Figure 1. NBR1 is phosphorylated by GSK3. (A) Schematic representation of NBR1 functional protein domains. Amino acid positions of phosphorylated threonine and serine residues (letter P in circle) on the human protein are shown. PB1, Phox, and Bemp1 domain; ZZ, ZZ-type zinc finger; CC1 and 2, coiled-coil domains; LIR1 and 2, LC3-interacting regions; J, juxta-UBA domain (membrane-interacting amphipathic α-helix), UBA, ubiquitin-associated domain. Alignment of NBR1 amino acids flanking GSK3 putative phosphorylation consensus site shows conservation of threonine and serine residues (indicated by stars) in different species. Gray color shows amino acids conserved in all depicted species, and numbers above stars indicate threonine and serine residue positions in the human NBR1 protein. H. sapiens, Homo sapiens; M. musculus, Mus musculus; G. gallus, Gallus gallus; X. tropicalis, Xenopus tropicalis; D. rerio, Danio rerio. (B) GSK3 in vitro kinase assays. GST-tagged GSK3A or GSK3B forms (wt, wild-type; inactive, GSK3A K148A or GSK3B K85A; active, GSK3A S21A or GSK3B S9A) and wild-type GST-tagged NBR1 proteins were purified from 293T cells and mixed with radioactive γ−³²P ATP. The upper panel shows radioactive ³²P signal and the lower panels show GST-tagged protein amounts revealed with an anti-GST antibody. Arrows indicate the bands corresponding to phosphorylation of NBR1 by GSK3, GSK3 autophosphorylation, and total proteins. (C) GSK3 in vitro kinase assays performed with purified active (S9A) or inactive (K85A) GST-GSK3B mutants mixed with wild-type or mutant GST-NBR1. Thr581, Thr586, and Ser590 were mutated to nonphosphorylable alanine (3Mut., T581A T586A S590A). Lower panel: adjusted phosphorylation signal calculated as the ratio of the relative amount of phosphorylated NBR1 (³²P signal) to total GST-NBR1. (D) Western blot showing the specificity of an antibody generated against human NBR1 phosphorylated at threonine 586. Human HeLa cells and murine C2C12 cells were transfected with a control siRNA targeting luciferase or human or mouse NBR1-specific siRNA. (E) Western blot showing the level of phospho-NBR1^T586 signal in gsk3a^−/− MEFs cotransfected with plasmids encoding GST or GST-GSK3A forms (wt, wild-type; inactive, K148A; active, S21A) and DsRed-NBR1 forms (wt, wild-type; nonphosphorylable, T586A). Phosphorylation of NBR1 by endogenous GSK3B is detectable in the absence of wild-type and active GST-GSK3A. The phosphorylation status of this signal was confirmed through treatment of gsk3a^−/− MEF protein extracts with lambda-phosphatase. (F) Confocal pictures showing the immunofluorescence staining of anti-phospho-NBR1^T586 in C2C12 myoblasts transfected with wild-type DsRed-NBR1 or the nonphosphorylable DsRed-NBR1 T586A S590A double mutant. Inserts correspond to a magnification of the dotted frame in each picture. Right panels are merged pictures showing the presence or absence of colocalization. Scale bar: 15 μm. Data in this figure are representative of at least 2 independent experiments.