Regulation of Hippo signalling by p38 signalling

Abstract

The Hippo signalling pathway has a crucial role in growth control during development, and its dysregulation contributes to tumorigenesis. Recent studies uncover multiple upstream regulatory inputs into Hippo signalling, which affects phosphorylation of the transcriptional coactivator Yki/YAP/TAZ by Wts/Lats. Here we identify the p38 mitogen-activated protein kinase (MAPK) pathway as a new upstream branch of the Hippo pathway. In Drosophila, overexpression of MAPKK gene licorne (lic), or MAPKKK gene Mekk1, promotes Yki activity and induces Hippo target gene expression. Loss-of-function studies show that lic regulates Hippo signalling in ovary follicle cells and in the wing disc. Epistasis analysis indicates that Mekk1 and lic affect Hippo signalling via p38b and wts We further demonstrate that the Mekk1-Lic-p38b cascade inhibits Hippo signalling by promoting F-actin accumulation and Jub phosphorylation. In addition, p38 signalling modulates actin filaments and Hippo signalling in parallel to small GTPases Ras, Rac1, and Rho1. Lastly, we show that p38 signalling regulates Hippo signalling in mammalian cell lines. The Lic homologue MKK3 promotes nuclear localization of YAP via the actin cytoskeleton. Upregulation or downregulation of the p38 pathway regulates YAP-mediated transcription. Our work thus reveals a conserved crosstalk between the p38 MAPK pathway and the Hippo pathway in growth regulation.

Keywords: F-actin; Hippo signalling; p38 signalling.

Figure 1

Overexpression of lic promotes Yki activities and induces Hippo target gene expression. (A–H) Immunostaining of Wg (A and B), ban-lacZ (C and D), ex-lacZ (E and F), and Diap1-lacZ (G and H) in the wing disc expressing UAS-GFP control or UAS-lic by en-Gal4 tub-Gal80^ts. Wg signal at the hinge region and the D/V boundary is indicated by white and yellow arrows, respectively. (I) Total and phosphorylated (S168) levels of Yki in the wing disc expressing UAS-lic by nub-Gal4. The right panel displays the ratio of p-Yki (S168) levels to total Yki levels. Data represented as mean ± SD (n = 3). Student's t-test, **P < 0.01. (J–M) Drosophila eyes expressing UAS-GFP (J), UAS-lic (K), UAS-yki (L), or UAS-lic and UAS-yki by GMR-Gal4 (M).

Figure 2

lic regulates Hippo signalling in ovary follicle cells and in the wing disc. (A–D) Immunostaining of Lic (A), ex-lacZ (at stages 7–9) (B), Cut (at stages 1–6) (C), and FasIII (at stages 1–6) (D) in the egg chamber containing lic^d13MARCM clones. (E and F) Immunostaining of Diap1 in the wing disc containing wild-type MARCM clones (E) or lic^d13 mutant MARCM clones expressing UAS-yki by Act-Gal4 (F). The MARCM clones are marked by the presence of GFP and outlined by white dashed lines. (G–J) Adult wings expressing UAS-GFP (100% penetrant, n = 35) (G), UAS-lic IR and UAS-GFP (100% penetrant, n = 35) (H), UAS-wts IR and UAS-GFP (100% penetrant, n = 33) (I), UAS-lic IR and UAS-wts IR (91.4% penetrant, n = 35) by en-Gal4 (J). (K–N) Adult wings expressing UAS-GFP (100% penetrant, n = 35) (K), UAS-lic IR and UAS-GFP (100% penetrant, n = 35) (L), UAS-hpo IR and UAS-GFP (100% penetrant, n = 30) (M), UAS-lic IR and UAS-hpo IR (85.7% penetrant, n = 35) by SalE-Gal4 (N). The expression domain of en-Gal4 or SalE-Gal4 in the adult wing was outlined with black dashed lines.

Figure 3

Lic acts upstream of Wts and Yki. (A and B) Immunostaining of FasIII in follicle cells expressing UAS-wts IR in control (A) or lic^d13 MARCM clones (B) (marked by the presence of GFP and outlined by white dashed lines). (C–E) Immunostaining of ex-lacZ in the wing disc expressing UAS-lic (C), UAS-wts (D), or UAS-lic and UAS-wts by en-Gal4 tub-Gal80^ts (E). (F–H) Immunostaining of ex-lacZ in the wing disc expressing UAS-lic (F), UAS-yki IR (G), or UAS-lic and UAS-yki IR by ci-Gal4 (H).

Figure 4

lic regulates Hippo signalling via p38b. (A–D) Immunostaining of Diap1-lacZ in the wing disc expressing UAS-GFP (A), UAS-p38b IR (B), UAS-lic (C), or UAS-lic and UAS-p38b IR by ap-Gal4 (D). (E) Immunostaining of Diap1-lacZ in the wing disc expressing UAS-lic by hh-Gal4 and containing p38b^156A clones (marked by the absence of GFP and outlined by white dashed lines). (F and G) Immunostaining of phosphorylated p38 in the wing disc expressing UAS-lic (F) or UAS-lic KD by ci-Gal4 (G).

Figure 5

The p38 pathway regulates the Hippo pathway via F-actin accumulation. (A–D) Staining of F-actin in the wing disc expressing UAS-GFP (A), UAS-lic (B), UAS-p38b IR (C), or UAS-lic and UAS-p38b IR by Ci-Gal4 (D). (E–H) Staining of F-actin (E and F) or ex-lacZ (G and H) in the wing disc expressing UAS-dia IR (E and G) or UAS-lic and UAS-dia IR by Ci-Gal4 (F and H).

Figure 6

Regulation of Hippo signalling by p38 signalling requires Jub. (A–F) Staining of Diap1-lacZ or F-actin in the wing disc expressing UAS-jub IR (A and B), UAS-lic and UAS-jub IR (C and D), or UAS-Mekk1 and UAS-jub IR (E and F). (G) Mobility shift of Jub in the presence of Lic and p38b measured by western blotting. (H) Mobility shift of Jub in the absence or presence of CIP measured by western blotting.

Figure 7

p38 signalling regulates Hippo signalling in mammalian cell lines. (A–E) MCF10A cells were transfected with indicated plasmids, and cells in B and E were treated with 1 μg/ml CytoD for 1 h. (A and B) Staining of F-actin. (C–E) Immunostaining of HA-YAP1. (F) Luciferase assays in cells expressing Gal4-TEAD reporter and the indicated plasmids. (G) Total and phosphorylated (S127) levels of HA-YAP1 in HEK293 cells expressing MKK3. (H) Transcription of CTGF and Cyr61 in HEK293T cells transfected with the indicated plasmids. (I) Transcription of CTGF and Cyr61 in A549 cells with siRNAs targeting p38α. All the experiments were performed with densely cultured cells. Data represented as mean ± SD (n = 3).