cAMP/PKA signalling reinforces the LATS–YAP pathway to fully suppress YAP in response to actin cytoskeletal changes
2013; Springer Nature; Volume: 32; Issue: 11 Linguagem: Inglês
10.1038/emboj.2013.102
ISSN1460-2075
AutoresMinchul Kim, Mi‐Ju Kim, Seung‐Hee Lee, Shinji Kuninaka, Hideyuki Saya, Ho Lee, Sookyung Lee, Dae‐Sik Lim,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoArticle3 May 2013free access cAMP/PKA signalling reinforces the LATS–YAP pathway to fully suppress YAP in response to actin cytoskeletal changes Minchul Kim Minchul Kim National Creative Research Initiatives Center, Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea Search for more papers by this author Miju Kim Miju Kim National Creative Research Initiatives Center, Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea Search for more papers by this author Seunghee Lee Seunghee Lee Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, Republic of Korea Neuroscience Section, Department of Pediatrics, Papé Family Pediatric Research Institute, and Vollum Institute, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Shinji Kuninaka Shinji Kuninaka Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo, Japan Search for more papers by this author Hideyuki Saya Hideyuki Saya Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo, Japan Search for more papers by this author Ho Lee Ho Lee Department of Convergence Technology, National Cancer Center, Goyang, Republic of Korea Search for more papers by this author Sookyung Lee Sookyung Lee Neuroscience Section, Department of Pediatrics, Papé Family Pediatric Research Institute, and Vollum Institute, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Dae-Sik Lim Corresponding Author Dae-Sik Lim National Creative Research Initiatives Center, Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea Search for more papers by this author Minchul Kim Minchul Kim National Creative Research Initiatives Center, Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea Search for more papers by this author Miju Kim Miju Kim National Creative Research Initiatives Center, Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea Search for more papers by this author Seunghee Lee Seunghee Lee Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, Republic of Korea Neuroscience Section, Department of Pediatrics, Papé Family Pediatric Research Institute, and Vollum Institute, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Shinji Kuninaka Shinji Kuninaka Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo, Japan Search for more papers by this author Hideyuki Saya Hideyuki Saya Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo, Japan Search for more papers by this author Ho Lee Ho Lee Department of Convergence Technology, National Cancer Center, Goyang, Republic of Korea Search for more papers by this author Sookyung Lee Sookyung Lee Neuroscience Section, Department of Pediatrics, Papé Family Pediatric Research Institute, and Vollum Institute, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Dae-Sik Lim Corresponding Author Dae-Sik Lim National Creative Research Initiatives Center, Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea Search for more papers by this author Author Information Minchul Kim1, Miju Kim1, Seunghee Lee2,3, Shinji Kuninaka4, Hideyuki Saya4, Ho Lee5, Sookyung Lee3 and Dae-Sik Lim 1 1National Creative Research Initiatives Center, Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea 2Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, Republic of Korea 3Neuroscience Section, Department of Pediatrics, Papé Family Pediatric Research Institute, and Vollum Institute, Oregon Health and Science University, Portland, OR, USA 4Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo, Japan 5Department of Convergence Technology, National Cancer Center, Goyang, Republic of Korea *Corresponding author. National Creative Research Initiatives Center, Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-D. Yuseong-Gu, Daejeon 305-701, Republic of Korea. Tel.:+82 42 350 2635; Fax:+82 42 350 2610; E-mail: [email protected] The