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. 2010 Jan 1;24(1):72-85.
doi: 10.1101/gad.1843810.

A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP)

Bin Zhao  1 , Li Li, Karen Tumaneng, Cun-Yu Wang, Kun-Liang Guan
Affiliations

A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP)

Bin Zhao et al. Genes Dev. .

Abstract

The Yes-associated protein (YAP) transcription coactivator is a key regulator of organ size and a candidate human oncogene. YAP is inhibited by the Hippo pathway kinase cascade, at least in part via phosphorylation of Ser 127, which results in YAP 14-3-3 binding and cytoplasmic retention. Here we report that YAP is phosphorylated by Lats on all of the five consensus HXRXXS motifs. Phosphorylation of Ser 381 in one of them primes YAP for subsequent phosphorylation by CK1delta/epsilon in a phosphodegron. The phosphorylated phosphodegron then recruits the SCF(beta-TRCP) E3 ubiquitin ligase, which catalyzes YAP ubiquitination, ultimately leading to YAP degradation. The phosphodegron-mediated degradation and the Ser 127 phosphorylation-dependent translocation coordinately suppress YAP oncogenic activity. Our study identified CK1delta/epsilon as new regulators of YAP and uncovered an intricate mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation (nuclear-cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation).

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Figures

Figure 1.
Figure 1.
Phosphorylation of S127 and S381 inhibits YAP oncogenic activity. (A) YAP is phosphorylated in vivo on all five Lats phosphorylation consensus sites (S61, S109, S127, S164, and S381). YAP expressed in MCF10A cells was tandem affinity-purified and analyzed by LC-MS/MS. Identified YAP phosphopeptides were shown. Phosphorylated residues are highlighted in bold. (M′) Methionine oxidation. In the phosphopeptide containing S163 and S164, the mass spectronomy data could not determine which one of them is phosphorylated. However, S164 phosphorylation was confirmed by later experiments and therefore is highlighted here. (B) Mutations of S61, S109, S127, and S164 to alanine increase YAP electrophoretic mobility. YAP wild type and mutants were expressed in HEK293 cells with or without Mst and Lats cotransfection as indicated. Cell lysates were resolved on SDS-PAGE gels containing 25 mM Phos tag-conjugated acrylamide followed by standard Western blotting with anti-Flag antibody. (C) Restoration of Ser 127 or Ser 381 is sufficient to inhibit YAP-5SA transformation activity. NIH-3T3 cell colony formation assays were performed using vector control or indicated YAP constructs. Colonies were visualized with crystal violet staining 2 wk after transfection. Expression of YAP wild type or mutants 24 h after transfection was shown by Western blot (Supplemental Fig. S1B). (D) Mutation of both Ser 127 and Ser 381 is required to activate YAP transforming activity. Experiments were similar to those in C with indicated YAP constructs. Expression of YAP wild type or mutants 24 h after transfection was shown by Western blot (Supplemental Fig. S1C). (E) Transformation of MCF10A cells by YAP-S127/381A and YAP-5SA. Indicated MCF10A stable cells were cultured in soft agar for 16 d before pictures were taken. Colonies were then visualized by crystal violet staining and colonies observable by the naked eye were counted and are shown in the right panel.
Figure 2.
Figure 2.
