Figure 2.

Cell-cycle analysis and protein modification of Fez1. (A and B) Cell-cycle analysis of synchronized MCF7 FEZ1 transfectants. FEZ1 transfectants were cultured in Tet-free medium and treated with double-thymidine block. After medium was exchanged for fresh medium, cells were fixed at indicated times and subjected to flow-cytometry analysis. Representative data for clone 54 is shown in A. Data in B are shown as ratios of G₂/M (Upper) or S (Lower) to G₁ population of clone 18 (solid line) or clone 54 (dotted line). ●, with induced Fez1; ○, without Fez1. (C and D) Endogenous Fez1 protein analysis in synchronized 293 cells in medium without (C) or with (D) 20 μM PKA inhibitor. After medium was exchanged for fresh medium, cellular proteins were extracted at indicated times and subjected to immunoblot analysis with anti-Fez1 or anti-actin antibody. P in figure denotes Fez1 with lower mobility. (E) Serine phosphorylation of Fez1 protein in MCF7 Tet-inducible FEZ1 transfectants (clone 54). After synchronizing, cells were incubated in fresh medium for indicated times. Lysates were immunoprecipitated with anti-Fez1 antibody, followed by immunoblot analysis with anti-phosphoserine (P-Ser) or anti-Fez1 antibody (Fez1). Cells were harvested at 0 (lane 1), 1 (lane 2), 2.5 (lane 3), 6 (lane 4), or 8 hr (lane 5) after relief from G₁/S block. (F) In vivo phosphorylation of Tet-inducible MCF7 FEZ1 transfectants (clone 54) with [³²P]orthophosphate. Cells were cultured in medium containing [³²P]orthophosphate with Tet (lane 1) and without Tet (lane 3). Lane 2, control vector transfectant. Lysates were immunoprecipitated with anti-Fez1 antibody, subjected to SDS/PAGE, dried, and exposed to film.