Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007we hope to establish the importance of the pathways in PGC regulation. The inhibitions will be tested inresponse to different PPAR members to determine their individual involvements. First, each PPARmember level will be recorded upon pathway stimulation, these results will then be compared to eachPPAR member level upon pathway inhibition.Determining Levels of RegulationOnce the MAPK pathways have been established, the phosphorylative actions MAPK is known for will beexamined as possible regulatory mechanisms of PPAR family members. Namely does MAPK-p38phosphorylate the PPAR family members? Does MAPK-Erk have such a function?We hypothesize that p38 phosphorylates PGC-1α and PGC-1β when PGC-1α is absent. We alsohypothesize that p38 phosphorylates the CREB/PRC/NRF complex. It is unclear what to expect in termsof a phosphorylative action of MAPK-Erk on PGC family members, however it would be interesting touncover whether MAPK-Erk is capable of upregulation as well as downregulation through phosphorylationand dephosphorylation or whether it is only capable of one side of regulation.The first step in determining phosphorylative regulation is to determine whether it occurs at all. This will bedone using PerkinElmer Phos-tools kit.The second step involves identifying phosphorylation sites. Such sites are known for PGC-1α and wepropose to search for similar sequences in PGC-1β and PRC through the BLAST search program.Thirdly, the importance of any occurring phosphorylation should be determined. For example, we wish todetermine whether phosphorylation of PPAR family members is required for NRF-1 binding, or CREBbinding to PRC. To examine this, we will use site directed mutagenesis on the phosphorylation sitespreviously identified and thus determine the effects of disturbed phosphorylation.Determination of phosphorylation sheds light on the mechanisms and pathways through whichmitochondrial biogenesis is regulated. If phosphorylation is identified then this allows control over anothervital step in the mitochondrial biogenesis pathways.Subtopic 3In this section we will be investigating the hypothesis that: ‘The regulation of mitochondrial biogenesis inthe cell cycle will be coordinated through regulation of NRF-1 and 2 activity, mediated by interactions ofthese transcription factors with PRC and CyclinD1/cdk4.’We would wish to establish interactions between NRF-1 and CyclinD1/cdk4 and PRC in order tocharacterize its activity during the cell cycle with respect to its regulation of mitochondrial biogenesis.Additionally, we would like to establish the timing and mechanisms of these interactions and whether theycan be mediated through alternative means. Interestingly, while PRC activates NRF-1 throughphosphorylation of a number of different sites, one of these (S47) is also used to inhibit its activity byCyclinD1/cdk4. Therefore, it is also interesting to investigate the importance of this phosphorylation site forthese two processes in regulating NRF-1 activity. These experiments will be conducted in synchronizedproliferating cells in order to see how these mechanisms relate to the cell cycle and in cells induced intothe cell cycle that experience a metabolic burst in order to establish possible differences in the activity ofthese factors under the different conditions.As a start, the expression patterns of the different compounds throughout the cell cycle have to beestablished, in order to look at activation and interaction. Although it is commonly known when thedifferent cyclins and cdk’s are expressed in the cell cycle, we want to re-investigate these expressionlevels, in combination with NRF-1, NRF-2 and PRC, to get a general overview of the whole transcriptionand translation processes of these compounds throughout the cell cycle. Moreover, we want to look atboth mRNA and protein expressions, to see whether present mRNA levels also mean protein expression.SCI 332 Advanced Molecular Cell Biology Research Proposal 66