PSME4 Degrades Acetylated YAP1 in the Nucleus of Mesenchymal Stem Cells

Figures

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  Figure 1. Apicidin prompts nuclear localization and subsequent nuclear degradation of YAP1. (a) Apicidin initiates acute loss of YAP1 as early as 6 h after treatment. (b) Overnight treatment with 3 μM apicidin (Api) in hTERT-MSCs (human mesenchymal stem cells immortalized by human telomerase reverse transcriptase) robustly reduced the YAP1 protein level while a notable amount of YAP1 remained after treatment with cycloheximide (CHX, 20 μg/mL) or actinomycin D (ActD, 10 μg/mL), which indicates that apicidin actively accelerates YAP1 clearance. (c) Apicidin reduced endogenous YAP1 even in the presence of chloroquine (CQ, 10 μM). The clearance of YAP1 was not linked to the lysosomal pathway. (d) YAP1 underwent proteasomal degradation predominantly upon apicidin treatment. The proteasome inhibitor MG132 (25 μM, 4 h) significantly attenuated acute loss of YAP1 by apicidin treatment. (e) Acute degradation of YAP1 by apicidin stimuli occurred in the nucleus. Overnight incubation with apicidin reduced YAP1 protein in both cytoplasm (first vs. second lane) and nucleus (fifth vs. sixth lane). MG132 predominantly preserved YAP1 degradation in the nucleus (seventh lane), which suggested that the…

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  Figure 2. Serine 127 phosphorylation fails to restrict YAP1 in the cytoplasm. (a) Phosphor-S127 YAP1 localized both in the cytoplasm and in the nucleus in hTERT-MSCs. Phos-tag phosphoprotein gel stain greatly exaggerated retardation of phosphorylated proteins in the SDS-PAGE. Proteins were separated by various grades of phosphorylation (bottom). Phosphor-specific antisera against S127 phosphor-YAP1 successfully probed nuclear YAP1 accumulated by MG132 treatment. The specificity of phosphor-S127 antibody was confirmed by the first lane, which was incubated with lambda phosphatase; phosphor-S127 antibodies did not detect anything (top). (b) Apicidin forced nuclear localization of YAP1 in the presence of S127 phosphorylation. Phosphor-S127 YAP1 antibody visualized nuclear YAP1. Abbreviations: Api, apicidin; λPPase, lambda phosphatase.

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  Figure 3. Acetylation governs YAP1 shuttling into the nucleus. (a) Apicidin induced acetylation on YAP1. Short-term (2 h) incubation of YAP1 augmented the acetylation level of YAP1 without alteration of YAP1 amounts, which allowed dissociation of YAP1 from the 14-3-3 molecule that tethered YAP1 in the cytoplasm. (b) YAP1 acetylation in response to 2 h of treatment with apicidin took place in the cytoplasm, but most acetyl-YAP1 was enriched in the nucleus. HDAC2 shows the nuclear fraction, whereas GAPDH represents cytoplasmic components. (c) Cytosolic HDAC(s) might participate in YAP1 acetylation. Tubastatin A phenocopied apicidin-dependent YAP1 degradation. Overnight treatment with Tubastatin A successfully reduced endogenous YAP1 as strongly as did treatment with apicidin, while nicotinamide failed to do so. Acetyl-tubulin delineated cytosol HDAC inhibition. Note that apicidin also induced tubulin acetylation slightly. (a,e) HDAC6 regulated YAP1 acetylation. Three days of knocking down with siRNA against HDAC6 significantly reduced YAP1 in the hTERT-MSCs (d), while 1 day of incubation after siRNA transfection accelerated nuclear localization of YAP1 (fourth lane) (e). HDAC2 depicts the…

