Tour-de-Force

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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
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007To establish mRNA levels, reverse-transcriptase real-time PCR (RT-PCR) will be used. To see whetherthis mRNA is also converted to proteins, Western Blotting will be used. All experiments in this subtopic willbe conducted at half an hour intervals commencing after cell synchronization.Characterizing Cyclin D 1 /cdk 4 interaction with NRF-1 and whether this extends to NRF-2Although previous research has shown that Cyclin D 1 /cdk 4 inhibits NRF-1 activity through phosphorylationof S47 (serine at location 47 on the protein) (Wang et al., 2006) it is useful to check whether thesemechanisms are active during the cell cycle and whether it may also be involved in regulating the activityof NRF-2 in a similar way. The phosphorylation of NRF-1 at S47 was found in the same study to berequired but not sufficient on its own for the full repression of NRF-1 activity. It would therefore beinteresting to establish which other sites may be phosphorylated in this process. In order to examine thiswe propose to conduct the following experiments.Identification of the nature and extent of phosphorylation of NRF-1 and NRF-2 by Cyclin D1/cdk4 and PRCTo establish whether (or rather at which points) NRF-1 and NRF-2 are phosphorylated at specific points inthe cell cycle we will conduct phosphoprotein analysis using mass spectrometry to identify specific sites ofphosphorylation. These analyses will be conducted at the time points in the cell cycle mentioned above.Due to the fact that we need to distinguish between NRF-1 that is phosphorylated by PRC and Cyclin D1 itis important for us not only to check if NRF-1 is phosphorylated, but the precise nature and extent of thisphosphorylation. In order to do this we propose to use the PerkinElmer Phos-tools kit along withPerkinElmer Phos-tag stains to highlight phosphorylated proteins due to their high binding affinities withphosphorylated serine, threonine, and tyrosine residues. Additionally, for this method it is required toenrich the phosphopeptides for adequate detection using PerkinElmer Phos-trap. We will then establishdifferences in phosphorylation of activated and inhibited NRF. This method allows us to do this, is highlysensitive to phosphoprotiens, and does not take too much time. For these reasons this method isappropriate for our experiment and provides relevant data that we could not as easily get from othersources.The role of localization of Cyclin D 1 /cdk 4 in NRF regulationWe will look at differences in phosphorylation of NRF-1 and NRF-2 in the cell cycle with respect to whenCyclin D 1 /cdk 4 is located in the nucleus and when it is moved out. We will also look at changes in thephosphorylation of NRF-1 and 2 following the phosphorylation and activation of PRC characterized insubtopic 2.Using evidence of the timing of PRC activation and NRF activation found in this subtopic we will be able tohypothesize whether PRC activation can override cyclin D 1 /cdk 4 inhibition or whether this must beremoved before PRC can exert its activity. If removal of the inhibitory signal is required it is likely thatcyclin D 1 /cdk 4 translocation to the cytoplasm will coincide with NRF activation.To check whether cyclin D1 inhibition of NRF is related to its nuclear localization we will construct amutant that has been shown to remain located in the nucleus by mutating cyclin D1 at this site: T286A.Differences in cyclin D1 binding between cdk4 & 6All cyclin D isoforms have been shown to bind with cdk4 to mediate cell cycle progression. Cyclin Disoforms are expressed in different levels in various tissue types, suggesting a partial or completeredundancy (Carthon et al., 2005). However, there has been no study conducted into the possibilities ofNRF-1 regulation by the other two cyclin D isforms through either cdk4 or cdk6. Only cyclins D1 and D3bind to cdk6, which means that cdk4, is the sole cdk for cyclin D2, whereas D1 and D3 can bind both.Cdk4 and cdk6 both exert the same functions in terms of cell cycle progression when bound to cyclin D.We want to examine when cyclin D/cdk complexes are formed, but more importantly, if there is a bindingspecificity for either cdk4 or cdk6 during certain points in the cell cycle.SCI 332 Advanced Molecular Cell Biology Research Proposal 67
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