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.

ACKNOWLEDGMENTS

We thank F. R. McKenzie (Nice, France) for the gift of GST-RBD plasmid, L. Carpenter (NIMR, London, United Kingdom) for the gift of purified GDP- and GTP-bound K-Ras, and J. Ureña (Barcelona, Spain) for the gift of anti-Grb2 antibody. We also thank Mathew Garnett (ICR, London, United Kingdom) for preparing the insect cell expression vectors for GST-H-RasV12 and GST-K-RasV12.

This work was supported by CICYT grant SAF97-014. Priam Villalonga is a recipient of a predoctoral fellowship from the CIRIT.