Fig. 5: LATS1 promotes Beclin-1 ubiquitination at lysine residues K32 and K263.
From: LATS1 but not LATS2 represses autophagy by a kinase-independent scaffold function
a HEK293T/17 cells transfected with indicated plasmids were collected 72 h later for immunoprecipitation with anti-Flag antibody and then immunoblotted for HA and Flag-tagged proteins and for LATS1/2. Input represents analysis of cell lysates before immunoprecipitation. Immunoblots represents three independent experiments. b HEK293T/17 cells were transfected with indicated siRNAs followed by transfection with HA-tagged wild-type or K27 (all lysins were mutated to arginine except K27) ubiquitin constructs. Seventy-two hours later, cell lysates were prepared and immunoprecipitated with Beclin-1 antibody and immunoblotted with antibodies against Beclin-1 and HA (HA-Ub). Input represents immunoblotting of cell lysates for Beclin-1 and LATS1 before immunoprecipitation. Immunoblots represent three independent experiments. c HEK293T/17 cells transfected with indicated plasmids. Seventy-two hours later, cell lysates were prepared and immunoprecipitated with anti-Flag antibody and immunoblotted with antibodies against HA and Flag-tagged proteins and LATS1/2. Input represents analysis of cell lysates before immunoprecipitation. GAPDH was used as loading control. Immunoblots represent three independent experiments. d Schematic representation of LATS1-mediated ubiquitination levels in the different domains of Beclin-1. Note that the MID construct containing the central coiled-coil ___domain is the minimal recipient ___domain for ubiquitination. e Mass spectrometric identification of ubiquitination sites in Beclin-1 induced by LATS1. HEK293T/17 cells were transfected with vectors encoding for HA-tagged wild-type ubiquitin, Flag-tagged-Beclin-1 with vectors encoding for empty vector and Myc-tagged LATS1. After immunoprecipitation with antibodies against Flag, samples were eluted for ubiquitination site identification by mass spectrometry. Related fold change of ubiquitinated peptide (ratio of LATS1/empty vector) is presented. Results were pooled from five independent experiments. f Mutation of lysine residues K32/263 blocks LATS1-induced ubiquitination of Beclin-1. Beclin-1 lysine K32 and K263 were mutated to arginine via site-directed mutagenesis. HEK293T/17 cells were transfected with indicated plasmids. Cells were collected 72 h later for immunoprecipitation with anti-Flag antibody and then immunoblotted for HA, Flag tagged proteins and LATS1/2. Input represents analysis of cell lysates before immunoprecipitation. Immunoblots represents three independent experiments. Note that the different proteins have been analyzed on different membranes.
