Protein kinase A activates the Hippo pathway to modulate cell proliferation and differentiation

Abstract

The Hippo tumor suppressor pathway plays an important role in tissue homeostasis that ensures development of functional organs at proper size. The YAP transcription coactivator is a major effector of the Hippo pathway and is phosphorylated and inactivated by the Hippo pathway kinases Lats1/2. It has recently been shown that YAP activity is regulated by G-protein-coupled receptor signaling. Here we demonstrate that cyclic adenosine monophosphate (cAMP), a second messenger downstream from Gαs-coupled receptors, acts through protein kinase A (PKA) and Rho GTPases to stimulate Lats kinases and YAP phosphorylation. We also show that inactivation of YAP is crucial for PKA-induced adipogenesis. In addition, PKA activation in Drosophila inhibits the expression of Yorki (Yki, a YAP ortholog) target genes involved in cell proliferation and death. Taken together, our study demonstrates that Hippo-YAP is a key signaling branch of cAMP and PKA and reveals new insight into mechanisms of PKA in regulating a broad range of cellular functions.

Keywords: Hippo; PKA; YAP; adipogenesis; proliferation.

Figure 1.

cAMP signaling induces YAP phosphorylation and inactivation. (A,B) MDA-MB-231 cells were treated with 10 μM epinephrine (A) or forskolin (B) for the indicated durations, and cell lysates were subjected to immunoblotting using the indicated antibodies. (C) Time course of YAP and CREB phosphorylation in response to epinephrine or forskolin (the value for time 0 was arbitrarily set). (D) MDA-MB-231 cells were treated with different PDE inhibitors—ibudilast (100 μM), IBMX (100 μM), rolipram (50 μM), or theophylline (1 mM)—for 1 h, and the phosphorylation status of YAP was determined by phos-tag gels. (E) HEK293A, MCF10A, U2OS, or MEF cells were treated with or without 10 μM forskolin for 1 h. YAP phosphorylation was assessed using phos-tag gels, and the same lysates were also used to blot for TAZ protein levels. (F) MCF10A cells were serum-starved overnight and treated with 10 μM forskolin for 1 or 4 h, mRNA was extracted, and the expression level of CTGF was determined using real-time RT–PCR.

Figure 2.

cAMP signaling to YAP phosphorylation is mediated by PKA. (A) Flag-YAP was cotransfected with or without HA-tagged wild-type or kinase-dead PKA catalytic subunit; after 24 h, cell lysates were prepared, and phosphorylation of Flag-YAP was determined. (B) Similar to A except that Flag-tagged wild-type or constitutively active Rap1b was transfected. (C) HEK293A cells were transfected with mutant PKA regulatory subunits (PKARIα or PKARIIα); after 16 h, cells were treated with or without 10 μM forskolin for 1 h, and YAP or CREB phosphorylation was assessed. (D) Stable cell lines (MDA-MB-231) expressing control shRNA or shRNAs targeting the PKA catalytic subunit (α isoform) were established and treated with or without 10 μM epinephrine or forskolin for 1 h. Cell lysates were subjected to immunoblotting to determine the level of YAP and CREB phosphorylation. (E) MDA-MB-231 cells were pretreated with or without PKA inhibitor KT5720 (5 μM) for 30 min and then stimulated with 10 μM epinephrine or forskolin for 1 h; YAP and CREB phosphorylation was then determined. (F) Similar to E except that primary hepatocytes were used, and glucagon was used to induce PKA activity.

Figure 3.