Mutation of Ser 381 increases YAP protein stability. (A) Mutation of S381 does not affect YAP subcellular localization. YAP wild type or mutants were expressed in HeLa cells without (left panels) or with (right panels) Mst2/ Lats2 cotransfection. YAP localization was visualized by immunofluorescence staining with anti-Flag antibody (red). HA-Mst2 and HA-Lats2 were shown by anti-HA staining (green). DNA was stained with DAPI (blue). (B) YAP protein level decreases with increasing cell density. NIH-3T3 and mouse embryonic fibroblast cells were cultured at different densities from sparse to confluent. Endogenous YAP protein levels were determined by Western blot with anti-tubulin as a loading control. Relative YAP protein levels were quantified by the ratio between YAP and tubulin, which was arbitrarily set to 1 at the lowest cell density. (C) Effect of cell density on YAP phosphorylation. NIH-3T3 cells were cultured at different densities from sparse to confluent. Endogenous YAP protein levels and YAP-S381 and YAP-S127 phosphorylations were determined by Western blot with specific antibodies. Anti-tubulin was used as a loading control. Relative YAP phosphorylation levels were quantified by the ratio between pYAP and total YAP protein, which were arbitrarily set to 1 at the lowest cell density. (D) YAP is unstable in high-density cell culture. NIH-3T3 cells were seeded at 1 × 106 per 10-cm dish (low density) or 3 × 106 per well (high density) in six-well plates and cultured for 24 h. Protein synthesis was blocked by treatment with 50 μg/mL CHX for the indicated time. Relative YAP protein levels were quantified by YAP/tubulin ratio, which was arbitrarily set to 1 at the 0 time point. When the same samples were resolved for a longer time, YAP showed a shift as we reported previously (Supplemental Fig. S1F) that was eliminated by λ phosphatase treatment (Supplemental Fig. S1G). (E) YAP is ubiquitinated at high cell density. NIH-3T3 cells cultured at low or high density were treated with 25 μM MG132 for 5 h before harvest. Endogenous YAP was immunoprecipitated and Western blot was done with anti-YAP or anti-Ub antibodies. (S) Shorter exposure; (L) longer exposure. (F) Mutation of S381 to alanine stabilizes YAP. NIH-3T3 cells were transfected with indicated YAP constructs. After 24 h, cells were plated into six-well plates and cultured to confluence. Protein synthesis was blocked by treatment with 50 μg/mL CHX for the indicated time. Transfected YAP protein levels were shown by anti-Flag Western blot, and anti-tubulin Western blot was used as a loading control. Relative YAP protein levels were quantified by the ratio between Flag-YAP and tubulin, which was arbitrarily set to 1 at the 0 time point. The dot in the last lane of Flag-YAP-S127A Western blot, as indicated by the asterisk, was due to an artifact on the membrane, and this lane is therefore not quantified.
Figure 3.
Figure 3.
Ser 381 and the phosphodegron are required for YAP–β-TRCP interaction. (A) Alignment of YAP sequence around S381 with the β-catenin phosphodegron sequence. The phosphodegron is underlined. Positions of phosphorylation residues are labeled. Kinases responsible (or hypothesized to be) for phosphorylation of each residue are shown. Dashed arrows indicate priming relationship. (B) YAP and β-TRCP coimmunoprecipitated with each other. Indicated plasmids were transfected into HEK293 cells. YAP or β-TRCP was immunoprecipitated with anti-HA or Flag antibody. Western blots were done to detect specific proteins as indicated on the left sides of each panel. (IP) Immunoprecipitation. (C) S381 and S384 in YAP are required for β-TRCP interaction. Indicated plasmids were transfected into HEK293 cells. β-TRCP was immunoprecipitated with anti-Flag antibody. Coimmunoprecipitated YAP was shown by anti-HA Western blot. (S) Shorter exposure; (L) longer exposure. (D) S387 is important for YAP–β-TRCP interaction. Experiments were similar to those in C. (E) D383 and G385 in the phosphodegron are required for YAP–β-TRCP interaction. Experiments were similar to those in C.
Figure 4.
Figure 4.