EMBO Journal (2013)32:1543-1555https://doi.org/10.1038/emboj.2013.102 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Actin cytoskeletal damage induces inactivation of the oncoprotein YAP (Yes-associated protein). It is known that the serine/threonine kinase LATS (large tumour suppressor) inactivates YAP by phosphorylating its Ser127 and Ser381 residues. However, the events downstream of actin cytoskeletal changes that are involved in the regulation of the LATS–YAP pathway and the mechanism by which LATS differentially phosphorylates YAP on Ser127 and Ser381 in vivo have remained elusive. Here, we show that cyclic AMP (cAMP)-dependent protein kinase (PKA) phosphorylates LATS and thereby enhances its activity sufficiently to phosphorylate YAP on Ser381. We also found that PKA activity is involved in all contexts previously reported to trigger the LATS–YAP pathway, including actin cytoskeletal damage, G-protein-coupled receptor activation, and engagement of the Hippo pathway. Inhibition of PKA and overexpression of YAP cooperate to transform normal cells and amplify neural progenitor pools in developing chick embryos. We also implicate neurofibromin 2 as an AKAP (A-kinase-anchoring protein) scaffold protein that facilitates the function of the cAMP/PKA–LATS–YAP pathway. Our study thus incorporates PKA as novel component of the Hippo pathway. Introduction The YAP (Yes-associated protein) transcriptional co-activator is a potent oncogene that drives cell proliferation and promotes survival. Overexpression of Yorkie, the Drosophila homologue of YAP, triggers massive overgrowth of fly imaginal discs (Huang et al, 2005). Similarly, transgenic mice overexpressing YAP rapidly develop tumours in multiple organs (Camargo et al, 2007; Dong et al, 2007). In humans, YAP is overexpressed as a result of genomic amplification of the 11q22 locus in several cancer types (Weber et al, 1996; Imoto et al, 2001, 2002; Dai et al, 2003; Baldwin et al, 2005; Bashyam et al, 2005; Hermsen et al, 2005; Snijders et al, 2005). YAP might also accumulate in human cancers through genomic amplification-independent mechanisms (Zhao et al, 2007). Moreover, YAP expression is able to transform normal human mammary epithelial cells in culture (Overholtzer et al, 2006). These studies establish the evolutionarily conserved role of YAP as an oncogene and underscore the importance of understanding how YAP's activity is regulated. The Hippo pathway has been implicated as the major negative regulator of YAP (Harvey and Tapon, 2007; Pan, 2010; Sudol and Harvey, 2010; Zhao et al, 2010a; Halder and Johnson, 2011). The core of this pathway consists of two sterile 20-like protein kinases, MST1 and MST2 (also known as STK4 and STK3, respectively), and the scaffolding protein SAV1 (salvador homologue 1). Together with SAV1, MST1/2 activate LATS (large tumour suppressor) kinases 1 and 2 by phosphorylating residues in their hydrophobic motif (HM) (Chan et al, 2005). Subsequently, with the help of Mob1A/B (mob kinase activator 1 A/B) scaffold proteins, activated LATS1/2 kinases phosphorylate and thereby inactivate YAP. Importantly, mice lacking core Hippo pathway genes have increased YAP activity and develop cancers in various epithelial tissues. For example, mice with specific deletion of Mst1/2 or Sav1 in the liver exhibit hepatomegaly followed by rapid progression to liver cancer (Zhou et al, 2009; Lee et al, 2010; Lu et al, 2010; Song et al, 2010). Likewise, deletion of Mst1/2 in the intestinal epithelium triggers colon cancer development, a phenotype that is rescued by YAP knockout (Zhou et al, 2011). Although mice deficient for intestinal Sav1 do not progress to spontaneous cancer development, they are susceptible to chronic damage-induced tumorigenesis; this phenotype is also abolished by YAP deletion (Cai et al, 2010). These studies highlight the importance of the Hippo pathway as a suppressor of YAP. In terms of events upstream of MST1/2 kinase, mounting evidence suggests that epithelial cell adhesion and polarity initiates the signalling event (Grusche et al, 2010). At the molecular