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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.

Show MeSH
Isolation of interacting proteins for the catalytic domain of Rho-kinase.(A) Domain structure of Rho-kinase and the constructs used for affinity column chromatography. (B) Strategy for isolation of protein kinase substrates. (C, D) Isolation of Rho-kinase-cat-interacting proteins from rat brain cytosol (C) and P2 (D) fractions. The cytosolic or P2 fraction of rat brain lysate was loaded onto a Glutathione-Sepharose column coated with either GST, GST-Rho-kinase-cat, GST-Rho-kinase-cat-KD or GST-PKN-cat. The bound proteins were eluted by addition of 1 M NaCl after washing with 50 mM NaCl. The eluates were analyze by SDS-PAGE, and visualized by silver staining. Arrowheads indicate the GST-tagged proteins used as baits.
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pone-0008704-g001: Isolation of interacting proteins for the catalytic domain of Rho-kinase.(A) Domain structure of Rho-kinase and the constructs used for affinity column chromatography. (B) Strategy for isolation of protein kinase substrates. (C, D) Isolation of Rho-kinase-cat-interacting proteins from rat brain cytosol (C) and P2 (D) fractions. The cytosolic or P2 fraction of rat brain lysate was loaded onto a Glutathione-Sepharose column coated with either GST, GST-Rho-kinase-cat, GST-Rho-kinase-cat-KD or GST-PKN-cat. The bound proteins were eluted by addition of 1 M NaCl after washing with 50 mM NaCl. The eluates were analyze by SDS-PAGE, and visualized by silver staining. Arrowheads indicate the GST-tagged proteins used as baits.

Mentions: To screen potential substrates of Rho-kinase, we examined whether the active catalytic fragment of Rho-kinase (Rho-kinase-cat) interacts with its substrates by affinity column chromatography. Rat brain cytosol or peripheral membrane (P2) fractions concentrated by ammonium sulfate precipitation were loaded onto a glutathione-sepharose affinity column on which GST, GST-Rho-kinase-cat, or GST-Rho-kinase-cat-KD, a kinase-deficient mutant of Rho-kinase, was immobilized (Figure 1A, B). GST-PKN-cat, another Rho effector belonging to the PKC subfamily in the AGC family of kinases, was also subjected to affinity column chromatography. The proteins bound to the affinity columns were then eluted by addition of 50 mM and 1 M NaCl, and then 10 mM glutathione. Numerous proteins were detected in the eluates from the GST-Rho-kinase-cat, GST-Rho-kinase-cat-KD and GST-PKN-cat columns (Figure 1C, D). The apparent pattern of eluted proteins in the eluate from the GST-Rho-kinase-cat column was similar to that from the GST-Rho-kinase-cat-KD column, and different from that off the GST-PKN-cat column.


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)

Isolation of interacting proteins for the catalytic domain of Rho-kinase.(A) Domain structure of Rho-kinase and the constructs used for affinity column chromatography. (B) Strategy for isolation of protein kinase substrates. (C, D) Isolation of Rho-kinase-cat-interacting proteins from rat brain cytosol (C) and P2 (D) fractions. The cytosolic or P2 fraction of rat brain lysate was loaded onto a Glutathione-Sepharose column coated with either GST, GST-Rho-kinase-cat, GST-Rho-kinase-cat-KD or GST-PKN-cat. The bound proteins were eluted by addition of 1 M NaCl after washing with 50 mM NaCl. The eluates were analyze by SDS-PAGE, and visualized by silver staining. Arrowheads indicate the GST-tagged proteins used as baits.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0008704-g001: Isolation of interacting proteins for the catalytic domain of Rho-kinase.(A) Domain structure of Rho-kinase and the constructs used for affinity column chromatography. (B) Strategy for isolation of protein kinase substrates. (C, D) Isolation of Rho-kinase-cat-interacting proteins from rat brain cytosol (C) and P2 (D) fractions. The cytosolic or P2 fraction of rat brain lysate was loaded onto a Glutathione-Sepharose column coated with either GST, GST-Rho-kinase-cat, GST-Rho-kinase-cat-KD or GST-PKN-cat. The bound proteins were eluted by addition of 1 M NaCl after washing with 50 mM NaCl. The eluates were analyze by SDS-PAGE, and visualized by silver staining. Arrowheads indicate the GST-tagged proteins used as baits.
Mentions: To screen potential substrates of Rho-kinase, we examined whether the active catalytic fragment of Rho-kinase (Rho-kinase-cat) interacts with its substrates by affinity column chromatography. Rat brain cytosol or peripheral membrane (P2) fractions concentrated by ammonium sulfate precipitation were loaded onto a glutathione-sepharose affinity column on which GST, GST-Rho-kinase-cat, or GST-Rho-kinase-cat-KD, a kinase-deficient mutant of Rho-kinase, was immobilized (Figure 1A, B). GST-PKN-cat, another Rho effector belonging to the PKC subfamily in the AGC family of kinases, was also subjected to affinity column chromatography. The proteins bound to the affinity columns were then eluted by addition of 50 mM and 1 M NaCl, and then 10 mM glutathione. Numerous proteins were detected in the eluates from the GST-Rho-kinase-cat, GST-Rho-kinase-cat-KD and GST-PKN-cat columns (Figure 1C, D). The apparent pattern of eluted proteins in the eluate from the GST-Rho-kinase-cat column was similar to that from the GST-Rho-kinase-cat-KD column, and different from that off the GST-PKN-cat column.

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