Fig. 4: Functional characterization of the divergence between LATS1 and LATS2.
From: LATS1 but not LATS2 represses autophagy by a kinase-independent scaffold function
a Schematic representation of the protein ___domain structure and truncated mutants of LATS1 used for mapping the LATS1 protein ___domain required for Beclin-1 interaction and stabilization. b Schematic illustration of the generation of a LATS2-LATS1 chimeric protein by replacing the center ___domain of LATS2 (blue bracket) with the analogous ___domain of LATS1 (red bracket; amino acids 151–554) inserted between the wild-type LATS2 N-terminal (amino acids 1–147) and C-terminal (amino acids 514–1088) domains. c Functional validation of the LATS2 chimera protein in Beclin-1 stabilization. HEK293T/17 cells were transfected with the same amount of vectors encoding for Flag-tagged Beclin-1 (Flag-BECN1) together with HA-tagged wild-type LATS1 or LATS2, or increasing amounts of vector encoding the HA-tagged LATS2-chimeric protein as indicated. Cells were collected 72 h after transfection and analyzed by immunoblotting for Flag-BECN1, LATS1, and LATS2. Immunoblotting for GAPDH was used as loading control. Results represent three independent experiments. d, e LATS2 chimera protein can fully rescue autophagic flux (p62) in endogenous LATS1-knockdown Huh7 cells in response to sorafenib. Huh7 cells transfected with siControl or siLATS1 were in addition transfected with empty vector (EV) or a vector encoding for LATS2 chimera. Cells were lysed 72 h after transfection and p62 and LATS1 protein levels were analyzed by immunoblotting. GAPDH was used as loading control. A representative immunoblot (d) and quantification of the relative p62 levels (e) (sorafenib 8 μM condition, normalized to GAPDH) from three independent experiments are shown. Statistical significance was calculated using one-way ANOVA.