level, a number of proteins, including neurofibromin 2 (NF2), Angiomotin and Kibra, have been implicated as potential upstream regulators of MST1/2 kinase (Hamaratoglu et al, 2006; Baumgartner et al, 2010; Genevet et al, 2010; Yu et al, 2010; Chan et al, 2011a, 2011b; Paramasivam et al, 2011; Wang et al, 2011; Zhao et al, 2011). However, the exact nature of the upstream event responsible for MST1/2 activation remains elusive. LATS1/2 can potentially phosphorylate five residues in YAP that follow the LATS consensus, HxRxxS/T (Zhao et al, 2007; Lee et al, 2008). Of these five sites, two—Ser127 and Ser381—seem to be the most critical for YAP inactivation since retention of phosphorylation at either of these two serines is sufficient to abolish the transforming ability of the YAP 5SA mutant (Zhao et al, 2009). Mechanistically, Ser127 phosphorylation mediates interaction with 14-3-3 proteins, which sequester YAP in the cytosol (Dong et al, 2007; Zhao et al, 2007). On the other hand, Ser381 phosphorylation triggers successive phosphorylation on Ser384 by casein kinase-1 followed by BTRC (beta-transducin repeat-containing E3 ubiquitin protein ligase)-mediated degradation (Zhao et al, 2010b). However, in cell-free systems Ser127 serves as the primary and preferred substrate for LATS, whereas phosphorylation of Ser381 is a minor reaction (Zhao et al, 2007; Lee et al, 2008). This raises the important question of whether and how these two phosphorylations are differentially regulated in intact cells. A recent report revealed a novel upstream regulator of YAP (Halder et al, 2012), demonstrating that, when stress fibres develop tension, YAP is imported into the nucleus and thus activated. Conversely, YAP is exported from the nucleus when stress fibres loose tension. Similarly, actin cytoskeletal damages, such as latrunculin B, cytochalasin D, or maintenance of cells in suspension, inactivates YAP (Dupont et al, 2011; Fernandez et al, 2011; Sansores-Garcia et al, 2011; Wada et al, 2011; Zhao et al, 2012). However, unlike the aforementioned canonical Hippo pathway, signalling events downstream of the actin cytoskeleton are poorly understood. Moreover, reports pertaining to the requirement of canonical Hippo pathway components in this context have been conflicting, possibly reflecting the use of siRNAs or dominant-negative approaches instead of genetically deficient cell lines. Moreover, although these studies commonly show that localization and Ser127 phosphorylation of YAP are affected by cytoskeletal damage, whether and how Ser381 participates in this signalling and which downstream components are actually involved have not been elucidated. In this study, we investigated these issues by taking advantage of genetically engineered mouse embryonic fibroblasts (MEFs) for each Hippo pathway component and a YAP phospho-Ser381-specific antibody that we generated. Our study reveals an unexpected role of cyclic AMP (cAMP)-dependent protein kinase (PKA) in promoting LATS-mediated Ser381 phosphorylation, and thus full inactivation, of YAP. Moreover, we demonstrate that NF2 plays a role as an AKAP (A-kinase-anchoring protein) in the cAMP/PKA–LATS–YAP pathway. Results The LATS/Mob1 complex, but not upstream canonical Hippo components, is essential for induction of YAP phosphorylation by cytoskeletal damage We first asked if Ser381 phosphorylation was induced by cytoskeletal damage. To do this, we first generated and confirmed the specificity of a YAP phospho-Ser381 antibody (Supplementary Figure S1). We found that YAP was rapidly phosphorylated at both Ser127 and Ser381 in NIH3T3 cells after latrunculin B treatment or cell detachment (Figure 1A). We noted that, unlike Ser127 phosphorylation, which is already abundant in unstressed cells, basal Ser381 phosphorylation was barely detectable and was induced in an almost all-or-none fashion by cytoskeletal damage. To examine whether this signalling event requires the canonical Hippo pathway, we exploited MEFs isolated from mice deficient for each pathway component. First, we generated Lats1- and Lats2-deficient MEFs by