Developing novel methods to search for substrates of protein kinases such as Rho-kinase

Protein phosphoiylation is a major and essential post-translational modification in eukaryotic cells that plays a critical role in various cellular processes. Recent progresses in mass spectrometry techniques have enabled the effective identification and analysis of protein phosphorylation. Mass spectrometry-based approaches in investigating protein phosphorylation are very powerful and informative and can further improve our understanding of protein phosphorylation as a whole, but they cannot determine the upstream kinases involved. We introduce several studies that attempted to uncover the relationships between various kinases of interest and substrates, including two methods we developed: an in vitro approach termed the kinase-interacting substrate screening (KISS) method and an in vivo approach termed the phosphatase inhibitor and kinase inhibitor substrate screening (PIKISS) method. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. (C) 2015 Elsevier B.V. All rights reserved.