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jcb Home » 2015 Archive » 22 June » 209 (6): 777.3
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Kinases KISS and tell

Ben Short
Ben Short
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DOI: 10.1083/jcb.2096iti3 | Published June 22, 2015
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Figure1

Rho-kinase (blue), Scrib (red), and Shroom2 (green) colocalize at the contractile edge of an epithelial wound.

Amano et al. describe a new method to identify the substrates of protein kinases.

There are ∼500 protein kinases in the human genome and, collectively, they regulate a wide variety of cellular processes by phosphorylating numerous substrate proteins. Most of these substrates remain unknown, however, so Amano et al. devised a new technique, called kinase-interacting substrate screening (KISS), to identify kinase targets.

The researchers first used the KISS approach to look for substrates of Rho-kinase. The catalytic domain of this enzyme was loaded onto affinity beads, which were then incubated with rat brain lysates in the presence or absence of ATP. Sensitive mass spectrometry techniques were then used to identify the bound substrate proteins and determine which of their residues were phosphorylated. In total, the researchers catalogued 356 phosphorylation sites on 140 different proteins, most of which had not previously been identified as Rho-kinase substrates.

Amano et al. verified several of these new substrates, including the cell polarity protein Scrib. Phosphorylation of a serine residue in Scrib’s C-terminal domain enhanced the protein’s assembly into a ternary complex with Rho-kinase and the actin-binding protein Shroom2. This complex preferentially localized to the free edges of epithelial cells, where it promoted myosin light chain phosphorylation and actomyosin contractility.

The researchers successfully applied KISS to eight other kinases, each of which phosphorylated a distinct set of proteins. Senior author Kozo Kaibuchi plans to deposit his team’s results in a public database.

References

    1. Amano, M., et al
    . 2015. J. Cell Biol. doi:10.1083/jcb.201412008
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Kinases KISS and tell
Ben Short
J Cell Biol Jun 2015, 209 (6) 777; DOI: 10.1083/jcb.2096iti3

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The Journal of Cell Biology: 217 (4)

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April 2, 2018
Volume 217, No. 4

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