systems in mammalian cells [14]. Autophagy is an intracellular degradation system for long-lived cytoplasmic constituents using lysosomes [15]. Based on the known mechanisms that substrates of autophagy are delivered into lysosomes, three major forms of autophagy have been described in mammalian cells including macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) [16]. Compared to other forms of autophagy, CMA is a selective lysosomal pathway best characterized in the degradation of individual cytoplasmic proteins [17]. For degradation via CMA, cytoplasmic proteins bear the KFERQ motif or a closely related sequence are recognized by the heat shock 70 k Da protein 8 (HSPA8) chaperone [17,18]. Following binding of the chaperone-substrate complex, the proteins are delivered to a lysosomal membrane receptor – lysosome-associated membrane protein 2A (LAMP-2A), and then translocate into the lysosomal lumen and degraded [17,19]. In the present study, we investigated the role of lysosome pathway in AF1Q degradation. We found that AF1Q protein levels are markedly increased by inhibition of lysosome. Our analysis of AF1Q amino acid sequence revealed a motif related to KFERQ in the protein, supporting AF1Q as a target for CMA-dependent proteolysis. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Changing the levels of CMA-related proteins affected the accumulation of AF1Q. Our data demonstrated that AF1Q can be recognized by HSPA8 through KFERQ-like pentapeptide motif and be degraded in lysosomes via CMA.

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USA), GV141-LAMP-2A (GENECHEM, China), GV141-HSPA8 (GENECHEM, China), and short hairpin RNA (shRNA) for ATG5 (GENECHEM, China). pEGFP-C3-AF1Q, pCDNA3.1-AF1Q90-6-myc (AF1Q90), and pCDNA3.1-AF1Q80-6-myc (AF1Q80) were kindly provided by Dr. Xiulian Sun, National Key Lab of Otolaryngology, Qilu Hospital, Shandong University, China. PLOC-AF1Q was kindly provided by Dr. William Tse, Mary Babb Randolph Cancer Center, West Virginia University School of Medicine, Morgantown, WV, USA. Corresponding empty plasmids were used for control transfections. Double-strand siRNA for ATG7 (50 –CAG UGG AUC UAA AUC UCA AAC UGA U-30 ) [20] (synthesized by Life Technologies), LAMP-2A (50 –GCU GUG CGG UCU UAU GCA U-30 ) [21] (synthesized by Shanghai GenePharma) and HSPA8 (50 –GUC UUC UAU GGU UCU GAC A-30 ) (Sigma, China) were used. Commercially available siRNA to random noncoding sequences were used for control transfections.

Cell culture, transfection and treatment K562 cells (Shanghai Institutes for Biological Sciences) were cultured in RPMI 1640 (Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, USA), 100 U/ml penicillin and 100 mg/ml streptomycin. HEK293 (Shanghai Institutes for Biological Sciences) and HeLa cells (Shanghai Institutes for Biological Sciences) were cultured in DMEM (Gibco, USA) supplemented with 10% FBS and penicillin/streptomycin. Cells were maintained in an incubator at 37 1C in an atmosphere containing 5% CO2 and 95% air. Transient transfection of cells was performed with lipofectamine 2000 (Invitrogen, USA), according to the manufacturer's recommendations. When appropriate, cells were treated with the following agents: 0.5 μM MG132 (Sigma, USA), 10 μM chloroquine (CQ, Sigma, USA), 10 mM 3-methyladenine (3-MA, Sigma, USA), and 50 mM 6-aminonicotinamide (6-AN) (Sigma, USA).

Antibodies and reagents For this study we used the following antibodies: rabbit anti-AF1Q, dilution of 1:100 (co-immunoprecipitation) and 1:2000 (western blot) (EPITOMICS, USA); rabbit anti-HSPA8, dilution of 1:2000 (western blot) and 1:200(immunofluorescence) (EPITOMICS, USA); rabbit anti-LAMP-2A, dilution of 1:1000(western blot) and 1:200(immunofluorescence) (abcam, USA); rabbit anti-LC3, dilution of 1:200(immunofluorescence) (Sigma, USA); rabbit antiATG5, dilution of 1:2000 (EPITOMICS, USA); rabbit anti-ATG7, dilution of 1:2000 (Sigma, USA); mouse anti-beta actin, dilution of 1:2000 (ZSGB-BIO, China), and peroxidase-conjugated affinipure goat anti-mouse (ZSGB-BIO, China), goat anti-rabbit (ZSGBBIO, China) dilution of 1:30000. Alexa Fluor 647 goat anti-rabbit IgG (HþL) antibody (Invitrogen, USA), dilution of 1:500. Polyvinylidene fluoride (PVDF) membranes (Millipore, USA), and enhanced chemiluminescence (ECL, Millipore, USA).

