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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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Reversal of the effect of Shoc2 knockdown by exogenous Shoc2 expression. (A) HeLa-eGRF1 cells were transfected with either Shoc2-specific siRNA or scrambled siRNA. Forty-two hours after transfection, cells were serum-starved for 6 h and stimulated with 5 ng/ml EGF, 100 μM 007, or 100 μM 007 and 1 μM ionomycin for 5 min, followed by immunoblotting analysis. (B) The activity of Raf1 and MEK as measured with the phospho-specific antibodies was normalized to the maximum values of scramble siRNA-transfected EGF-stimulated cells. The fraction of phospho-ERK was calculated from the ratio of phospho-ERK versus ERK shown in A. Averages are shown ± SD (n = 3). (C) HeLa-eGRF1 cells stably expressing siRNA-resistant Myc-tagged Shoc2 were transfected with either Shoc2-specific siRNA or scrambled siRNA. Forty-two hours after transfection, cells were serum-starved for 6 h and stimulated with 5 ng/ml EGF, 100 μM 007, or 100 μM 007, and 1 μM ionomycin for 5 min, followed by immunoblotting analysis. (D) The activities of Raf1, MEK, and ERK were measured as in C. Averages are shown ± SD (n = 3).
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Figure 5: Reversal of the effect of Shoc2 knockdown by exogenous Shoc2 expression. (A) HeLa-eGRF1 cells were transfected with either Shoc2-specific siRNA or scrambled siRNA. Forty-two hours after transfection, cells were serum-starved for 6 h and stimulated with 5 ng/ml EGF, 100 μM 007, or 100 μM 007 and 1 μM ionomycin for 5 min, followed by immunoblotting analysis. (B) The activity of Raf1 and MEK as measured with the phospho-specific antibodies was normalized to the maximum values of scramble siRNA-transfected EGF-stimulated cells. The fraction of phospho-ERK was calculated from the ratio of phospho-ERK versus ERK shown in A. Averages are shown ± SD (n = 3). (C) HeLa-eGRF1 cells stably expressing siRNA-resistant Myc-tagged Shoc2 were transfected with either Shoc2-specific siRNA or scrambled siRNA. Forty-two hours after transfection, cells were serum-starved for 6 h and stimulated with 5 ng/ml EGF, 100 μM 007, or 100 μM 007, and 1 μM ionomycin for 5 min, followed by immunoblotting analysis. (D) The activities of Raf1, MEK, and ERK were measured as in C. Averages are shown ± SD (n = 3).

Mentions: In agreement with the suppression of Raf1 activation by Shoc2 knockdown, the activation of both MEK and ERK was also suppressed by Shoc2 knockdown in cells stimulated with EGF or 007 plus ionomycin (Figure 5A and Figure 5B). This effect of Shoc2 knockdown was counteracted by the expression of Shoc2 cDNA, which was resistant to the siRNA used in this experiment, validating the specificity of siRNA against Shoc2 (Figure 5, C and D).


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)

Reversal of the effect of Shoc2 knockdown by exogenous Shoc2 expression. (A) HeLa-eGRF1 cells were transfected with either Shoc2-specific siRNA or scrambled siRNA. Forty-two hours after transfection, cells were serum-starved for 6 h and stimulated with 5 ng/ml EGF, 100 μM 007, or 100 μM 007 and 1 μM ionomycin for 5 min, followed by immunoblotting analysis. (B) The activity of Raf1 and MEK as measured with the phospho-specific antibodies was normalized to the maximum values of scramble siRNA-transfected EGF-stimulated cells. The fraction of phospho-ERK was calculated from the ratio of phospho-ERK versus ERK shown in A. Averages are shown ± SD (n = 3). (C) HeLa-eGRF1 cells stably expressing siRNA-resistant Myc-tagged Shoc2 were transfected with either Shoc2-specific siRNA or scrambled siRNA. Forty-two hours after transfection, cells were serum-starved for 6 h and stimulated with 5 ng/ml EGF, 100 μM 007, or 100 μM 007, and 1 μM ionomycin for 5 min, followed by immunoblotting analysis. (D) The activities of Raf1, MEK, and ERK were measured as in C. Averages are shown ± SD (n = 3).
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Figure 5: Reversal of the effect of Shoc2 knockdown by exogenous Shoc2 expression. (A) HeLa-eGRF1 cells were transfected with either Shoc2-specific siRNA or scrambled siRNA. Forty-two hours after transfection, cells were serum-starved for 6 h and stimulated with 5 ng/ml EGF, 100 μM 007, or 100 μM 007 and 1 μM ionomycin for 5 min, followed by immunoblotting analysis. (B) The activity of Raf1 and MEK as measured with the phospho-specific antibodies was normalized to the maximum values of scramble siRNA-transfected EGF-stimulated cells. The fraction of phospho-ERK was calculated from the ratio of phospho-ERK versus ERK shown in A. Averages are shown ± SD (n = 3). (C) HeLa-eGRF1 cells stably expressing siRNA-resistant Myc-tagged Shoc2 were transfected with either Shoc2-specific siRNA or scrambled siRNA. Forty-two hours after transfection, cells were serum-starved for 6 h and stimulated with 5 ng/ml EGF, 100 μM 007, or 100 μM 007, and 1 μM ionomycin for 5 min, followed by immunoblotting analysis. (D) The activities of Raf1, MEK, and ERK were measured as in C. Averages are shown ± SD (n = 3).
Mentions: In agreement with the suppression of Raf1 activation by Shoc2 knockdown, the activation of both MEK and ERK was also suppressed by Shoc2 knockdown in cells stimulated with EGF or 007 plus ionomycin (Figure 5A and Figure 5B). This effect of Shoc2 knockdown was counteracted by the expression of Shoc2 cDNA, which was resistant to the siRNA used in this experiment, validating the specificity of siRNA against Shoc2 (Figure 5, C and D).

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