Calmodulin Binds to K-Ras, but Not to H- or N-Ras, and Modulates Its Downstream Signaling

Abstract

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.

K-Ras4B phosphorylation at Ser181 is inhibited by calmodulin and modulates K-Ras activity and function

“…As shown in Figure 2a, no significant differences were observed, in NIH3T3 cells, at least after 10 min of PDGF addition between wild-type K-Ras and K-RasS181A, but PMA treatment activated K-Ras only if CaM was inhibited and if Ser181 could be phosphorylated. In our previous work we also demonstrated that CaM inhibition is not sufficient to induce Ras activation (Villalonga et al, 2001, indicating that PKC activity is also required. Consequently, phosphorylation of K-Ras is essential for its activation by PKC when CaM is inhibited.…”

“…We have shown that CaM inhibition specifically enhances K-Ras activation provided protein kinase C (PKC) is active. It is interesting that we have also demonstrated that K-Ras is a CaM-binding protein, although it is only able to bind CaM if it is GTP-bound and not phosphorylated at Ser181, suggesting an interplay between CaM binding to K-Ras, K-Ras phosphorylation and K-Ras activity (Villalonga et al, 2001Lopez-Alcala et al, 2008).…”

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation

“…In Swiss 3T3 cells, calmodulin binds the GTP-bound K-RasB isoform and downmodulates ERK phosphorylation induced by EGF, bombesin, PDGF and serum. 20 In colon adenocarcinoma cells, CaMKII can bind and directly phosphorylate MEK1, resulting in ERK activation, cell cycle progression and cell proliferation. 21 The CaMKII interplay with multiple proteins of the MAPK signaling pathway increases the complexity of the crosstalk between signals and makes the role of this kinase largely cell context-dependent.…”

Suprachiasmatic Nucleus Circadian Oscillatory Protein, a Novel Binding Partner of K-Ras in the Membrane Rafts, Negatively Regulates MAPK Pathway

“…In addition to these regulatory steps, the presence of Ras isoforms may contribute to a well ordered framework of Ras-mediated signaling. Despite a high degree of sequence similarity among Ras isoforms, accumulating evidence points to a preferential activation of specific effectors by each Ras isoform (9,10). This issue seems to be associated with protein clustering in membrane microdomains such as rafts (11,12).…”