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  Figure 4. Lysine 494/497 are acetylation targets of YAP1. (a) Acetylation of YAP1 was not detected when two lysines, 494 and 497, were mutated (2KA). Output with histone H3 after immunoprecipitation with acetyl-lysine was used for internal control. Acetyl-histone H3 delineated intracellular effect of apicidin treatment. (b,c) Acetylation facilitated nuclear localization. Enhanced green fluorescent protein (EGFP) was fused at the C terminus of YAP1 to visualize location of YAP1. Wildtype YAP1-EGFP started to accumulate in the nucleus as early as 1 h after treatment with apicidin, which was greatly increased after 5 h. Nuclear targeting of the acetyl-dead form of YAP1 was significantly lower than that of wild-type. HPF, high-power field. (d) Acetyl-dead mutant of YAP1 remained intact even in the presence of apicidin. Simultaneous treatment with apicidin and cycloheximide failed to degrade acetylation-resistant mutant of YAP1, while wildtype YAP1 was cleared. The gray circle indicates vehicle (or tet-off) condition, and the red rhombus denotes doxycycline (or tet-on) treatment. (c) Two-way ANOVA and Tukey’s HSD multiple comparison were performed. ** p < 0.01, *** p < 0.001.

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  Figure 5. PSME4 degrades YAP1 in a polyubiquitination-independent manner. (a) Proteasome activator subunit 4 (PSME4) participates in YAP1 degradation in apicidin-treated cells. siRNA against F-box protein (FBXW) 7 or suppressor of cytokine signaling (SOCS) 6 failed to preserve acute loss of YAP1. A notable amount of YAP1 was visualized when PSME4 was targeted. (b) Apicidin did not induce YAP1 polyubiquitination. MG132 (25 μM, 4 h) successfully mitigated YAP1 degradation but failed to induce YAP1 polyubiquitination. (c) PSME4 shRNA attenuated YAP1 degradation (seventh vs. eighth lane). (d) Preserved YAP1 mediated by PSME4 knockdown mainly accumulated in the nucleus (sixth vs. eighth lane). Gray circle: vehicle. Red square: apicidin. Two-way ANOVA with Tukey’s HSD post hoc test. Abbreviations: Ctrl, control. DOX, doxycycline. Ub, ubiquitin.

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  Figure 6. PSME4 governs MSC differentiation via YAP1. (a) YAP1 protein was more abundant in primary cultures of MSCs from PSME4-null individuals compared with wildtype littermates. (b) Aberrant transcription of YAP1. YAP1 mRNA was significantly lowered in PSME4 knockout (KO) bone marrow (BM) MSCs. (c) Survival rate after direct injection of MSCs either from PSME4 KO or wildtype littermate was measured for 2 weeks. When MSCs from PSME4 KO mice were injected, survival was paradoxically decreased. Statistical significance was obtained by applying Mantel–Cox log-rank test. Cohort size: PBS n = 13, MSCs from WT littermate n = 15, MSCs from PSME4 KO n = 10. (d,e) Cardiac function, ejection fraction (d), and fractional shortening (e) were not improved in rtTA-shPSME4 MSCs delivered group. Cohort size: PBS n = 11, MSCs/rtTA shControl n = 13, MSCs/rtTA shPSME4 n = 14. (f–h) Cardiac remodeling was not impaired. Cardiac fibrosis (g,h) and wall thickness after ischemic insults (h) were not improved in the group with PSME4-knockdown hTERT-MSCs injection. Gray rhombus: PBS injection group. Blue circle: rtTA-shControl hTERT-MSCs injection group. Red square: rtTA-shPSME4 hTERT-MSCs delivered group.…

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  Figure 7. Working hypothesis. Hippo pathway in the resting MSC: LATS1/2 phosphorylated YAP1 S127. 14-3-3 recognizes phosphor-YAP1 and restricts YAP1 in the cytoplasm. HDAC6 indirectly interferes with nuclear translocalization of YAP1 (left). Acute regulation of YAP1 occurs under apicidin treatment. Apicidin blocks cytosolic HDAC6 and facilitates acetylation indirectly. Apicidin transactivates p21, which induces transcriptional arrest of YAP1. Acetylation of YAP1 dissociates YAP1 from 14-3-3 regardless of phosphorylation status. Acetylated-YAP1 is shuttled into the nucleus after being released from the cytosolic tethering molecule, 14-3-3. Nuclear YAP1 initiates target gene expression and recognition by PSME4, a subunit of the nuclear proteasome. PSME4 degrades acetyl-YAP1 in the nucleus (right).