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A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

Amano M, Tsumura Y, Taki K, Harada H, Mori K, Nishioka T, Kato K, Suzuki T, Nishioka Y, Iwamatsu A, Kaibuchi K - PLoS ONE (2010)

Bottom Line: Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates.Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo.This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Japan.

ABSTRACT

Background: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored.

Methodology/principal findings: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo.

Conclusions/significance: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

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Related in: MedlinePlus

Effect of mutations of DCX at phosphorylation sites on microtubule organization in HeLa cells.(A) Effect of overexpression of DCX-WT, -T42A and -T42E in HeLa cells. DCX mutants with substitutions at phosphorylation sites were expressed in HeLa cells. HeLa cells expressing GFP-DCX or its mutants were fixed in methanol for 10 min at room temperature. After washing, the cells were immunostained with anti-GFP and anti-tubulin Abs. Colors indicate GFP (green) and tubulin (red). These results are representatives of at least three independent experiments. Scale bar, 10 µm. (B) The percentages of cells with highly bundled microtubules. Data are means ± SD of three independent experiments. Asterisks indicate that there is a significant difference from the value of control cells (p<0.05).
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pone-0008704-g006: Effect of mutations of DCX at phosphorylation sites on microtubule organization in HeLa cells.(A) Effect of overexpression of DCX-WT, -T42A and -T42E in HeLa cells. DCX mutants with substitutions at phosphorylation sites were expressed in HeLa cells. HeLa cells expressing GFP-DCX or its mutants were fixed in methanol for 10 min at room temperature. After washing, the cells were immunostained with anti-GFP and anti-tubulin Abs. Colors indicate GFP (green) and tubulin (red). These results are representatives of at least three independent experiments. Scale bar, 10 µm. (B) The percentages of cells with highly bundled microtubules. Data are means ± SD of three independent experiments. Asterisks indicate that there is a significant difference from the value of control cells (p<0.05).

Mentions: Phosphorylation of DCX by Cdk5 or PKA decreases the ability of DCX to bind to microtubules [9], [10]. To examine the effects of phosphorylation of DCX at the Rho-kinase site, DCX mutants with a substitution at the phosphorylation site were expressed in HeLa cells (Figure S2). GFP was diffusely distributed in HeLa cells (Figure 6A, a). GFP-tagged DCX-WT and the -T42A mutant were localized to microtubules (Figure 6A, d and g) and caused highly bundled microtubules in a certain population of cells (Figure 6A, e and h, and 6B), whereas GFP-DCX-T42E, a phospho-mimicking form, was still localized to the microtubules (Figure 6A, j), but lost its ability to bundle microtubules (Figure 6A, k, and 6B), suggesting that phosphorylation of DCX at Thr-42 weakens its microtubule-bundling activity.


A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

Amano M, Tsumura Y, Taki K, Harada H, Mori K, Nishioka T, Kato K, Suzuki T, Nishioka Y, Iwamatsu A, Kaibuchi K - PLoS ONE (2010)

Effect of mutations of DCX at phosphorylation sites on microtubule organization in HeLa cells.(A) Effect of overexpression of DCX-WT, -T42A and -T42E in HeLa cells. DCX mutants with substitutions at phosphorylation sites were expressed in HeLa cells. HeLa cells expressing GFP-DCX or its mutants were fixed in methanol for 10 min at room temperature. After washing, the cells were immunostained with anti-GFP and anti-tubulin Abs. Colors indicate GFP (green) and tubulin (red). These results are representatives of at least three independent experiments. Scale bar, 10 µm. (B) The percentages of cells with highly bundled microtubules. Data are means ± SD of three independent experiments. Asterisks indicate that there is a significant difference from the value of control cells (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806833&req=5

pone-0008704-g006: Effect of mutations of DCX at phosphorylation sites on microtubule organization in HeLa cells.(A) Effect of overexpression of DCX-WT, -T42A and -T42E in HeLa cells. DCX mutants with substitutions at phosphorylation sites were expressed in HeLa cells. HeLa cells expressing GFP-DCX or its mutants were fixed in methanol for 10 min at room temperature. After washing, the cells were immunostained with anti-GFP and anti-tubulin Abs. Colors indicate GFP (green) and tubulin (red). These results are representatives of at least three independent experiments. Scale bar, 10 µm. (B) The percentages of cells with highly bundled microtubules. Data are means ± SD of three independent experiments. Asterisks indicate that there is a significant difference from the value of control cells (p<0.05).
Mentions: Phosphorylation of DCX by Cdk5 or PKA decreases the ability of DCX to bind to microtubules [9], [10]. To examine the effects of phosphorylation of DCX at the Rho-kinase site, DCX mutants with a substitution at the phosphorylation site were expressed in HeLa cells (Figure S2). GFP was diffusely distributed in HeLa cells (Figure 6A, a). GFP-tagged DCX-WT and the -T42A mutant were localized to microtubules (Figure 6A, d and g) and caused highly bundled microtubules in a certain population of cells (Figure 6A, e and h, and 6B), whereas GFP-DCX-T42E, a phospho-mimicking form, was still localized to the microtubules (Figure 6A, j), but lost its ability to bundle microtubules (Figure 6A, k, and 6B), suggesting that phosphorylation of DCX at Thr-42 weakens its microtubule-bundling activity.

Bottom Line: Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates.Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo.This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Japan.

ABSTRACT

Background: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored.

Methodology/principal findings: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo.

Conclusions/significance: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

Show MeSH
Related in: MedlinePlus