Revealing Secrets of Cellular Phosphorylation Systems Using Biological Mass Spectrometry

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All organisms contain proteins (e.g. enzymes) that perform the necessary chemical reactions that allow cellular life to exist. In order to restrict protein function to when it is needed by the cell, nature has developed molecular systems that can change the three-dimensional shape of proteins in a reversible fashion, and thus turn protein function “on” and “off”. Protein phosphorylation is a common chemical modification utilized by all living organisms to alter the shape and function of proteins. The enzymes that mediate the addition and removal of the phosphate molecules are called kinases and phosphatases respectively. Since a delicate balance of reversible phosphorylation is essential for nearly all cellular processes to function properly, the activities of all kinases/phosphatases are under tight control. One of the most powerful analytical instruments to study phosphorylation systems is a mass spectrometer. Mass spectrometry-based proteome sequencing techniques are the protein equivalent to genome sequencing techniques, and thus have the potential to discover fundamental aspects of phosphorylation systems. Accordingly, the long-term goal of our research program is to integrate mass spectrometry, biochemistry, and cell biology techniques to better understand the cellular role of poorly studied kinases and phosphatases predicted to regulate important cellular processes. We propose to utilize advanced mass spectrometry techniques towards the study of human YVH1 (hYVH1), a scantily studied member of the atypical dual specificity phosphatase (DUSP) subfamily. Also known as DUSP12, YVH1 orthologues are widely conserved in the animal and plant kingdom, and recent studies from our laboratory and others have provided evidence that hYVH1 is a regulator of protein synthesis, cell survival, and cellular DNA content. Moreover, we recently reported the results of a hYVH1 mass spectrometry study that discovered hYVH1 associates with various ribonucleoprotein particles (RNP) and functions as a novel stress granule disassembly factor. This initial mass spectrometry analysis also provided preliminary evidence that a well characterized tyrosine kinase known as Src, may control the activities of hYVH1. We have confirmed that Src can phosphorylate hYVH1 in human cells and have sequenced a novel phospho-tyrosine site using mass spectrometry. We are now interested in thoroughly characterizing the functional significance of Src-mediated phosphorylation of hYVH1. Specifically, we are interested in determining how this phosphorylation modification changes the shape of hYVH1 in order to alter the function of hYVH1 in the cell. The methods employed within our proposal will be transferrable to the study of other poorly understood phosphatases while the findings will deepen our understanding of the role of hYVH1 in cellular regulation.