Ser585-mediated but not Tyr577-mediated signaling selectively affects Akt activation. (A) CTL-EN cells expressing either GMRα alone, the wt GM-CSF receptor (GMRαβc), or the indicated βc mutants were factor deprived for 12 hours in RPMI containing 0.5% FCS, after which the cells were stimulated with 50 ng/mL GM-CSF for 0, 5, 15, and 30 minutes. Following stimulation, cells were lysed and cleared lysates were subjected to SDS-PAGE and immunoblotted sequentially using anti–phospho-tyrosine antibodies (4G10), anti–phospho-JAK2, anti–phospho-STAT5, anti–phospho-Akt, anti–phospho-ERK, and anti-ERK antibodies as described in “Materials and methods.” To examine the activation of ras in response to GM-CSF (B), cells were factor deprived, stimulated with GM-CSF, and lysed as described above. Cell lysates were then subjected to a pull-down experiment using either 10 μg GST (middle panel) or GST-RBD (top panel). The relative amounts of ras in the lysates were determined by Western blot analysis (bottom panel). The pulldowns were washed and the association of ras was examined by Western blot analysis.
