Figure 4.

Active c-Abl Phosphorylates TFEB on Tyrosine

(A) Schematic diagram showing that c-Abl-ERT2 under tamoxifen treatment phosphorylates in tyrosine (P-Tyr) its target proteins.

(B) Representative Western blot of HeLa TFEB-GFP cells transfected with c-Abl-ERT2 and c-Abl-ERT KD plasmids and treated with Tamoxifen for 8 h. GFP was immunoprecipitated using beads-anti-GFP and then used a anti-phospho-tyrosine antibody. n = 3 independent experiments.

(C) Autoradiography of an in vitro phosphorylation assay. TFEB-Flag IP was incubated with human recombinant c-Abl active and ATP-γ-³²P for 0 h, 0.5 hr, 1 h, and 2 h. ATP-γ-³²P incubation for 2 h without recombinant c-Abl active is showed as control (ct).

(D) Immunoprecipitated (IP) CRKII was incubated with human recombinant c-Abl active and ATP-γ-³²P for 2 h.

(E) Tyrosine 75 (Y75) and Y173 are highly conserved across different species and are included in the c-Abl phosphorylation motif YX_1-5P, which included the tyrosine (Y) and after one to five different amino acids, it recognize a proline (P).

(F) Representative confocal microscopy images and quantification of subcellular localization of TFEB-GFP mutants and control plasmids. n = 3 independent experiments. Scale bars, 10 μM.

(G) Western blot of HeLa cells transfected with a TFEB-GFP wild type plasmid or with plasmids carrying the Y75 or Y173 mutations. Western blot membranes were incubated with a specific antibody against S211, S138, and S142.