Co-immunoprecipitation and western blot analysis

Materials and methods Expression plasmid, shRNA and siRNA The following plasmids were used: pCMV-myc-ATG5 (pCMVATG5, addgene, USA), pCMV-myc-ATG7 (pCMV-ATG7, addgene,

Cells treated under different experimental conditions were washed twice with ice-cold PBS containing 1 mM Na3VO4, and lysed in cell lysis buffer for western bolt and immunoprecipitation (IP) (Beyotime, China) for 30 min on ice. Insoluble material was removed by centrifugation at 13,000g for 20 min at 4 1C. Protein


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concentrations in the supernatant were quantified with the bicinchoninic acid assay protein reagent kit (Novoprotein, China) according to a standardized curve. The cell lysates (1 mg) were incubated with normal rabbit IgG and protein AþG (Beyotime, China) at 4 1C for 1 h with gentle shaking. The pretreated lysates were incubated with anti-AF1Q with gentle shaking at 4 1C overnight and then further incubated with protein AþG agarose for 2 h. These precipitates were washed four times with ice-cold PBS. Then, proteins were released by boiling in sample buffer, followed by immunoblotting analysis. The whole cell lysates (20 μg) or precipitates were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) or TricineSDS-PAGE and transferred onto PVDF membranes. Non-specific sites were blocked with 5% nonfat milk in TBS/0.1% Tween-20 and incubated with appropriate primary antibodies overnight at 4 1C, followed by incubation with horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG at room temperature for 1 h. After washing, immunoreactive bands were detected by ECL. Immunoblots were visualized by FluorChem E Chemiluminescent Western Blot Imaging System (Cell Biosciences, Santa Clara, CA). The results were quantified using the Image J software (National Institutes of Health, USA). For the quantification of specific bands, the same size square was drawn around each band to measure the intensity and then the value was adjusted by the intensity of the background near that band. The results were expressed as a relative ratio of the target protein to reference protein β-actin. The relative ratio of the target protein of control group is standardized as 1.

RNA extraction and quantitative real-time PCR Total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. To detect mRNA expression, cDNA was synthesized from total RNA using M-MLV RTase cDNA synthesis kit (Takara, Japan). Quantitative real-time PCR (qRT-PCR) was performed using CFX96TM Real-Time System (Bio- Rad, USA) with SYBR Green PCR Master Mix (Toyobo, Japan). The housekeeping gene β-Actin was used as an internal control. Primer sequences were as follows (50 -30 ): AF1Q forward: GGA CCC TGT GAG TAG CCA GTA; AF1Q reverse: CTT GCC CGA TCA TTT TGC CA; β-Actin forward: CGG GAC CTG ACT GAC TAC CT; β-Actin reverse: AAG CAT TTG CGG TGG A.

Immunofluorescence microscopy After being transfected with specific plasmids for 24 h, cells were plated onto 15 mm polylysine-coated coverslips and cultured for 24 h. Cells were fixed with immunol staining fix solution (Beyotime, China) at room temperature for 10 min, incubated in 0.5% Triton X-100 for 5 min, washed 3 times in PBS, then incubated in normal goat serum (ZSGB-BIO, China) for 1 h at room temperature. Cells were incubated with primary antibodies at 4 1C overnight. The coverslips were washed 3 times in PBS, and then incubated with Alexa Fluor 647 goat anti-rabbit IgG (HþL) antibody at room temperature for 1 h, and DAPI for 5 min at room temperature. Images were acquired by laser scanning confocal microscope (PerkinElmer, UltraVIEW VoX) at 400 magnification.

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Statistical analysis Experiments were performed three times and quantitative data are expressed as mean7SD. Statistical analysis was conducted using Student's t-test or one-way analysis of variance, with posthoc Student LSD (Least Significant Differences) pairwise comparisons applied as appropriate. Unless otherwise specified, Po0.05 was considered significant.

