Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.

We previously showed that K-Ras is a calmodulin-binding protein. Involvement of this interaction in anterograde and retrograde transport of K-Ras was then suggested. To test this we have analyzed here the domains of K-Ras essential for the interaction with calmodulin. At least three different regions in the K-Ras molecule were important; they are the hypervariable region, the ␣-helix between amino acids 151 and 166, and the Switch II. Within the hypervariable region, both the hydrophobic farnesyl group and the positive-charged amino acids were essential for the interaction between K-Ras and calmodulin in cellular extracts. Consistently, K-Ras S181D, which mimics phosphorylation of Ser-181 of K-Ras, also completely abolished binding to calmodulin. K-Ras mutants correctly farnesylated that did not bind calmodulin were all located at plasma membrane, showing that calmodulin interaction was not required for the transport of K-Ras to plasma membrane. In NIH3T3 cells, K-Ras and calmodulin colocalized mainly in the plasma membrane even after the addition of Ca 2؉ ionophore, indicating that interaction did not directly lead to K-Ras internalization. Furthermore, using a K-Ras with impaired binding to calmodulin but with membrane localization, we could demonstrate in striatal neurones that interaction between K-Ras and calmodulin was not required for Golgi K-Ras translocation induced by Ca 2؉

Ras proteins are small guanosine triphosphatases involved in the regulation of important cellular functions such as proliferation, differentiation, and apoptosis. Understanding the intracellular trafficking of Ras proteins is crucial to identify novel Ras signaling platforms. In this study, we report that epidermal growth factor triggers Kirsten Ras (KRas) translocation onto endosomal membranes (independently of calmodulin and protein kinase C phosphorylation) through a clathrin-dependent pathway. From early endosomes, KRas but not Harvey Ras or neuroblastoma Ras is sorted and transported to late endosomes (LEs) and lysosomes. Using yellow fluorescent protein–Raf1 and the Raichu-KRas probe, we identified for the first time in vivo–active KRas on Rab7 LEs, eliciting a signal output through Raf1. On these LEs, we also identified the p14–MP1 scaffolding complex and activated extracellular signal-regulated kinase 1/2. Abrogation of lysosomal function leads to a sustained late endosomal mitogen-activated protein kinase signal output. Altogether, this study reveals novel aspects about KRas intracellular trafficking and signaling, shedding new light on the mechanisms controlling Ras regulation in the cell.

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