The scaffolding protein KSR1 (kinase suppressor of Ras 1) assembles extracellular-regulated kinase (ERK) and its upstream activators at the plasma membrane. In a proteomic analysis of KSR protein complexes, Dougherty et al. found that the related KSR2 not only is an ERK scaffold like KSR1, but also selectively binds to the calcium-activated phosphatase calcineurin. Calcineurin directly interacted with KSR2 through an LxPV (Leu-X-Pro-Val) motif not found in KSR1; this interaction was abolished by mutation of this motif to alanine residues (which was referred to as LxPVm) or pretreatment with the calcineurin inhibitor cyclosporin A (CsA). In COS-7 cells expressing KSR1, wild-type KSR2 (WT-KSR2), or LxVPm-KSR2, all three KSR proteins were found in the cytosol of serum-starved cells and relocated to the plasma membrane on serum treatment, which would be expected to activate ERK signaling through growth factor pathways. However, only WT-KSR2 relocated to the plasma membrane in response to treatments that generate high intracellular Ca2+ concentrations, such as histamine or the calcium ionophore ionomycin and the phorbol ester PMA (phorbol 12-myristate 13-acetate). Phosphorylated and activated ERK was detected in cells treated with epidermal growth factor regardless of transfection condition, but the combination of ionomycin and PMA increased ERK activity only in cells transfected with WT-KSR2, an effect that could be blocked by CsA. Phosphorylation of KSR2 at Ser198, Thr287, and Ser310 was increased by ionomycin and PMA treatment; intriguingly, only mutation of Ser310 to alanine (which inhibited KSR2 interaction with 14-3-3) resulted in constitutive membrane localization of KSR2. Glucose treatment of the pancreatic β cell line INS1 or KCl treatment of the neuroblastoma cell line NG108, both of which stimulate ERK activity, decreased phosphorylation of KSR2 at Ser198, Thr287, and Ser310, increased association of activated ERK with endogenous KSR2, and promoted localization of KSR2 at the plasma membrane. RNA interference directed against KSR2 in NG108 cells decreased ERK activation and neurite outgrowth in response to KCl treatment, effects that were more efficiently rescued by re-expression of wild-type KSR2 than that of the LxVPm mutant. Thus, KSR2 couples intracellular calcium increases to elevated ERK signaling.
M. K. Dougherty, D. A. Ritt, M. Zhou, S. I. Specht, D. M. Monson, T. D. Veenstra, D. K. Morrison, KSR2 is a calcineurin substrate that promotes ERK cascade activation in response to calcium signals. Mol. Cell 34, 652–662 (2009).[PubMed]