Date of Award

Fall 2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

First Advisor

Mark C. Hall

Committee Chair

Mark C. Hall

Committee Member 1

Harry Charbonneau

Committee Member 2

Clint Chapple

Committee Member 3

Laurie L. Parker

Abstract

Protein phosphorylation is perhaps the most ubiquitous posttranslational modification in eukaryotes and recent studies suggest that upwards of 75% of human proteins are phosphorylated. Many proteins are phosphorylated at multiple sites, often controlled by multiple kinases and phosphatases. Multisite phosphorylation can differentially affect the functional and regulatory cellular outcomes. For example, dephosphorylation of a protein at a particular site may inhibit nuclear localization of a protein while dephosphorylation of a different site may be necessary for enzymatic activation of a protein. Thus, multisite protein phosphorylation can complicate our understanding of the biological significance and the functional consequences of protein phosphorylation. A clearer understanding of the functional consequences associated with an individual proteins phosphorylation status requires methods that can quantitatively monitor each phosphorylation site on a protein independent of one another.

Here, I present a general method for quantitatively studying multisite protein phosphorylation. I demonstrate that label-free selected reaction monitoring (SRM) mass spectrometry is comparable to conventional assays for measuring reaction rates and steady-state kinetic parameters of phosphatases and kinases. Furthermore, I demonstrate that this method can be employed to measure the activity of an enzyme towards multiple substrates in a single reaction, suggesting that this approach is a useful tool for studying substrate specificity of kinases and phosphatases. I also demonstrate that this method can be used to simultaneously measure relative rates of dephosphorylation at individual phosphorylation sites on intact protein substrates in the context of a whole cell extract.

Failure to properly regulate protein phosphorylation during the cell cycle compromises genome integrity and can lead to cancer and other disease states. The protein phosphatase Cdc14 has been implicated in the general reversal of cyclin dependent kinase (Cdk) phosphorylation at the end of cell division. However, the molecular mechanisms responsible for ordered Cdk substrate dephosphorylation are poorly understood. The protein Fin1 is a multisite phosphorylated protein and is a known substrate of Cdk and Cdc14. The SRM assay affords one the ability to examine the dephosphorylation of individual phosphorylation sites on an intact protein substrate, a task that cannot be accomplished using a conventional phosphatase assay. Therefore, using the SRM assay, I examined the dephosphorylation of Fin1 in response to Cdc14 treatment. This approach to studying protein dephosphorylation revealed that Cdc14 differentially dephosphorylates Fin1 such that the three phosphoserine sites that were monitored were readily dephosphorylated while the phosphothreonine site was resistant to Cdc14 activity. Importantly, these results suggested that Cdc14 may specifically reverse only a subset of Cdk-dependent phosphorylation events. These findings sparked an investigation into the specificity of Cdc14 and eventually lead to the identification of the first experimentally defined consensus sequence for Cdc14.

Using our experimentally defined consensus sequence for Cdc14, we searched the yeast proteome for potential Cdc14 substrates. From this list, I identified the protein Yen1, a Holliday junction (HJ) resolvase, as a potential Cdc14 substrate. HJs are DNA intermediates that form during homologous recombination (HR) in response to repair of DNA double strand breaks. Yen1 has been shown to resolve HJs in a cell cycle and phosphorylation dependent manner; however, the protein kinase and phosphatase responsible for modulating the resolvase activity of Yen1 in vivo were previously unidentified. Here I demonstrate that Yen1 is indeed a bona fide physiological Cdc14 substrate and that dephosphorylation of distinct clusters of Cdk sites on Yen1 by Cdc14 is important for 1) modulating the nucleocytoplasmic localization of Yen1 and 2) triggering enzymatic activation of Yen1. I show that dephosphorylation of Yen1 is essential for its biological function in recombination based repair of DNA damage. These findings suggest that Cdc14 plays a previously unknown role in DNA repair and maintenance of genome stability.

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