Results CMA is involved in degradation of AF1Q We first explored the contribution of the autophagy–lysosome system to the degradation of AF1Q. K562 leukemia cells and HEK293 cells were treated with MG132 (a proteasome inhibitor), 3-MA (to inhibit macroautophagy) or CQ (to inhibit total lysosomal proteolysis). We found that AF1Q was degraded in lysosomes, as intracellular AF1Q levels increased when lysosomal proteolysis were blocked (Fig. 1A). Lysosomal degradation of AF1Q did not occur through macroautophagy, because AF1Q was not increased in cells treated with 3-MA (Fig. 1A), which is a macroautophagy inhibitor. We also confirmed that a portion of cellular AF1Q was degraded by proteasomes (Fig. 1A) [2]. Furthermore, treatment with both CQ and MG-132 showed a cumulative effect in the stabilization of AF1Q (Fig. 1A). Since we showed that lysosomal degradation of AF1Q did not occur through macroautophagy as stated above, we reasoned whether inducing CMA would stimulate AF1Q degradation. We analyzed the effect of 6-AN, a commonly used activator of CMA [22], on AF1Q levels. In both K562 and HEK293 cells, 6-AN treatment decreased the levels of AF1Q (Fig. 1B). Starvation is a classical method to activate both macroautophagy and CMA [23]. Switching to Hanks balanced salt solution (HBSS), which contains no nutrients required by the growing cells, markedly enhanced levels of Beclin 1, which is a macroautophagy regulatory protein, and LAMP-2A, which is a receptor protein of CMA (Fig. 1C). The elevation of Beclin 1 was earlier than that of LAMP-2A in the time course experiment, indicating that macroautophagy actually occurred before the CMA. Also, we found that switching to HBSS for 24 h but not 6 h markedly reduced the accumulation of AF1Q both in K562 and HEK293 cells (Fig. 1D). Our data suggest that CMA activation by either treated with 6-AN or switched to HBSS can decrease AF1Q levels. Next, we examined the effect of these treatments on AF1Q mRNA levels by qRT-PCR. We found that treating with 3-MA, CQ, MG132 or 6-AN have no effect on AF1Q mRNA levels in both K562 and HEK293 cells (Fig. 1E). However, switching to HBSS for 12 h or 24 h can increase AF1Q mRNA levels in both K562 and HEK293 cells (Fig. 1E).

Macroautophagy does not contribute to AF1Q degradation To explore whether macroautophagy participated in AF1Q degradation, we analyzed AF1Q levels by altering the levels of ATG5 and ATG7, which are essential for autophagosome formation [24,25]. To inhibit macroautophagy, HEK293 cells were transfected with ATG7 siRNA or ATG5 shRNA vector, which resulted in 37% or 62% inhibition of the corresponding proteins expression (Fig. 2A and B). To induce macroautophagy, HEK293 cells were


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transfected with ATG7 (pCMV-Atg7) or ATG5 (pCMV-Atg5) overexpression vector, which resulted in 48% or 36% increase of the corresponding proteins expression (Fig. 2C and D). There was a reduction in levels of AF1Q when endogenous ATG7 or ATG5 were knocked down (Fig. 2E and F), whereas overexpression of ATG5 or ATG7 increased the accumulation of AF1Q (Fig. 2G and H). The results further corroborated that the degradation of AF1Q did not occur through macroautophagy.

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AF1Q Interacts with HSPA8 and LAMP-2A Previous studies show that the substrates of CMA contain a pentapeptide KFERQ-like motif [17]. Sequence alignment of AF1Q amino acid (aa) sequence with a KFERQ motif using the Clustal Omega tool revealed that 84–88 aa of AF1Q contain the putative KFERQ-like motif (Fig. 3A). Thus, AF1Q is a potential target for CMA-dependent proteolysis.

