Project 1. Characterizing Phosphorylation Sites of Saccharomyces cerevisiae Transcription Factors, Rap1 and GcM Using Nanospray Mass Spectrometry Two Saccharomyces cerevisiae transcription factors, Raplp and Gcrlp, have been partially characterized along with their shared participation in glycolytic enzymes and ribosomal protein (RP) gene transcription. As an essential DMA binding protein required for transcriptional activation and repression, Raplp has a co-activator, Gcrlp. Much evidence has been gathered that indicates the Rap1p/Gcr1p complex participates in transcription directly by binding to their target genes. In vivo, the promoters of most genes encoding glycolytic and ribosomal proteins (RP) contain sites occupied by Rap 1p (1). Transcriptomic analysis using microarray hybridization of defective gene expression in gcrIA cells indicates that 11 glycolytic genes, 110 ribosomal genes, 4 translocational components, and 67 other genes exhibit at least a twofold decrease in expression (2). The transcription of RP and glycolytic genes are the two most abundantly transcribed classes of glucose-induced target genes in yeast cells. While much of the role of Raplp and Gcrlp in transcriptional regulation is known, little has been done to characterize the regulation of these two transcription factors. There is a small amount of experimental evidence that one or both of these factors have phosphorylated residues, but the requirement of phosphorylation for the perinuclear gene regulation by Raplp and Gcrlp has not been established. It is thought that these two transcription factors are regulated through the TOR (Target of Rapamycin) and the protein kinase A (PKA) signaling pathways. Exquisitely regulated and timed reversible phosphorylation is used to activate or inhibit pathway proteins throughout the cell cycle. Common intracellular signal transduction events occur with up to one third of all proteins at any one point in the cell cycle being phosphorylated. Reversible phosphorylation plays a critical role in transmitting signals from outside the cell. While the glycolytic enzymes genes and RP genes are constitutively expressed throughout the cell cycle, knowledge of phosphorylation of the activators of these genes might yield the finer points of their regulation. The goal of this project is to determine the role of phosphorylation of S. cerevisiae transcription factors, Raplp and Gcrlp, in the activation of glycolytic enzyme and ribosomal protein genes.
The specific aims are:
Specific Aim 1 : To test the hypothesis that Rap1 p and Gcr1 p are phosphoproteins.
Specific Aim 2 : To test the hypothesis that phosphorylation is either increased or decreased on Gcrlp when cells are arrested at G1 phase of the cell cycle and at G2 phase of the cell cycle.
Specific Aim 3 : To test the hypothesis that the TOR pathway and a PKA signaling pathway are responsible for the phosphorylation of Gcrlp and Raplp. Data gathered should yield information to further reveal subtle control elements in the regulatory pathways of these two factors. Interestingly, a recent Psi-Blast analysis of Gcr1 yielded evidence for a human homolog, MLL3, that is found on a region of chromosome 7 that is frequently deleted in myeloid leukemia (3). A comparison of the sequences of MLL3 and Gcrlp indicates that MLL3 also has serine-proline-rich regions in similar areas of the amino acid sequence. Revealing a part of the story of the regulation of Gcr1 p could yield insight into MLLS's role in this disease.
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