The Hippo pathway is an evolutionarily conserved pathway that controls cell proliferation, organ size, tissue regeneration and stem cell self-renewal. Lats2 knockdown, or expression of c-Abl mutants SC75741 supplier that escape inhibition by Lats2, enabled DNA damage-induced apoptosis of densely plated cells, while Lats2 overexpression inhibited apoptosis in sparse cells. These findings explain a long-standing enigma of why densely plated cells are radioresistant. Furthermore, they demonstrate that the Hippo pathway regulates cell fate decisions in response to DNA damage. by activated Lats2, but not the T197A mutant, suggesting that Lats2 phosphorylates c-Abl at the Thr197 site (Figure 5d). The activity of the myc-Lats2 used in the assay was confirmed by its ability to phosphorylate Yap (Supplementary Figure 4a). Furthermore, a myc-Lats2 kinase mutant did not phosphorylate c-Abl (Supplementary Figure 4b). To determine whether the Thr197 phosphorylation site is important for the inhibition of c-Abl by Lats2, we tested the ability of wild type constitutively active c-Abl (1-81), and the T197A c-Abl mutant to phosphorylate Yap when transfected together with Lats2. The T197A mutant was more Rabbit polyclonal to ABCA6 resistant to Lats2 inhibition than the wild type, and phosphorylated Yap SC75741 supplier even in the presence of Lats2 (Figure 5e). These results suggest that phosphorylation of c-Abl by Lats2 has a role in the inhibition of c-Abl. Figure 5 Lats2 inhibits c-Abl activity, and phosphorylates c-Abl at its SH2 domain. (a) Schematic representation of c-Abl. The SH3, SH2, SC75741 supplier kinase, DNA binding (DBD) and G and F-actin (G-Act, F-Act) binding domains are indicated. The putative Lats phosphorylation … The Hippo kinase Lats2 binds the c-Abl SH3 domain Our results (Figure 4b) indicating that multiple Lats2 domains were capable of interacting with c-Abl, and that Lats2 possesses a number of consensus motifs for SH3 domain binding (Figure 4a), suggested that Lats2 inhibition of c-Abl could be mediated through Lats2 binding to the SH3 domain of c-Abl. To validate this, we made a point mutation in the SH3 domain of c-Abl (W118A) at a position shown to reduce SH3 domain binding to PXXP motifs,36 and tested the interaction between c-Abl and Lats2 by co-IP. Lats2 co-immunoprecipitated approximately 2-fold less efficiently with the W118A mutant c-Abl than with the wild type, indicating lower binding with this mutant (Figure 6a). We next compared the sensitivity of the constitutively active c-Abl 1-81, and the T197A and W118A mutants, to inhibition by Lats2. Lats2 inhibited wild-type c-Abl 1-81, as indicated by a decrease in phosphorylation of p73 (pTyr99) and c-Abl autophosphorylation (pTyr245), but the T197A and W118A mutants escaped inhibition (Figures 6b and c). These results indicate that Lats2 inhibits c-Abl, and the SH3 domain is involved in mediating the interaction between Lats2 and c-Abl. Figure 6 c-Abl SH3 mutants are resistant to inhibition by Lats2. (a) Mutation of c-Abl at W118 impairs binding to Lats2. HEK293 cells were transfected with the constructs indicated, SC75741 supplier with 0.25, 0.5, and 1?kinase assays HEK293 cells were transfected separately with Flag-c-Abl constructs or Flag-Yap1, with SC75741 supplier myc-Lats2 transfected alone or together with untagged Mst1 and WW45, or transfected with control plasmids. Cells were harvested 24C30?h post-transfection, and were lysed in Buffer A (10?mM Tris-HCl pH 7.5, 250?mM NaCl, 300?mM sucrose, 3?mM MgCl2, 1?mM EDTA, 0.5% Triton X-100, 1?mM DTT, protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktails II and III (Sigma, for Tyrosine and Serine/Threonine phosphatases). Cells were incubated for 20?min on ice and centrifuged at 13?000for 10?min. Proteins were immunoprecipitated using anti-Flag agarose beads (Sigma), or anti-myc antibody with Protein A/G sepharose (Santa Cruz). Immunoprecipitates (IPs) were washed four times with Buffer A, then twice with kinase buffer (50?mM Tris-HCl, pH 7.5, 10?mM MgCl2, 1?mM EGTA, 2?mM DTT, and 0.01% Brij 35). Flag-tagged proteins were eluted by incubating beads in kinase buffer with 0.1?mg/ml Flag peptide for 10?min at 30?C. Flag beads were centrifuged at 3000 g, and eluted proteins were transferred to another tube. For the assay, eluted Flag-tagged proteins or control were added to the tubes containing the immunoprecipitated control or myc-Lats2 (not eluted from the beads). BSA was added to 200?g/ml, and ATP to 200?M. Reactions were incubated at 30?C for 30?min, and stopped by the addition of Laemmli protein gel sample buffer, and boiling for 1?min. Samples were analyzed by SDS-PAGE and immunoblotting. Acknowledgments We thank M Oren and Y Aylon for helpful discussion and for the myc-Lats2 constructs and reagents. We thank XJ Yang for the HA-Lats2, Mst1 and WW45 constructs. We thank G Superti-Furga for the pSLX c-Abl constructs. STI571 was kindly provided by Novartis Pharmaceuticals. We thank R Keshet for providing the pBabe Flag-Yap constructs, and M Elbaz for her technical assistance. This work was supported by grants from the Israel Science Foundation (grant.