retroviral Cre infection of Lats1−/−;Lats2fl/fl MEFs. In cells deficient for all LATS isoforms, YAP was unphosphorylated under basal conditions and cytoskeletal damage was unable to induce YAP phosphorylation (Figure 1B). Next, we asked if the interaction of LATS with Mob1 was necessary for YAP phosphorylation. To overcome the problem of cellular senescence associated with multiple passaging, we immortalized Lats1−/−;Lats2fl/fl MEFs with SV40 LT. Then, either wild-type or Mob1-binding-defective versions of LATS1 were introduced followed by retroviral Cre infection (Hergovich et al, 2006). Cells reconstituted with LATS1 R690A or R696A mutants were also unable to phosphorylate YAP (Figure 1C). These results genetically confirm that an intact LATS/Mob1 complex is indispensable for both basal and cytoskeletal damage-induced YAP phosphorylation. Figure 1.The LATS/Mob1 complex is essential for induction of YAP Ser127 and Ser381 phosphorylation by cytoskeletal damage. (A) Phosphorylation at Ser127 and Ser381 by actin cytoskeletal damage. NIH3T3 cells were treated with 5 μM latrunculin B or seeded onto poly-HEMA-coated dishes and incubated for the indicated times. (B) Indispensability of LATS1/2 for YAP phosphorylation. Lats1−/−;Lats2fl/fl MEFs were transduced with either empty or Cre retroviruses. Selected cells were treated as indicated. (C) Indispensability of intact LATS/Mob1 complex for YAP phosphorylation. SV40 LT-immortalized Lats1−/−;Lats2fl/fl MEFs were complemented with either LATS1 WT, LATS1 R660A or R696A mutants. After Cre infection, cells were treated as indicated. Lanes 13 and 14 were infected with empty virus in place of Cre to measure LATS2 deletion efficiency. (D) Dispensability of MST1/2 for YAP phosphorylation. Mst1fl/fl;Mst2+/+vector, Mst1fl/fl;Mst2−/− vector, and Mst1fl/fl;Mst2−/− Cre MEFs were infected and treated as indicated. Download figure Download PowerPoint Mst1/2-deficient cells were obtained similarly by retroviral Cre infection into Mst1fl/fl;Mst2−/− MEFs. Mst1/2-null MEFs were fully competent to induce YAP phosphorylation, both basally and in response to cytoskeletal damage (Figure 1D), consistent with previous reports (Zhou et al, 2009; Song et al, 2010; Zhao et al, 2012). Also, YAP was normally phosphorylated in Sav1-null MEFs (Supplementary Figure S2). We conclude that the upstream components of the canonical Hippo pathway, Mst1/2 and Sav1, are dispensable for cytoskeletal damage-induced YAP phosphorylation; thus, some other upstream signal(s) is involved in this context. PKA activity is required for Ser381 phosphorylation of YAP after cytoskeletal damage Cell detachment has been reported to increase the concentration of cAMP, which contributes to anchorage-dependent mitogenic signalling (Howe and Juliano, 2000). To determine if cAMP signalling is necessary for the induction of YAP phosphorylation by cytoskeletal damage, we pre-treated cells with the PKA inhibitor, H-89. Interestingly, PKA inhibition selectively decreased YAP Ser381 phosphorylation without affecting YAP Ser127 phosphorylation (Figure 2A). To confirm these results using an independent approach, we inhibited PKA activity using the dominant-negative PKA mutant (dnPKA), a mutant regulatory subunit 1α that is defective for cAMP binding. Overexpression of this mutant renders the PKA holoenzyme complex insensitive to cAMP stimuli, and thus inhibits downstream signalling (Clegg et al, 1987). Expression of dnPKA, like H-89, also specifically inhibited YAP Ser381 phosphorylation (Figure 2B). We further confirmed the involvement of PKA in YAP Ser381 phosphorylation by using PKI, PKA-specific peptide inhibitor (Day et al, 1989). In order to deliver PKI peptide inside the cell, we linked 11 Arg to N terminus of PKI (11R-PKI). Such polybasic peptides can be efficiently taken up by the cell (Matsushita et al, 2001). Pre-treatment of 11R-PKI also attenuated YAP Ser381 phosphorylation induced by latrunculin B (Supplementary Figure S3). Of note, the adenylate cyclase inhibitor DDA (2′,5′-dideoxyadenosine), which inhibits adenylate cyclase only when signalling via the G-protein