Fig. 4. Phosphorylation is not required for activation of ERM proteins but required for the maintenance of the active state in A431 cells. (A) Serum-starved A431 cells cultured in the absence (a,b) or presence (c-f) of staurosporine (10 minutes at 100 nM) were stimulated with EGF for 30 seconds and stained with anti-ERM mAb, CR22 (a,c) and anti-CPERM mAb (b,d). Although CPERMs were almost completely dephosphorylated by staurosporine treatment, microvillar elongation and recruiting of ERM proteins appeared normal. EGF-induced microvilli-like structures in the presence of staurosporine contained both ERM proteins stained with anti-ERM pAb, TK89 (e) and actin filaments stained with rhodamine phalloidin (f). Bars, a-d, 10 µm; e,f, 10 µm. (B) Translocation of ERM proteins in A431 cells after EGF stimulation. Serum-starved cells cultured in the absence (a-e) or presence (f-j) of 25 nM staurosporine for 1 hour were stimulated with EGF. Cells were fixed with TCA and stained with anti-ERM mAb, CR22. Times after EGF stimulation (0 minute: a,f; 2 minutes: b,g; 5 minutes: c,h; 10 minutes: d,i; 30 minutes: e,j) are indicated. Note that ERM proteins recruited to cell surface structures such as microvilli and ruffling membranes after EGF stimulation from the cytoplasm were relocated to the cytoplasm much faster in the presence of staurosporine than in its absence. Bar, 20 µm.