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Ras and calcium signaling pathways converge at Raf1 via the Shoc2 scaffold protein.

Yoshiki S, Matsunaga-Udagawa R, Aoki K, Kamioka Y, Kiyokawa E, Matsuda M - Mol. Biol. Cell (2010)

Bottom Line: Increase in Ca(2+) concentration has been shown to modulate the Ras-dependent activation of Raf1; however, the mechanism underlying this effect remains elusive.Furthermore, the Ca(2+)-dependent activation of Raf1 was found to be abrogated by knockdown of Shoc2, a scaffold protein that binds both Ras and Raf1.These observations indicated that the Shoc2 scaffold protein modulates Ras-dependent Raf1 activation in a Ca(2+)- and calmodulin-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
Situated downstream of Ras is a key signaling molecule, Raf1. Increase in Ca(2+) concentration has been shown to modulate the Ras-dependent activation of Raf1; however, the mechanism underlying this effect remains elusive. Here, to characterize the role of Ca(2+) in Ras signaling to Raf1, we used a synthetic guanine nucleotide exchange factor (GEF) for Ras, eGRF. In HeLa cells expressing eGRF, Ras was activated by the cAMP analogue 007 as efficiently as by epidermal growth factor (EGF), whereas the activation of Raf1, MEK, and ERK by 007 was about half of that by EGF. Using a biosensor based on fluorescence resonance energy transfer, it was found that activation of Raf1 at the plasma membrane required not only Ras activation but also an increase in Ca(2+) concentration or inhibition of calmodulin. Furthermore, the Ca(2+)-dependent activation of Raf1 was found to be abrogated by knockdown of Shoc2, a scaffold protein that binds both Ras and Raf1. These observations indicated that the Shoc2 scaffold protein modulates Ras-dependent Raf1 activation in a Ca(2+)- and calmodulin-dependent manner.

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Shoc2 association with Ras by the calmodulin inhibitor or calcium ionophore. HeLa-eGRF1 cells expressing CFP-KRas and YFP (A and C) or Shoc2-YFP (B and D) were stimulated with 100 μM W-13 + 100 μM 007 (A and B) or 1 μM ionomycin + 100 μM 007 (C and D) and time-lapse–imaged as described in the text. The ratio of corrected FRET versus CFP was averaged over each cell and plotted against time. Shown are averages ± SD (left panel) and data of each cell (right panel; n = 11). The asterisks show the time points in which difference from time = 0 was significant by Student's t test (p < 0.001).
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Figure 7: Shoc2 association with Ras by the calmodulin inhibitor or calcium ionophore. HeLa-eGRF1 cells expressing CFP-KRas and YFP (A and C) or Shoc2-YFP (B and D) were stimulated with 100 μM W-13 + 100 μM 007 (A and B) or 1 μM ionomycin + 100 μM 007 (C and D) and time-lapse–imaged as described in the text. The ratio of corrected FRET versus CFP was averaged over each cell and plotted against time. Shown are averages ± SD (left panel) and data of each cell (right panel; n = 11). The asterisks show the time points in which difference from time = 0 was significant by Student's t test (p < 0.001).

Mentions: To directly assess this possibility, we examined the Shoc2 binding to Ras upon W-13 treatment by the intermolecular FRET method with CFP-KRas and Shoc2-YFP (Figure 7). We found that the level of FRET increased significantly upon treatment with 007 and W-13 or 007 and ionomycin, indicating the Shoc2 association with KRas. In conclusion, the results shown here strongly argue that calmodulin inhibits Shoc2 association with Ras and thereby formation of the Ras-Shoc2-Raf1 complex and that Ca2+ enhances Ras to Raf1 signaling by suppressing calmodulin's inhibition of Shoc2 binding to Ras. We performed similar experiments by using CFP-HRas and obtained essentially the same results (data not shown).


Ras and calcium signaling pathways converge at Raf1 via the Shoc2 scaffold protein.

Yoshiki S, Matsunaga-Udagawa R, Aoki K, Kamioka Y, Kiyokawa E, Matsuda M - Mol. Biol. Cell (2010)

Shoc2 association with Ras by the calmodulin inhibitor or calcium ionophore. HeLa-eGRF1 cells expressing CFP-KRas and YFP (A and C) or Shoc2-YFP (B and D) were stimulated with 100 μM W-13 + 100 μM 007 (A and B) or 1 μM ionomycin + 100 μM 007 (C and D) and time-lapse–imaged as described in the text. The ratio of corrected FRET versus CFP was averaged over each cell and plotted against time. Shown are averages ± SD (left panel) and data of each cell (right panel; n = 11). The asterisks show the time points in which difference from time = 0 was significant by Student's t test (p < 0.001).
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Related In: Results  -  Collection

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Figure 7: Shoc2 association with Ras by the calmodulin inhibitor or calcium ionophore. HeLa-eGRF1 cells expressing CFP-KRas and YFP (A and C) or Shoc2-YFP (B and D) were stimulated with 100 μM W-13 + 100 μM 007 (A and B) or 1 μM ionomycin + 100 μM 007 (C and D) and time-lapse–imaged as described in the text. The ratio of corrected FRET versus CFP was averaged over each cell and plotted against time. Shown are averages ± SD (left panel) and data of each cell (right panel; n = 11). The asterisks show the time points in which difference from time = 0 was significant by Student's t test (p < 0.001).
Mentions: To directly assess this possibility, we examined the Shoc2 binding to Ras upon W-13 treatment by the intermolecular FRET method with CFP-KRas and Shoc2-YFP (Figure 7). We found that the level of FRET increased significantly upon treatment with 007 and W-13 or 007 and ionomycin, indicating the Shoc2 association with KRas. In conclusion, the results shown here strongly argue that calmodulin inhibits Shoc2 association with Ras and thereby formation of the Ras-Shoc2-Raf1 complex and that Ca2+ enhances Ras to Raf1 signaling by suppressing calmodulin's inhibition of Shoc2 binding to Ras. We performed similar experiments by using CFP-HRas and obtained essentially the same results (data not shown).

Bottom Line: Increase in Ca(2+) concentration has been shown to modulate the Ras-dependent activation of Raf1; however, the mechanism underlying this effect remains elusive.Furthermore, the Ca(2+)-dependent activation of Raf1 was found to be abrogated by knockdown of Shoc2, a scaffold protein that binds both Ras and Raf1.These observations indicated that the Shoc2 scaffold protein modulates Ras-dependent Raf1 activation in a Ca(2+)- and calmodulin-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
Situated downstream of Ras is a key signaling molecule, Raf1. Increase in Ca(2+) concentration has been shown to modulate the Ras-dependent activation of Raf1; however, the mechanism underlying this effect remains elusive. Here, to characterize the role of Ca(2+) in Ras signaling to Raf1, we used a synthetic guanine nucleotide exchange factor (GEF) for Ras, eGRF. In HeLa cells expressing eGRF, Ras was activated by the cAMP analogue 007 as efficiently as by epidermal growth factor (EGF), whereas the activation of Raf1, MEK, and ERK by 007 was about half of that by EGF. Using a biosensor based on fluorescence resonance energy transfer, it was found that activation of Raf1 at the plasma membrane required not only Ras activation but also an increase in Ca(2+) concentration or inhibition of calmodulin. Furthermore, the Ca(2+)-dependent activation of Raf1 was found to be abrogated by knockdown of Shoc2, a scaffold protein that binds both Ras and Raf1. These observations indicated that the Shoc2 scaffold protein modulates Ras-dependent Raf1 activation in a Ca(2+)- and calmodulin-dependent manner.

Show MeSH
Related in: MedlinePlus