The Hippo pathway induces YAP S381 phosphorylation and YAP–β-TRCP interaction. (A) pYAP (S381/384) antibody preferentially recognizes S381/384-phosphorylated YAP. YAP wild type or mutants were coexpressed with Mst2 and Lats2 in HEK293 cells. Flag-YAP was then immunoprecipitated and one set of the immunoprecipitates was treated with λ protein phosphatase as indicated. The immunoprecipitated YAP phosphorylation and protein levels were then detected by anti-pYAP (S381/384) antibody and anti-Flag antibody, respectively. (B) Mst2 and Lats2 induce YAP-S381/384 phosphorylation. Indicated plasmids were cotransfected into HEK293 cells. (KR)Kinase-inactive mutants. YAP phosphorylation was shown by a pYAP (S381/384)-specific antibody. (C) Lats knockdown significantly attenuates YAP-S381/384 phosphorylation. HA-YAP was expressed in HEK293 cells. Lats1 and Lats2 knockdown was achieved by two rounds of siRNA transfection with 24-h intervals. YAP phosphorylation was shown by a pYAP (S381/384)-specific antibody. Anti-HA Western blot indicates YAP protein level. Lats knockdown efficiency was shown by an antibody against both Lats1 and Lats2. (D) Mst2 and Lats2 stimulate YAP–β-TRCP interaction. Indicated plasmids were transfected into HEK293 cells. β-TRCP was immunoprecipitated with anti-Flag antibody, and coimmunoprecipitated YAP was shown by anti-HA Western blot. (E) Dominant-negative Mst2 and Lats2 inhibit YAP–β-TRCP interaction. Indicated plasmids were transfected into HEK293 cells. (KR) Kinase-inactive mutants. YAP was immunoprecipitated with anti-Flag antibody, and coimmunoprecipitated β-TRCP was shown by anti-HA Western blot. (F) Lats knockdown inhibits YAP–β-TRCP interaction. Indicated plasmids were cotransfected with or without Lats1/2 siRNA. Lats1/2 siRNA transfection was repeated once after 24 h. β-TRCP was immunoprecipitated with anti-Flag antibody, and coimmunoprecipitated YAP was shown by anti-HA Western blot. Lats knockdown efficiency was shown by an antibody against both Lats1 and Lats2.
Figure 5.
Figure 5.
YAP-S381 phosphorylation primes it for S384 phosphorylation by CK1δ/ɛ and stimulates β-TRCP binding. (A) YAP phosphorylation is inhibited by CK1δ/ɛ inhibitor. HA-YAP was transfected into HEK293 cells. Cells were treated with CK1δ/ɛ inhibitor IC261, and phosphorylation of S381 and S384 was detected in Western blot by a phosphospecific antibody. (B) CK1ɛ increases YAP-S384 phosphorylation in a kinase activity-dependent manner. HEK293 cells were transfected and Western blot was done as indicated. (KR) Kinase-inactive form of CK1ɛ. Quantification of the ratio between pYAP (S381/384) and Flag-YAP showed a 40% increase of the phosphorylation by coexpression of CK1ɛ. (C) In vitro phosphorylation of YAP by CK1ɛ requires S381 priming phosphorylation by Lats. Flag-Lats2 and HA-CK1ɛ were expressed in HEK293 cells separately and were immunoprecipitated with resin conjugated with anti-Flag or anti-HA antibody. GST-YAP substrates were expressed and purified from Escherichia coli and were first subjected to Lats kinase assay with cold ATP as indicated. Aliquots of the reaction mixture supernatant (without the resin-bound Lats kinase) were taken to the second-step kinase assay with immunoprecipitated HA-CK1ɛ and 32P-ATP. Resin after the first-step reaction was prepared for Western blot for Lats protein level. All autoradiography and other Western blots were from samples after the second-step reaction. (D) CK1δ/ɛ inhibitor dose-dependently inhibited the interaction between YAP and β-TRCP. HA-YAP and Flag-β-TRCP were expressed in HEK293 cells as indicated. Cells were treated with the indicated concentration of IC261 for 1 h before harvest. Flag-β-TRCP was immunoprecipitated, and coimmunoprecipitated YAP was shown by anti-HA Western blot. (E) CK1δ/ɛ increases YAP–β-TRCP interaction in a kinase activity-dependent manner. HEK293 cells were transfected with indicated plasmids and were treated with IC261 as indicated. Flag-β-TRCP was immunoprecipitated, and coimmunoprecipitated YAP was shown by anti-HA Western blot.
Figure 6.