Gs subunit is involved (Florio and Ross, 1983), failed to block YAP Ser381 phosphorylation (Supplementary Figure S4A). This result is consistent with an early study that reported that Gs does not participate in cAMP production induced by cytoskeletal damage (Watson, 1990). Figure 2.Requirement of cAMP/PKA signalling for induction of YAP Ser381 phosphorylation by cytoskeletal damage. (A) Effect of H-89 on YAP phosphorylation by cytoskeletal damage. NIH3T3 cells were pre-treated with 20 μM H-89 for 1 h followed by addition of latrunculin B or seeding onto poly-HEMA-coated dishes. (B) Effect of dnPKA on YAP phosphorylation by cytoskeletal damage. NIH3T3 cells were infected with Flag–dnPKA retroviruses. Selected cells were treated with latrunculin B or seeded onto poly-HEMA-coated dishes. (C, D) Effect of PKA agonist on YAP phosphorylation. NIH3T3 cells were stimulated with 20 μM forskolin and 500 μM IBMX for the indicated times. YAP phosphorylation (C) and localization (C, D) were determined. WCL, whole cell lysate; Nuc, nuclear lysate. Scale bar, 10 μm. (E) LATS activation by PKA agonist or cell detachment. LATS activation loop (AL) phosphorylation was determined in cells treated with the indicated stimuli. (F) Requirement of LATS1/2 for YAP phosphorylation by PKA agonist. Lats1−/−;Lats2fl/fl MEFs were transduced with either empty or Cre retroviruses. Selected cells were treated with forskolin/IBMX. *, nonspecific signal. Download figure Download PowerPoint Next, we attempted to activate cAMP/PKA signalling by treating cells with the adenylate cyclase agonist, forskolin/IBMX (3-isobutyl-1-methylxanthine). This treatment induced both Ser127 and Ser381 phosphorylation of YAP and excluded YAP from the nucleus (Figures 2C and D). We noted that a 1-h treatment with cAMP agonist did not cause obvious cytoskeletal damage, ruling out secondary effects (Supplementary Figure S4B). cAMP can signal through the cAMP-activated guanine nucleotide exchange factors (GEFs) Epac1/2 in parallel with PKA (de Rooij et al, 1998). However, cells stimulated with compound 007, an Epac1/2-selective agonist, failed to induce YAP phosphorylation (Supplementary Figure S4C). Forskolin/IBMX as well as cell detachment activated LATS kinase, as evidenced by activation-loop (AL) phosphorylation (Figure 2E). YAP phosphorylation induced by activated PKA signalling was mediated by LATS kinases since forskolin/IBMX was unable to induce YAP phosphorylation in Lats1/2-null MEFs (Figure 2F). Importantly, Lats1/2-null MEFs normally showed CREB phosphorylation at Ser133, indicating that PKA is still active in the absence of Lats1/2. Taken together, these data suggest that cAMP/PKA signal to LATS1/2 to promote YAP phosphorylation, and this signalling has stronger effect on Ser381 phosphorylation. YAP Ser127 and Ser381 phosphorylation are differentially sensitive to a reduction in LATS1/2 The observation that PKA inhibition selectively affected Ser381 phosphorylation was surprising. However, because PKA agonists were also able to increase YAP Ser127 phosphorylation, we reasoned that PKA generally enhanced LATS activity but Ser127 phosphorylation did not necessarily require PKA. In contrast, PKA might be necessary for efficient Ser381 phosphorylation by further activating LATS. This model agrees well with the biochemical observation that Ser381 is a poor substrate of LATS in cell-free systems. If so, we hypothesized that Ser381 phosphorylation might be more sensitive to a reduction in LATS expression level, whereas Ser127 phosphorylation would be relatively unaffected. To test this idea, we partially depleted LATS1/2 in RPE cells by siRNA transfection. In our experimental system, about 75% of LATS1 and 90% of LATS2 were depleted. Interestingly, latrunculin B treatment showed that Ser127 phosphorylation was almost completely unaffected by the knockdown level we achieved. However, in the same sample, Ser381 phosphorylation markedly decreased (Supplementary Figure S5). We conclude that only a trace amount of LATS is capable of inducing Ser127 phosphorylation with high efficiency in intact