PKA increases YAP phosphorylation by stimulating kinase activity of Lats1/2. (A) Myc-tagged wild-type or S127A or 5SA mutant YAP were transfected into HEK293A cells, and, after 16 h, cells were treated with or without 10 μM forskolin for 1 h. YAP phosphorylation was assessed by phos-tag gel. (B) MDA-MB-231 cells were transfected with control, MST1/2, or Lats1/2 siRNAs. Two days later, cells were treated with 10 μM epinephrine or forskolin for 1 h. Cell lysates were subjected to immunoblotting to assess knockdown efficiency and YAP phosphorylation. The arrowhead indicates MST2 position. (C,D) Flag-YAP was cotransfected into HEK293A cells with or without K/R mutants (kinase-dead) of MST2 (C) or Lats2 (D); after 16 h, cells were stimulated with 10 μM forskolin for 1 h, and phos-tag gels were used to determine the phosphorylation status of Flag-YAP. (E) MDA-MB-231 cells were untreated or treated with 10 μM forskolin for 1 h, endogenous Lats1 was immunoprecipitated and subjected to kinase assay using GST-YAP as substrate, and phosphorylation of GST-YAP by Lats1 was monitored by YAP phosphorylation at S127.

Figure 4.

Rho GTPases mediate the effect of PKA on YAP phosphorylation. (A) MDA-MB-231 cells were treated with 10 μM forskolin for 1 h, and cell lysates were subjected to immunoblotting. Phosphorylation of MLC2, CREB, and YAP was determined. (B) Flag-YAP was cotransfected into HEK293A cells with wild-type or constitutively active RhoA, and, after 16 h, cells were stimulated with 10 μM forskolin for 1 h before Western blotting. (C) Flag-YAP was cotransfected into HEK293A cells with or without GFP-tagged RhoGDI, and, after 16 h of incubation in serum-free medium, cells were treated with or without KT5720 for 1 h. Phosphorylation of Flag-YAP and endogenous TAZ was determined.

Figure 5.

YAP/TAZ mediate the effect of cAMP in adipogenesis. (A) 3T3-L1 cells were treated with 10 μM forskolin or 100 μM IBMX for 1 h, or serum-starved 3T3-L1 cells were treated with 5 μM KT5720 for 1 h, and YAP phosphorylation and TAZ protein levels were determined. (B) 3T3-L1 cells were incubated under adipocyte differentiation conditions with IBMX or KT5720. (Tro) Troglitazone. IBMX increased, whereas KT5720 repressed, adipogenesis, as assessed by oil red staining. (C,D) 3T3-L1 cells were transfected with control or YAP and TAZ siRNAs (siYT), and the knockdown efficiency was determined by immunoblotting (shown in C). (D) These cells are also subjected to adipogenesis. (E) 3T3-L1 cells were transfected with control or YAP and TAZ siRNAs. Cells were treated with Tro and IBMX in the presence of vehicle (DMSO) or KT5720 as indicated. Adipocyte differentiation was measured by oil red staining. (F) Overexpression of YAP abolished IBMX- or forskolin-induced adipogenesis. (G) Following differentiation (as in F), cells were lysed, and the expression of adipogenesis marker genes was determined by real-time RT–PCR; the mRNA level was normalized to that of cells incubated in growth medium.

Figure 6.

PKA inhibits Yki in Drosophila. (A) In Drosophila S2R⁺ cells, knockdown of PKA-C1 by RNAi increased Yki/Sd reporter activity. (B) Relative transcript levels of ex, CycE, and Diap1 genes in wild-type (blue), C5-Gal4/UAS-PKA-C1 RNAi (red), and C5-Gal4/UAS-PKA-C1 (green) larval wing discs. (C) Yki phosphorylation was increased in C5-Gal4/UAS-PKA-C1 larval wing discs. (D–F) en-Gal4/UAS-PKA-C1 UAS-GFP larval wing discs exhibiting expression of GFP marker (D, green), Diap1 protein (E, red), and Caspase3 (F, white). (G) Merge of D–F.

Figure 7.

Regulation of the Hippo pathway by cAMP-PKA signaling. Upon stimulation of Gα_s-coupled GPCR, activation of PKA by cAMP leads to inhibition of Rho GTPases, which indirectly inhibit Lats kinase activity. Stimulation of Gα_12/13- or Gα_q/11-coupled receptors antagonize the effect of cAMP or PKA on YAP phosphorylation by inducing Rho GTPases. Inhibition of YAP and TAZ mediates functions of cAMP and PKA on adipogenesis, cell proliferation, and apoptosis.