Figure 6.
SCF, Mst/Lats, and CK1δ/ɛ promote YAP ubiquitination through S381 and phosphodegron phosphorylation. (A) The Hippo pathway components and SCF stimulate YAP ubiquitination. Flag-YAP, Myc-Ub, the Hippo pathway components Mst2 and Lats2, and the SCF complex components β-TRCP, SKP1, and CUL1 were cotransfected into HEK293 cells as indicated. Cells were treated with MG132 as indicated before harvest. Flag-YAP was immunoprecipitated, and Western blots were done with specific antibodies. (L) longer exposure for Flag-YAP; (S) shorter exposure for Flag-YAP. (B) YAP ubiquitination is dependent on S381, 384, and 387 phosphorylation and on intact phosphodegron. YAP wild type and mutants were cotransfected with other plasmids into HEK293 cells as indicated. Cells were treated with 25 μM MG132 for 5 h before harvest. Flag-YAP was immunoprecipitated, and Western blots were done with indicated antibodies. (C) YAP ubiquitination is inhibited by the CK1δ/ɛ inhibitor IC261. HEK293 cells were transfected as indicated. Cells were pretreated with 10 μM IC261 for 1 h and then treated with MG132 as indicated before harvest. Flag-YAP was immunoprecipitated, and Western blots were done with indicated antibodies.
Figure 7.
Figure 7.
Inhibition of YAP oncogenic transformation ability through SCFβ-TRCP-mediated YAP degradation depends on the Hippo pathway, CK1δ/ɛ, and the phosphodegron. (A) YAP degradation is inhibited by dominant-negative Cul1. Flag-YAP was transfected into NIH-3T3 cells at low expression level alone (left panels) or together with dominant-negative Cul1 (right panels), and cells were cultured to confluence. Cells were treated with 50 μg/mL CHX for the indicated time. Anti-Flag Western blot was used to show YAP protein levels and anti-tubulin Western blot was used as a loading control. (B) The Hippo pathway and CK1ɛ destabilize YAP in a S381-dependent manner. Flag-YAP wild type or S127/381A mutant were transfected alone or together with the Hippo pathway components Mst2/Lats2, or CK1ɛ into NIH-3T3 cells. Cells were cultured at low density so that most cells do not have contact with each other. CHX chase and Western blots were done as in A. (C) Mutation of S384 or D383 in the phosphodegron stabilizes YAP. YAP wild type or mutants were transfected into NIH-3T3 cells. Cells were cultured to confluence, and CHX chase and Western blots were done as in A. (D). Regulation of YAP stability by proteasome is dependent on functional Hippo pathway, as well as S381 and the phosphodegron of YAP. Flag-YAP wild type or mutants were transfected alone or together with kinase-inactive Mst2-KR and Lats2-KR as indicated. Cells were cultured to confluence and were treated with 25 μM MG132 for the indicated time before harvest. Western blots were done as in A. (E) Mutation of the phosphodegron together with S127 is sufficient for YAP to transform NIH-3T3 cells. NIH-3T3 cell colony formation assays were performed using indicated YAP constructs. Colonies were visualized with crystal violet staining.
Figure 8.
Figure 8.
A model of YAP inhibition by the Hippo pathway and CK1. When the Hippo pathway is activated, YAP is phosphorylated on both S127 and S381 in the HXRXXS motifs. Phosphorylation of S127 results in 14–3–3 binding and cytoplasmic retention of YAP. Therefore, YAP can be inhibited by spatial separation from its nuclear target transcription factors, such as TEAD. This mechanism of regulation is reversible. Phosphorylation of S381 primes YAP phosphorylation by CK1δ/ɛ, resulting in activation of a phosphodegron, which then recruits the SCFβ-TRCP E3 ubiquitin ligase, leading to YAP degradation. This mechanism of regulation is irreversible. Through these two mechanisms, YAP activity is under both spatial and temporal control, which coordinately inhibit YAP oncogenic activity.
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