The effects of RhoA phosphorylation (⁸⁸S and ¹⁰⁰T) on RhoA interaction with ERK and on EGF-induced ERK phosphorylation.

<p><b>(A)</b> The effects of RhoA phosphorylation on its interaction with ERK. COS-7 cells were serum starved and treated with EGF (50 ng/ml) for 15 min. The cell lysates were incubated with GST-fused wild type and mutant RhoA proteins bound to glutathione-sepharose beads. ERK pulldowns were analyzed by immunoblotting with antibodies to ERK. GST fusion protein loading was verified by Amido Black staining of the nitrocellulose membrane. <b>(B)</b> The effects of RhoA phosphorylation on EGF-induced ERK phosphorylation. COS-7 cells were transfected with expression constructs encoding GFP-tagged wild type and mutant RhoA proteins and these proteins were overexpressed. After serum starvation, cells were stimulated with EGF for 15 min. ERK phosphorylation and activation was determined by immunoblotting cell lysates with antibodies to p-ERK and p-ELK1, respectively. The expression of GFP-RhoA wild type and mutant proteins was determined by immunoblotting with antibodies to GFP.