Figure 4.

Interaction of Fez1 with Microtubules. (A) Association of Fez1 with assembled tubulin. Forty μg each of disassembled (lanes 1 and 3) or assembled (lanes 2 and 4) tubulin, which was extracted from brain, was electrophoresed and stained with Coomassie blue (lanes 1 and 2) or immunoblotted with anti-Fez1 antibody (lanes 3 and 4). (B) Double immunofluorescence with mouse anti-tubulin and fluorescein-labeled anti-mouse antibody (Tubulin), and with rabbit anti-Fez1 and rhodamine-labeled anti-rabbit antibody (Fez1). Yellow areas depict colocalization of the two fluorochromes (Tubulin + Fez1). Fetal kidney 293 cells were cultured in medium without (293) or with (293 + Noc) nocodazole (40 μg/ml) before staining. 293/AS (similar to Fig. 5 A–C), 293 cell transfectants expressing ecdysone-inducible antisense FEZ1, cultured with ecdysone derivative ponasterone A to inhibit endogenous Fez1 expression. (Bar = 5 μm.) (C) Fez1-negative cervical cancer HeLaS3 cells transfected with full-length (Wt) or N-terminal truncated (C-term, lacking N-terminal 388-aa portion) FEZ1 cDNA in pcDNA4V5 vector. MTS cell-growth assay (Upper) was performed, similar to Fig. 1B. Immunofluorescence (Lower) was performed with anti-V5 tag and fluorescein-labeled anti-mouse antibody. wt, wild-type FEZ1; C-term, truncated FEZ1; Control, without primary antibody. (Bar = 5 μm.) (D) In vitro kinase assay of Fez1 proteins. Purified recombinant GST-Fez1 proteins (wt, S29P, and Q501Stop) were subjected to the analysis. PKA was incubated with wt (lane 1), mutated (Q501Stop) (lane 2), or mutated (S29P) protein (lane 3) at 30°C for 45 min for [γ-³²P]ATP labeling. Samples were subjected to SDS/PAGE, dried and exposed to film. (E) In vitro kinetics of Fez1 phosphorylation by PKA. One μg each of purified GST-wt or S29P mutated Fez1 protein (Mut) was subjected to kinase assay using [γ-³²P]ATP, without or with (+ Hep) heparin at 20 μg/ml. Samples were separated by SDS/PAGE, dried, and exposed to film for quantitative analysis with the PhosphorImager (Molecular Dynamics). y axis, mol of phosphate per mol of Fez1. (F and G) Involvement of Fez1 with tubulin polymerization in vitro. Purified tubulin and purified MAP2 were incubated at 37°C for indicated times with GST-Fez1 (F, ●), PKA-phosphorylated GST-Fez1 (F, ⊗), GST-mutated Fez1 (S29P) (G, ■), and PKA-phosphorylated GST-mutated Fez1 (S29P) (G, square with cross). Controls in F and G include reactions with tubulin and MAP2 (□), with tubulin, MAP, and GST (diamonds with cross), or reaction buffer alone (○). Absorbance at 350 nm was measured with spectrophotometer, and the increase is shown.