Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor

Abstract

LATS2 kinase functions as part of the Hippo pathway to promote contact inhibition of growth and tumor suppression by phosphorylating and inhibiting the transcriptional coactivator YAP. LATS2 is activated by the MST2 kinase. How LATS2 is activated by MST2 in response to changes in cell density is unknown. Here we identify the angiomotin-family tight junction protein AMOTL2 as a novel activator of LATS2. Like AMOTL2, the other angiomotin-family proteins AMOT and AMOTL1 also activate LATS2 through a novel conserved domain that binds and activates LATS2. AMOTL2 binds MST2, LATS2, and YAP, suggesting that AMOTL2 might serve as a scaffold protein. We show that LATS2, AMOTL2, and YAP all localize to tight junctions, raising the possibility that clustering of Hippo pathway components at tight junctions might function to trigger LATS2 activation and growth inhibition in response to increased cell density.

FIGURE 1:

AMOTL2 interacts with LATS2 and YAP2 and promotes LATS2-mediated YAP phosphorylation. (A) YAP2, LATS2-FLAG, and the indicated plasmids were transfected into HEK293 cells. Cell lysates were analyzed by Western blotting to detect YAP2 phosphorylation levels using anti-pYAP-S127 antibody (top). The blot was then reprobed to obtain the levels of other proteins. (B) HEK293 cells were transfected with LATS2-FLAG along with either AMOTL2 or an AMOTL2 mutant lacking the C-terminal PDZ motif (AMOTL2-ΔPDZ). Cell lysates were subjected to immunoprecipitation (IP) of LATS2 with FLAG antibody or immunoglobulin G (IgG) control. The immunoprecipitates and cell lysates were subjected to immunoblot analysis with anti-Myc and anti-FLAG antibodies. The levels of AMOTL2 and LATS2 proteins in the starting lysates are shown in the bottom half (Input). (C) The AMOTL2-Myc plasmid was transfected along with either full-length GFP-LATS2 (1–1088) or the indicated GFP-LATS2 deletion constructs into HEK293 cells. LATS2 was immunoprecipitated (IP) from cell lysates with anti-GFP antibodies. The immunoprecipitates and cell lysates were subjected to immunoblot with anti-Myc and anti-GFP antibody. The levels of AMOTL2 and LATS2 proteins in the starting lysates are shown in the bottom half (Input). (D) LATS2-FLAG along with full- length AMOTL2-Myc (1–780) or the indicated Myc tagged AMOTL2 deletion constructs were transfected into HEK293 cells. AMOTL2 was immunoprecipitated (IP) from cell lysates using anti-Myc antibody, and the immunoprecipitates and cell lysates were subjected to immunoblot with anti-FLAG and anti-Myc antibodies. (E) YAP2, LATS2-FLAG, and the indicated AMOTL2-Myc deletion constructs were transfected into HEK293 cells, and the resulting cell lysates were subjected to immunoblot analysis with anti-pYAP-S127 antibody (top). The levels of YAP, AMOTL2-Myc, LATS2-FLAG, and tubulin are shown (bottom).

FIGURE 2:

LATS2 and YAP colocalize with the tight junction marker ZO-1 in Caco2 cells. (A) Caco2 cells grown to confluence and processed for indirect immunofluorescence using antibodies against LATS2 (i, ii) with E-cadherin (i) or Zo1 (ii) (top half) or YAP2 (iii, iv) with Zo1 (iii) or LATS2 (iv) (bottom half). The merged green, red, and blue (DAPI/DNA) signals are shown. The boxed region was deconvolved to show LATS2 and YAP2 localization along the apical–basolateral axis (right). (B) Endogenous levels of LATS1, LATS2, and YAP phosphorylation (pYAP-S127) were analyzed by Western blotting of lysates from the indicated cell lines.

FIGURE 3:

AMOTL2 promotes LATS2 kinase activity and binds to the active form of LATS2. (A) Cell lysates were prepared from HEK293 cells (control) or HEK293 cells transfected with LATS2-FLAG and the indicated plasmids. LATS2-FLAG was immunoprecipitated using anti-FLAG antibodies. The immunoprecipitates were subjected to vitro kinase assays using bacterially expressed GST-YAP2 as a substrate, and LATS2 phosphorylation of GST-YAP2 (top) was measured using a PhosphorImager. Simultaneously, equal volumes of kinase assay samples were subjected to Western blot analysis to detect the levels of GST-YAP2 and LATS2-FLAG. (B) LATS2-FLAG was coexpressed with the indicated AMOTL2 deletion constructs in HEK293 cells and processed for in vitro kinase assays and Western blot analysis as described in A. (C) LATS2-FLAG was coexpressed with indicated plasmids in HEK293, and the lysates were subjected to Western blot with anti-pLATS2-S872, anti-pLATS2-T1041, and other antibodies as marked. (D) AMOTL2-Myc, LATS2-FLAG, and GFP-MST2 plasmids were coexpressed in HEK293 cells, and cell lysates were immunoprecipitated either with anti-Myc or control IgG antibodies. The immunoprecipitates (IP) and cell lysates (Input) were subjected to immunoblot with the indicated antibodies. (E) LATS2-FLAG or kinase-dead LATS2 (LATS2^KD-FLAG) were coexpressed with AMOTL2-Myc in HEK293 cells. Cell lysates were immunoprecipitated with anti-FLAG antibodies and immunoprecipitates (IP) and cell lysates (Input) were subjected to immunoblot with the indicated antibodies. (F) AMOTL2-Myc was coexpressed with the indicated phosphorylation-site mutants of LATS2, and cell lysates were processed for immunoprecipitation and Western blot as described in E.

FIGURE 4:

AMOT family proteins are both positive and negative regulators of LATS2 signaling. (A) YAP2 was coexpressed with LATS2-FLAG and the indicated Myc-tagged AMOT proteins in HEK293 cells. Cell lysates were processed and subjected to immunoblot detection of YAP2 phosphorylation (pYAP-S127). The levels of AMOT proteins (Myc), YAP2, LATS2, and tubulin in the lysates are shown. The numbers under the blots show the ratio of the pixel densities of phospho-YAP to YAP. (B) LATS2-FLAG was coexpressed with Myc-tagged AMOT proteins as indicated, and the cell lysates were processed for LATS2 in vitro kinase assays and Western blot analysis as described in Figure 3A. (C) FLAG-YAP2 was transfected with Myc-tagged AMOT plasmids in HEK293 cells as indicated. Cell lysates were subjected to immunoprecipitation of AMOT proteins with anti-Myc antibodies, and the immunoprecipitates and lysates were probed with FLAG and Myc antibodies. (D) AMOT130-Myc, YAP2, and LATS2-FLAG were coexpressed with various amounts of AMOT80 as indicated in HEK293 cells, and the level of YAP2 phosphorylation was analyzed by Western blot. The levels of YAP2, AMOT130, AMOT80, LATS2, and tubulin are shown. (E) AMOTL2-Myc, YAP2, and LATS2-FLAG were coexpressed with various amounts of AMOT80 as indicated in HEK293 cells, and the level of YAP2 phosphorylation was analyzed by Western blot. The levels of YAP2, AMOTL2, AMOT80, LATS2, and tubulin are shown.

FIGURE 5:

Reduction in AMOT130 and AMOTL2 levels inhibits LATS2 signaling. (A) AMOTL2 was immunoprecipitated from lysates of MCF10A cells or MCF10A cells stably expressing luciferase control (GL2) or AMOTL2 shRNA vectors, and then the levels of AMOTL2 were analyzed using anti-AMOTL2 antibodies. (B) MCF10A cells stably expressing the indicated shRNA constructs described in A were processed for immunofluorescence for F-actin (top) and anti-YAP (bottom). Nuclei were stained with DAPI. (C) Lysates of MCF10A cells or MCF10A cells stably expressing the indicated shRNA constructs (see A) were subjected to Western blot analysis for detection of endogenous YAP phosphorylation using anti–pYAP-S127, and the blot was subsequently reprobed for YAP and tubulin. (D) Lysates of MCF10A cells alone or MCF10A cells stably expressing the indicated shRNA vectors (see A) were subjected to immunoprecipitation of endogenous LATS2 using anti-LATS2 antibodies and processed for in vitro kinase assays and Western blot analysis as described in Figure 3A. IgG immunoprecipitates from MCF10A cells (IgG) were used as a control. (E) The levels of kinase activity of LATS2 from the indicated MCF10A lines were quantified using ImageJ and normalized to the amount of LATS2. The values are the average of three independent experiments. Error bars, SD of the relative kinase activity; **p < 0.0003 (based on p value calculation by t-test analysis). (F) AMOTL2-, AMOT-, LATS2-, or luciferase (GL2)-specific siRNA pools were transfected into HEK293 cells along with LATS2-FLAG, HA-MOB1, and YAP2 as indicated. Cell lysates were prepared 72 h after transfection and analyzed by Western blot for detection of pYAP-S127 and other proteins as marked.