Related Experiment Videos
Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling
P Villalonga1, C López-Alcalá, M Bosch
1Departament de Biologia Cellular i Anatomia Patològica, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain.
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.
Insights
Calmodulin inhibits Ras activation, a key pathway in cell signaling. Inhibiting calmodulin boosts Ras/ERK pathway activation, particularly K-RasB, suggesting a direct interaction regulates signaling.
Area of Science:
- Cellular Biology
- Molecular Biology
- Signal Transduction
Background:
- Ras/Raf/MEK/ERK pathway activation drives cellular responses.
- Calmodulin (CaM) is crucial for down-regulating this pathway, partly by inhibiting Ras activation.
- Previous studies indicated CaM's inhibitory role in Ras activation.
Purpose of the Study:
- To investigate the role of calmodulin in Ras/ERK pathway regulation.
- To identify calmodulin-binding proteins involved in Ras signaling.
- To elucidate the mechanism of calmodulin's interaction with Ras.
Main Methods:
- Calmodulin affinity chromatography was used to identify binding proteins.
- Ras and Raf proteins were analyzed for calmodulin binding in cellular lysates.
- K-RasB isoform-specific binding and activation were assessed.
Main Results:
- Calmodulin inhibition synergized with various stimuli to enhance ERK activation.
- Ras and Raf proteins bound to calmodulin, with Ras binding favored by GTP-bound Ras.
- Only K-RasB isoform directly bound to calmodulin, and its activation was preferentially enhanced by calmodulin inhibition.
- Calmodulin kinase II calmodulin-binding domain inhibited the calmodulin-K-RasB interaction.
Conclusions:
- GTP-bound K-RasB directly interacts with calmodulin.
- This direct interaction is a key mechanism for modulating Ras signaling.
- Calmodulin acts as a direct regulator of K-RasB activity.