(A) PKCζ phosphorylates Numb in a dose-dependent manner. Purified GST-Numb immobilized on glutathione sepharose beads was incubated with purified His-PKCζ. Bound protein complex was detected by blot with a monoclonal antibody phospho-Ser/Thr (MPM2) or antibody against phospho-Numb (p-Numb (S284)).
(B) Specificity of anti-phospho-Numb (S284). HEK 293T cells were transfected with plasmids expressing wt Numb or Numb mutant (S284A). Lysates were precipitated with anti-Numb. Numb IPs were incubated with or without active PKCζ, and blotted with anti-p-Numb (S284) and anti-Numb, respectively.
(C) BDNF stimulates Numb phosphorylation by PKCζ in vivo. GCPs from P7 mice were treated with 50 ng/ml BDNF for 5 minutes in the presence or absence of phosphatase inhibitor calyculin-A and/or 10 μM PKC inhibitor chelerythrine. Lysates were blotted with anti-p-Numb (S284) and anti-Numb, respectively.
(D) Phosphorylated Numb (green) colocalizes with TrkB (red) in the leading processes of GCPs along Bergmann glia in response to BDNF. Staining with p-Numb (S284) antibody and TrkB antibody. DAPI stains nuclei (blue). Scale bar: 5 μm
(E) Numb mutations at PKCζ phosphorylation sites reduce Numb interaction with TrkB. HEK 293T cells co-transfected with plasmid expressing TrkB-GFP and plasmid expressing wild type Numb (wt) or Numb mutant for all three aPKC phosphorylation sites (mut), and treated with vehicle or BDNF. Lysates precipitated with anti-GFP and blotted with anti-Numb and anti-GFP, respectively.
(F) Model for Numb function in cell migration. In response to a BDNF gradient, Numb associates with activated TrkB to regulate receptor endocytosis and recruit aPKC. Activated aPKC then phosphorylates Numb. Phosphorylation of Numb serves in a feed-forward loop to potentiate binding of Numb to TrkB and thereby fosters formation of TrkB/Numb endosomal complexes. Endocytosis is required for Tiam1-mediated Rac activation and localization of signaling endosomes to leading processes and/or for recycling TrkB to plasma membrane in leading processes.