Fig. 1 – CMA is involved in degradation of AF1Q. (A) K562 cells and HEK293 cells were treated with 0.5 μM MG132, 10 mM 3-MA, 10 mM CQ or both 0.5 μM MG132 and 10 mM CQ for 8 h, AF1Q levels were determined with immunoblot analysis. (B) K562 cells and HEK293 cells were treated with 50 mM 6-AN for 8 h, AF1Q levels were determined with immunoblot analysis. (C) K562 cells and HEK293 cells were switched to HBSS for 3,6,12 and 24 h, Beclin1 and LAMP-2A levels were determined with immunoblot analysis. (D) K562 cells and HEK293 cells were switched to HBSS for 6, 12 and 24 h, AF1Q levels were determined with immunoblot analysis. (E) K562 cells and HEK293 cells were treated with above-mentioned treatment, AF1Q mRNA levels were determined with qRT-PCR. ***, po0.001. Please cite this article as: P. Li, et al., Degradation of AF1Q by chaperone-mediated autophagy, Exp Cell Res (2014), http://dx.doi.org/ 10.1016/j.yexcr.2014.05.013


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Fig. 2 – Macroautophagy does not contribute to AF1Q degradation. (A, B, E, F) HEK293 cells were transiently transfected with ATG7 siRNA or ATG7 overexpression vector, pCMV-Atg7, for 72 h, immunoblot was performed with the indicated antibodies. (C, D, G, H) HEK293 cells were transiently transfected with ATG5 shRNA vector or ATG5 overexpression vector, pCMV-Atg5, for 72 h, immunoblot was performed with the indicated antibodies. **, po0.01; ***, and po0.001.

To characterize the association between AF1Q clearance and CMA function, we also analyzed the co-localization of AF1Q with HSPA8 and LAMP-2A, two core components of the CMA machinery, in HeLa cells. We found that AF1Q was co-localized with HSPA8 and LAMP-2A in cells expressing exogenous AF1Q (Fig. 3B). However, we also found that AF1Q was co-localized with macroautophagy marker LC3 (Fig. 3B). To further verify the protein– protein interaction of AF1Q with CMA machinery, co-IP of HSPA8 and LAMP-2A with AF1Q was performed. We found that exogenous AF1Q was co-IP with both exogenous HSPA8 and LAMP-2A from HEK293 cells (Fig. 3C and D). The results confirm that AF1Q protein can interact with HSPA8 and LAMP-2A protein involved in CMA.

HSPA8 and LAMP-2A negatively regulate AF1Q levels We further examined the effects of CMA component proteins on AF1Q accumulation. To inhibit CMA, HEK293 cells were transfected with HSPA8 siRNA or LAMP-2A siRNA, which resulted in 42% or 58% inhibition of the corresponding proteins expression (Fig. 4A and B). To induce CMA, HEK293 cells were transfected with LAMP-2A or HSPA8 overexpression vector, which resulted in 49% or 65% increase in the LAMP-2A expression (Fig. 4C and D). There was an increase in levels of AF1Q when endogenous HSPA8 was knocked down (Fig. 4E). Similarly, knock down of LAMP-2A also increased accumulation of AF1Q (Fig. 4F), whereas overexpression of LAMP-2A or

HSPA8 decreased the accumulation of AF1Q (Fig. 4G and H). These data are consistent with the role of HSPA8 and LAMP-2A in CMAdependent removal of AF1Q.

Identification of CMA-targeted motif in AF1Q protein To confirm that the putative motifs may be the target of the CMA complex, an 80–90 aa deletion AF1Q expression plasmid (AF1Q80) was constructed. The AF1Q80 or the full-length plasmid (AF1Q90) was co-transfected with LAMP-2A siRNA (to inhibit CMA). In HeLa cells, LAMP-2A siRNA reduced LAMP-2A expression when co-transfected with either AF1Q80 or AF1Q90 (Fig. 5A). Knockdown of LAMP-2A was ineffective in increasing protein levels of AF1Q80 in comparison to AF1Q90, suggesting that the clearance of AF1Q80 can be inhibited by the deletion of the CMA recognition motif (Fig. 5B).

Discussion In this study we presented experimental evidence for the degradation of AF1Q in lysosomes via CMA. We confirmed that AF1Q protein, a well-established substrate of the ubiquitin– proteasome system, was increased upon treatment with proteasome inhibitor MG132 [2]. Most importantly, our data illustrated that CMA pathway plays a critical role in the AF1Q degradation.


