Kinase suppressor of Ras (KSR) is a molecular scaffold that interacts with the 
components of the Raf/MEK/ERK kinase cascade and positively regulates ERK 
signaling. Phosphorylation of KSR1, particularly at Ser(392), is a critical 
regulator of KSR1 subcellular localization and ERK activation. We examined the 
role of phosphorylation of both Ser(392) and Thr(274) in regulating ERK 
activation and cell proliferation. We hypothesized that KSR1 phosphorylation is 
involved in generating signaling specificity through the Raf/MEK/ERK kinase 
cascade in response to stimulation by different growth factors. In fibroblasts, 
platelet-derived growth factor stimulation induces sustained ERK activation and 
promotes S-phase entry. Treatment with epidermal growth factor induces transient 
ERK activation but fails to drive cells into S phase. Mutation of Ser(392) and 
Thr(274) (KSR1.TVSA) promotes sustained ERK activation and cell cycle 
progression with either platelet-derived growth factor or epidermal growth 
factor treatment. KSR1(-/-) mouse embryo fibroblasts expressing KSR1.TVSA 
proliferate two times faster and grow to a higher density than cells expressing 
the same level of wild-type KSR1. In addition, KSR1.TVSA is more stable than 
wild-type KSR1. These data demonstrate that phosphorylation and stability of the 
molecular scaffold KSR1 are critical regulators of growth factor-specific 
responses that promote cell proliferation.