The KSR1 scaffold translocates from the cytosol to the plasma membrane upon Ras activation and coordinates the assembly of a large multiprotein complex that functions to regulate the intensity and duration of ERK cascade signaling. In the past fiscal year, we have identified a hydrophobic motif in the proline-rich sequence of MEK1 and MEK2 that is required for constitutive binding to the KSR1 scaffold and find that KSR1 forms a ternary complex with B-Raf and MEK in response to growth factor treatment that enhances B-Raf-mediated MEK activation. Strikingly, we have also found that docking of active ERK to the KSR1 scaffold allows ERK to phosphorylate KSR1 and B-Raf on feedback sites and that these feedback events attenuate ERK cascade signaling by promoting the dissociation of the B-RAF/KSR1/MEK complex and causing KSR1 to be released from the plasma membrane. This year our laboratory has also taken a proteomic approach to further investigate the functional properties of the two mammalian KSR scaffolds, KSR1 and KSR2. These studies have revealed that both KSR1 and KSR2 interact with the core kinase components of the ERK cascade and have a common function in promoting RTK-mediated ERK signaling. Remarkably, these studies also found that the protein phosphatase calcineurin selectively interacts with KSR2 and that KSR2 uniquely contributes to Ca2+-mediated ERK signaling. In response to increased Ca2+ levels, we found that calcineurin dephosphorylates KSR2 on specific sites and, as a a result, regulates the membrane-localization and scaffolding activity of KSR2. Moreover, we found that depletion of KSR2 impairs Ca2+-mediated ERK activation and signaling in two different cell lines that express KSR2, INS1 pancreatic β-cells and NG108 neuroblastoma cells. These findings identify KSR2 as a Ca2+-regulated ERK scaffold and reveal a new mechanism whereby Ca2+ impacts Ras to ERK pathway signaling.
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