Activity and Regulation of AGC Kinases from Physcomitrella patens and Tomato

The AGC group of protein kinases (named for protein kinases A, G, and C) is found in all eukaryotes studied so far, and its members coordinate essential cellular processes including translation, metabolism, hormone response, growth, and survival. AGC kinases are intensively studied in mammals because of their connection with human diseases like cancer, diabetes, and neurological disorders. Some aspects of AGC kinase function are organism-specific, but others are conserved in highly divergent species. Several AGC kinases are regulated by the conserved 3-phosphoinositide dependent protein kinase-1 (PDK1), which is itself an AGC kinase. PDK1 regulates its substrates through phosphorylation at a conserved site in their activation loop. Here, I identify and characterize a PDK1 homologue from the moss Physcomitrella patens (PpPDK1). I show PpPDK1 phosphorylates plant AGC kinases in the activation loop, but unexpectedly lacks a lipid-binding domain, suggesting that its regulation differs from other species. In contrast to mammalian cells, PpPDK1 is not an essential gene, suggesting that AGC kinase pathways in P. patens are sufficient for survival even in the absence of activation by PpPDK1. I analyze putative PDK1 sequences from 100 different eukaryotic species, finding that many PDK1s differ from the "conventional" PDK1 found in humans. Phylogenetic analysis of these sequences suggests a complicated evolutionary history for PDK1, with the potential for unexpected functional and regulatory features. I also investigate the regulation of Adi3, an AGC kinase from tomato, through phosphorylation by PDK1. I identify a novel putative PDK1 phosphorylation site outside the kinase domain, which appears to increase Adi3 activity on a substrate. Finally, I produce a mutant version of Adi3 that can selectively utilize bulky ATP analogues. This analogue-sensitive protein may be used in a future search for direct Adi3 substrates. Together, my experiments provide insight into two members of the AGC group of protein kinases, one (PDK1) that is conserved in all eukaryotes and one (Adi3) that appears to be present only in plants. These experiments give a new perspective in our view of plant AGC kinase function and regulation