Model eucaryotes including Saccharomyces cerevisiae are emerging as key elements for identifying genes that modulate longevity and aging and for determining molecular mechanisms of protein action in these processes. The S. cerevisiae Sch9 protein kinase plays a role in chronological life span but how it does so is unclear, since upstream activators and downstream substrates remain unknown. We recently identified the first Sch9 substrate, Lsp1, a negative regulator of the Pkc1-MAP kinase cascade.
In Specific Aim 1 we will use the Lsp1 phosphopeptide sequence(s) to search for other substrates that may play roles in chronological life span. We will also determine the identity of two proteins that we have found to be phosphorylated in vitro by purified Sch9. Finally, we will use biochemical approaches to search for other Sch9 substrates. Our other novel discoveries are that sphingolipid long chain bases (LCBs) activate Sch9 kinase activity in vitro and that LCB levels rise over 100-fold as cells pass from log to stationary phase. Based upon these results we seek to determine in Specific Aim 2 if LCBs regulate Sch9 activity in vivo thereby controlling chronological life span.
In Specific Aim 3 we will determine if Lsp1 plays a role in chronological life span and whether or not it regulates the activity of Sch9. The same will be done for other Sch9 substrates identified in Aim 1. Finally, in Specific Aim 4 we will determine if Lsp1 and a related protein Pil1, also a negative regulator of the Pkc1-MAP kinase cascade that terminates with the Slt2 protein kinase, play a role in replicative life span. Slt2 has recently been shown to control replicative life span via phosphorylation of Sir3, a known regulator of life span. The results of these studies will establish a role for sphingolipids in life span regulation, provide the first mechanistic insight into the control of chronological life span by Sch9 and generate new leads for understanding how Sch9 governs life span. Since Sch9 is a homolog of human Akt/PKB protein kinases, our results may identify specific roles for Akts/PKBs in human aging and longevity.
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