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Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins
Published on: December 27, 2016
TOUSLED is a nuclear serine/threonine protein kinase that requires a coiled-coil region for oligomerization and
1Department of Plant Biology, University of California, Berkeley, California 94720, USA. jlr@nature.berkeley.edu
Abstract:
The TOUSLED (TSL) gene is essential for the proper morphogenesis of leaves and flowers in Arabidopsis thaliana. Protein sequence analysis predicts TSL is composed of a carboxyl-terminal protein kinase catalytic domain and a large amino-terminal regulatory domain. TSL fusion proteins, expressed in and purified from yeast, were used to demonstrate TSL protein kinase activity in vitro. TSL trans-autophosphorylates on serine and threonine residues, and phosphorylates exogenous substrates. Using the yeast two-hybrid system, TSL was found to oligomerize via its NH2-terminal domain. A deletion series indicates that a region containing two alpha-helical segments predicted to participate in a coiled-coil structure is essential for oligomerization. TSL localizes to the nucleus in plant cells through an essential NH2-terminal nuclear localization signal; however, this signal is not necessary for protein kinase activity. Finally, deletion mutants demonstrate a strict correlation between catalytic activity and the ability to oligomerize, arguing that activation of the protein kinase requires interaction between TSL molecules.
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