cells. In contrast, Ser381 phosphorylation requires sufficient LATS and PKA activity. PKA phosphorylates LATS and thereby enhances its activity To address the question of how PKA increases the ability of LATS to phosphorylate YAP on Ser381, we tested whether PKA enhances LATS activity through direct phosphorylation. Using a cell-free system, we found that purified PKACα catalytic subunits phosphorylated immunoprecipitated LATS1 and LATS2 as monitored using either radioisotope labelling or antibodies against phosphorylated PKA substrate (Figure 3A). To determine if PKA phosphorylates LATS in intact cells, we transfected NIH3T3 cells with HA-tagged LATS2 and treated transfected cells with cytoskeletal-damaging agents or forskolin/IBMX followed by immunoprecipitation with an anti-HA antibody. Western blotting of HA immunoprecipitates with an antibody against a phosphorylated PKA substrate revealed that LATS2 was phosphorylated (Figure 3B). We also obtained similar results using a stable RPE clone expressing SBP (streptavidin-Flag-S tag)-fused LATS2 (Figure 3C, lanes 1–4). H-89 treatment abolished the increase in the signal of the phosphorylated PKA substrate antibody, confirming that this signal was indeed due to PKA activity (Figure 3C, lanes 5–8). Importantly, H-89 did not affect AL phosphorylation and even increased phosphorylation in the HM of LATS (Supplementary Figure S6). It is not yet clear why PKA inhibition increased phosphorylation of the LATS HM. It may be that PKA affects the activity of a phosphatase or kinase that targets this motif. Figure 3.Phosphorylation of LATS1/2 by PKA enhances LATS kinase activity in cell-free systems and in intact cells. (A) PKA phosphorylates LATS in cell-free system. HA–LATS1 KD (kinase dead) or HA–LATS2 KD was immunoprecipitated from transfected 293T cells. Immunoprecipitated beads were incubated with purified PKACα and radiolabelled ATP. Reaction products were analysed isotopically or by western blotting with phospho-PKA substrate antibodies. *, nonspecific signal. (B) PKA phosphorylates LATS in intact cells. HA–LATS2-transfected NIH3T3 cells were treated with the indicated stimuli for 1 h. After immunoprecipitation with anti-HA antibody, phosphorylation by PKA was examined using a phospho-PKA substrate antibody. WCL, whole cell lysate. (C) RPE cells stably expressing SBP-LATS2 were pre-treated with 20 μM H-89 for 1 h, followed by an additional 1-h treatment with the indicated stimuli. LATS2 was pulled down using Streptavidin agarose bead and assayed as in panel (B). (D) LATS2 pre-incubated with PKA has increased kinase activity. HA–LATS2 WT or KD mutant was immunoprecipitated from 293T cells and reacted with PKACα. PKACα was extensively washed out, followed by incubation with 1 μg GST–YAP (full-length) protein and 200 μM unlabelled ATP. Reaction products were analysed by SDS–PAGE and immunoblotting. In lanes 6–9, HA–Mob1A was co-transfected to increase overall kinase activity. In lane 5, GST–YAP was reacted with PKACα to examine possible background phosphorylation of YAP by PKA. SE, short exposure; LE, long exposure. Download figure Download PowerPoint We then tested if PKA-mediated LATS phosphorylation increased LATS enzymatic activity using a sequential ‘cold’ kinase assay. The first kinase reaction was carried out using PKA and immunoprecipitated LATS2. After washing out recombinant PKA, the second kinase reaction was run using a GST-fusion protein of recombinant, full-length YAP as a substrate. Pre-incubation with PKA increased LATS2 kinase activity towards GST–YAP, monitored using antibodies against YAP phospho-Ser127 and phospho-Ser381 (Figure 3D). Overall activity was enhanced by co-transfection of Mob1A. Using purified PKA and YAP in in vitro kinase assays, we noted that PKA induced robust YAP Ser381 phosphorylation (Figure 3D, lane 5). However, we ruled out the possibility that residual PKA was responsible for the additional phosphorylation since PKA pre-incubation also enhanced activity towards Ser127, which was poorly phosphorylated by PKA alone. In addition, PKA pre-incubation increased LATS