Melbourne Protein Group Student Symposium

AbstractThe phytosulfokine receptor 1, PSKR1 is a membrane-localised leucine-rich repeat receptor-like kinase that also possesses intrinsic guanylate cyclase (GC) activity. The GC activity is conferred, in part, by the presence of a GC catalytic centre that is embedded within its kinase domain. This unusual domain architecture represents a novel class of GC-linked receptor kinases. This novel class of kinases was unearthed using sequence homology-guided bioinformatic data mining tools. Only four members of this new class of kinases have been shown to possess both kinase and GC activity. Currently, there is a paucity of information as to how this dual catalytic activity is regulated in these molecules; therefore we set out to explore the regulatory factors that modulate the dual catalysis in this unusual family of receptor kinases. Our functional studies on PSKR1 demonstrate that calcium acts as a molecular switch regulating this dual catalysis. Functional analysis of PSKR1 at different calcium concentrations showed that calcium inhibits the kinase activity of PSKR1 in a concentration dependant manner whilst on the other hand, enhancing the GC activity of PSKR1. Our previous studies have also demonstrated that, cyclicGMP (a product of GC activity) inhibits the kinase activity of PSKR1. Taken together, our observations indicate that calcium and cyclicGMP act as molecular switches of PSKR1-mediated signalling. Furthermore, our structural analysis of the cytoplasmic domain of PSKR1 suggests that it exists as a reversible dimer in solution. This observation may represent a physiological conformation of PSKR1. In a separate experiment, using tandem mass spectrometry, we have mapped out the phosphorylation pattern of the cytoplasmic domain of PSKR1. Our findings show that the cytoplasmic domain of PSKR1 has 14 phosphorylation sites in its cytoplasmic domain including 3 phospho-tyrosines. This current study presents a mechanistic model of how calcium and phosphorylation act as bimodal switches regulating the dual catalysis in PSKR1.