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Fig. 3 – AF1Q Interacts with LAMP-2A and HSPA8. (A) Sequence alignment with KFERQ motif using Clustal Omega tool. A putative CMA recognition motif KFERQ was identified at the region corresponding to 84–88 aa of AF1Q. (B) HeLa cells were transfected with pEGFP-C3-AF1Q for 48 h and processed for immunofluorescence with the anti-HSPA8, anti-LAMP-2A and anti-LC3 antibodies. The scale bar¼ 10 lm. (C,D) HEK293 cells were co-transfected with AF1Q and HSPA8 or LAMP-2A overexpression vector for 48 h, cells lysates were immunoprecipitated with an anti-AF1Q antibody or control normal rabbit IgG (rIgG), and immunoblot was performed with anti-HSPA8 or anti-LAMP-2A antibody. Empty indicates an empty lane, input indicates whole cell lysates.

Autophagy is an important intracellular proteolysis system that includes macroautophagy, microautophagy and CMA [16]. All of the substrates of autophagy are transported to lysosomes where the substrates are degraded by proteolytic enzymes [16]. Blockade of lysosomes means blockade of all types of autophagic degradation. When treating K562 and HEK293 cells with CQ to inhibit lysosomal proteolysis, AF1Q were dramatically increased. However, when we treated cells with macroautophagy inhibitor 3-MA, no obvious change was found in AF1Q levels. These results

indicated that lysosomal degradation of AF1Q did not occur through macroautophagy. To induce CMA, cultured K562 and HEK293 cells were treated with 6-AN [22]. 6-AN, an analog of niacin, acts by competition with niacin in pathways utilizing NAD(Pþ), being metabolized to 6ANAD (Pþ) [26]. 6ANAD(Pþ) can act as a competitive inhibitor of NAD (Pþ)-requiring processes, such as the synthesis of NADPH, and in turn, results in an oxidative environment within cells [26,27]. Oxidation of CMA substrates facilitates their translocation into



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Fig. 4 – HSPA8 and LAMP-2A negatively regulate AF1Q levels. (A, C, E, G) HEK293 cells were transiently transfected with HSPA8 siRNA or LAMP-2A overexpression vector, GV141-HSPA8, for 72 h, immunoblot was performed with the indicated antibodies. (B, D, F, H) HEK293 cells were transiently transfected with LAMP-2A siRNA or LAMP-2A overexpression vector, GV141-LAMP-2A, for 72 h, immunoblot was performed with the indicated antibodies. *, po0.05; **, po0.01; ***, and po0.001.

Fig. 5 – Identification of CMA-targeted motif in AF1Q protein. (A, B) HeLa cells were co-transfected with an 80–90 aa deletion AF1Q expression plasmid (AF1Q80) or the full-length plasmid (AF1Q90) and LAMP-2A siRNA for 72 h, immunoblot was performed with the indicated antibodies. ***, and po0.001.

lysosomes for degradation via CMA and CMA itself is also activated during oxidative stress [28]. AF1Q was decreased when cells were treated with 6-AN. Starvation, which increased the degradation of AF1Q as mentioned above, could activate both CMA and macroautophagy [23]. After serum removal, activation of macroautophagy occurred before the activation of CMA reaches maximal activity of 4–6 h after starvation, and then gradually declined to basal levels [29]. Prolonged starvation can activate CMA activity and continue as long as starvation persists [30]. Here, we confirm that the time-course of induction of Beclin 1 and LAMP-2A was consistent with the activation of macroautophagy and CMA with

different time frames as previous. Our study found that starvation for 24 h markedly reduced the levels of AF1Q but not during the first 6 or 12 h. These data suggest that CMA may be involved in degradation of AF1Q, but not macroautophagy. In macroautophagy, autophagosome completion is mediated by 2 ubiquitin-like conjugation systems: the ATG12/ATG5 and LC3-PE (phosphatidylethanolamine) conjugation systems [31]. It is known that LC3-PE process is mediated by ATG7 [31]. To explore whether macroautophagy participated in the AF1Q degradation, we analyzed AF1Q levels by regulating macroautophagy-related molecules. Inhibition of macroautophagy (ATG7 or ATG5 down-expression) did not