kinase activity even when we added PKI (5-24) in the second kinase reaction buffer to block residual PKA activity (Supplementary Figure S7). This result suggests that PKA increases LATS activity through direct phosphorylation of LATS. PKA phosphorylates LATS2 at (R/K)(R/K)xS/T motifs, thereby mediating cytoskeletal damage-induced YAP phosphorylation We then tried to identify the PKA target residues on LATS. PKA optimally phosphorylates RRxS/T motifs whereas Arg can be replaced with Lys in some instances. We noticed that LATS2 have four such sequences; Ser172 and Ser380 belonging to the optimal RRxS/T motif and Ser592 and Ser598 belonging to weaker consensus. Thus, we generated LATS2 mutant bearing alanine substitutions in all four serine residues (LATS2 4SA). This mutant was not phosphorylated in vitro by PKA as examined by phospho-PKA substrate western blot (Figure 4A). Signals from both antibodies, one raised against phosphorylated RRxS/T motifs (Ab #2) and another raised against phosphorylated RxxS/T motifs (Ab #1), were absent for LATS2 4SA. More importantly, LATS2 4SA was not phosphorylated by PKA in intact cells after cell detachment, while AL and HM were normally phosphorylated in LATS2 4SA (Figure 4B). LATS2 4SA interacted with Mob1 with comparable affinity to LATS2 WT (Supplementary Figure S8). These results confirm that LATS2 4SA mutant is specifically defective in phosphorylation by PKA while retaining other known regulations. Figure 4.Identification of PKA target sites on LATS2 and their contribution to cytoskeletal damage-induced YAP phosphorylation. (A) LATS2 4SA mutant is not phosphorylated by PKA in vitro. Flag–LATS2 WT or LATS2 4SA were prepared by immunoprecipitation from transfected 293T cells. Flag immunoprecipiates were incubated with cold ATP and PKACα. Reaction products were analysed by two antibodies against phosphorylated PKA substrate. (B) LATS2 4SA mutant is not phosphorylated by cell detachment. NIH3T3 cells were transfected with empty vector, Flag–LATS2 WT, or LATS2 4SA. Transfected cells were suspended for 1 h followed by Flag immunoprecipiation. Flag immunoprocipiates were fractionated by SDS–PAGE and analysed with indicated phospho-specific antibodies. SE, short exposure; LE, long exposure. (C) LATS2 4SA-reconstituted cells attenuate YAP phosphorylation. SV40 LT-immortalized Lats1−/−;Lats2fl/fl MEFs were complemented with either LATS2 WT or LATS2 4SA mutant. After Cre infection, cells were treated as indicated. Lanes 13 and 14 were infected with empty virus in place of Cre to measure LATS2 deletion efficiency. The different mobility of human LATS2 (complemented products) and murine Lats2 (endogenous product before deletion) ensures efficient excision of Lats2 in lanes 1–12. (D) Reduced kinase activity of LATS2 4SA mutant. 293T cells were transfected with indicated Flag-tagged LATS2 constructs. LATS2 was immunoprecipiated by Flag antibody followed by time-course kinase assay as indicated. Download figure Download PowerPoint To test whether PKA-mediated LATS2 phosphorylation is required for actin cytoskeletal damage-induced YAP phosphorylation, we reconstituted immortalized Lats1/2-null MEFs with either LATS2 WT or LATS2 4SA mutant followed by Cre infection. These cells were harvested at early time points (15 min, 30 min and 1 h) after latrunclin B treatment or detachment. Remarkably, YAP phosphorylation was attenuated in cells reconstituted with LATS2 4SA (Figure 4C). Of note, in this experimental setup where only LATS2 4SA mutant is expressed in cell, Ser127 as well as Ser381 phosphorylation was affected. Nevertheless, Ser127 phosphorylation level eventually catches up to that of LATS2 WT-reconstituted cells. In contrast, Ser381 phosphorylation was markedly reduced at all time points examined. Lastly, we performed kinase assay using immunoprecipitated LATS2 WT or 4SA mutant, and found that LATS2 4SA mutant have significantly lower activity (Figure 4D). These results prove that PKA-mediated LATS phosphorylation is required for full kinase activity and efficient YAP phosphorylation induced by actin cy
Referência(s)