Kinase-interacting substrate screening is a novel method to identify kinase substrates

Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 protei...

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Bibliographic Details
Published in:The Journal of cell biology 2015-06, Vol.209 (6), p.895-912
Main Authors: Amano, Mutsuki, Hamaguchi, Tomonari, Shohag, Md Hasanuzzaman, Kozawa, Kei, Kato, Katsuhiro, Zhang, Xinjian, Yura, Yoshimitsu, Matsuura, Yoshiharu, Kataoka, Chikako, Nishioka, Tomoki, Kaibuchi, Kozo
Format: Article
Language:English
Subjects:
Cellular biology
Contractile Proteins - metabolism
High-Throughput Screening Assays - methods
Humans
Kinases
Membrane Proteins - metabolism
Phosphorylation
Proteins
rho-Associated Kinases - metabolism
Substrate Specificity
Substrates
Tumor Suppressor Proteins - metabolism
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Summary:Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases.
ISSN:0021-9525
1540-8140
DOI:10.1083/jcb.201412008