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prevent AF1Q degradation, whereas activation of macroautophagy (ATG5 or ATG7 overexpression) prevented degradation of this protein. Along with the fact that AF1Q was not increased upon 3-MA treatment, the results from pharmacological and genetic approaches indicated that macroautophagy did not contribute to the degradation of AF1Q. It has been reported that blockage of macroautophagy leads to constitutive activation of CMA [32], thus we further confirmed that CMA participated in AF1Q degradation. HSPA8 is a CMA chaperone which preferentially interacts with CMA substrate proteins and delivers proteins moving into lysosome [18]. LAMP-2A is a transmembrane receptor protein that can directly import CMA substrate proteins across the lysosomal membranes, and then translocate into the lysosomal lumen and degraded [19]. To investigate the role of CMA in degradation of AF1Q, we examined the relation between two core components of the CMA machinery, HSPA8 and LAMP-2A, and AF1Q. Immunofluorescence microscopy data indicated that AF1Q co-localized with HSPA8 and LAMP-2A. Co-IP results confirmed that AF1Q could interact with HSPA8 and LAMP-2A. Moreover, inhibition of CMA (HSPA8 or LAMP-2A down-expression) increased AF1Q levels, whereas activation of CMA (HSPA8 or LAMP-2A overexpression) decreased AF1Q levels. These data suggest that AF1Q is degraded by an HSPA8 and LAMP-2A dependent mechanism through CMA. However, we also found that AF1Q co-localized with macroautophagy marker LC3. Previous research revealed that both LC3 and AF1Q are co-localized with γ-tubulin [2,33], indicating that these two proteins may be co-localized in the centrosomal area for some unknown functions. CMA pathway requires the presence of a cis-acting pentapeptide KFERQ-like motif that targets the protein to the lysosome [17]. This motif is recognized by the chaperone HSPA8, which interacts with the substrate proteins in the cytosol [17]. In silico analysis of the AF1Q amino acids sequence revealed six motifs (84–88 aa of AF1Q) related to KFERQ in the protein, supporting AF1Q as a potential target for CMA-dependent proteolysis. Furthermore, a CMA recognition motif deletion construct (an 80–90 aa deletion, AF1Q80) was achieved. The results showed that deletion of amino acids in 80–90 reduced clearance of AF1Q. These data suggest that a KFERQ-like motif located in the range of 80–90 aa is a functional site for recognition by HSPA8. The present study identified a functional putative KFERQ-like motif in AF1Q that bound AF1Q to HSPA8 and allowed uptake by lysosomes via LAMP-2A. In summary, this study provides evidence that AF1Q is degraded in lysosomes via CMA. It is important to confirm the involvement of the CMA pathway in AF1Q clearance, as this pathway is relatively selective for its substrates. Thus, CMA disorder may contribute to AF1Q related disease such as leukemia. We propose that the role of CMA in leukemia should be researched in future.

Acknowledgments This work was supported by National Nature Science Foundation of China (81070422, 30871088, and 81200377), Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP, Ministry of Education) (20100131110060) and the Independent Innovation Fund of Shandong University (yzc12161). This work was also partly supported by National Qianren Scholar Program Special Funding of

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Shandong University and Taishan Scholar Special Funding of Shandong Province.

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Autophagy promotes ferroptosis by degradation of ferritin.

Degradation of plant peroxisomes by autophagy.

Hif-2α promotes degradation of mammalian peroxisomes by selective autophagy.

Dissection of ubiquitinated protein degradation by basal autophagy.

Bulk RNA degradation by nitrogen starvation-induced autophagy in yeast.

Autophagy and other vacuolar protein degradation mechanisms.

Autophagy in sepsis: Degradation into exhaustion?

mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy.

Disruption of chaperone-mediated autophagy-dependent degradation of MEF2A by oxidative stress-induced lysosome destabilization.

Essential control of mitochondrial morphology and function by chaperone-mediated autophagy through degradation of PARK7.

RAD6 promotes homologous recombination repair by activating the autophagy-mediated degradation of heterochromatin protein HP1.

G-protein-coupled receptors regulate autophagy by ZBTB16-mediated ubiquitination and proteasomal degradation of Atg14L.

Degradation of misfolded proteins by autophagy: is it a strategy for Huntington's disease treatment?

Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death.

Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage.

Autophagy regulates spermatid differentiation via degradation of PDLIM1.

Glucocorticoid impairs cell-cell communication by autophagy-mediated degradation of connexin 43 in osteocytes.

Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis.

The progression of peroxisomal degradation through autophagy requires peroxisomal division.

Metformin increases degradation of phospholamban via autophagy in cardiomyocytes.

MLLT11 in the progression of ovarian cancer.

AF1q is differentially expressed in maturing neurons during development.

AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation.

AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation.