Enrique Congrains: mujer y marginalidad

A I 6. Redistribution of Ras with tipifarnib treatment in 293T cells. mKate labeled K-Ras

cells were mounted on a 4 well slide treated with tipifarnib (TF) for 48 hours, fixed using standard immunofluorescence techniques, nuclei were stained, and images were acquired on a confocal microscope. mKate and the nuclear stain were overlaid.

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Summary part A

As Ras acquires several post-translational modifications on its C-terminus, an N- terminal tag was necessary to distinguish endogenous from transduced protein. We devised an N-terminal mKate tag which naturally fluoresces. Incorporating a fusion protein would also allow for separation of transduced and non-transduced (or transfected) cells for analysis. To verify the tag was not interfering with the ability of Ras to function, a Ras activation assay was performed. An N-terminal mKate fusion of Ras was functional as full length fusion proteins were detected at ~45 kDa. Two bands of transduced proteins were detected at ~45 and 42 kDa. To ensure the 42 kDa protein was not prenylated Ras and the 45 kDa protein was unprenylated Ras we used pharmalogical inhibitors to block both prenylation enzymes in order to detect only unprenylated protein. Unprenylated protein migrated slower than 45 kDa. Therefore we suspect the 42 kDa fusion protein detected is a truncated mKate-Ras fusion protein. Active Ras mKate fusion proteins are active as demonstrated by the Ras pull down showing an increase of active Ras-RBD complexes in WT N-Ras and K-Ras transduced 293T cells.

Stably transduced WT N-Ras and K-Ras cells were assayed for Ras activation status, cell proliferation, and immunofluorescence to detect localization of transduced protein. We determined fluorescence intensity is proportional to the amount of transduced N- or K-Ras protein. This transduced protein still appears functionally active as shown by a Ras pull down with low, medium, and high mKate fluorescing cells. Endogenous levels of Ras increased in WT N-Ras transduced cells and decreased in K-Ras transduced cells. This suggests forced expression of WT K-Ras might be toxic to 293T cells and the cells respond by decreasing the activity of endogenous Ras.

As mkate is naturally fluorescent, confocal imaging can detect where the fusion protein is located within a cell. When cells were sorted by low, medium, and high fluorescent intensity, the light emission from low and medium sorted mkate positive cells were not visible by confocal microscopy. However, high fluorescent sorted K-Ras cells did have detectable mKate signal. In untreated cells, K-Ras signal was located diffusely throughout the cell and high punctate signal was also detected in cells. This punctate signal is likely to be located in endosomes where Ras is being degraded to compensate for the higher rate of protein produced in the cells. It was evident that K-Ras transduced cells treated with tipifarnib for 48 hours relocalizes K-Ras to the membrane where it can be functionally active. These findings confirm with tipifarnib treatment Ras signaling is upregulated and localized to the PM to intensify Ras downstream signaling.

91 The mKate fluorescent tag was undetectable in low and medium N- and K-Ras sorted cells. An N-terminal truncated GFP fusion to H-Ras has been confirmed to have fluorescent signal and to not interfere with H-Ras localization (124). This fusion may allow for sorting low, medium, and high fluorescent populations for better microscopy images. This mKate fusion tag was created as it is supposed to overcome imaging limitations. This tag has been used for numerous applications including cell labeling and whole body in vivo imaging. However mKate expression did not allow for detectable imaging when sorted in low and medium fluorescent intensity when fused to N- or K-Ras. For in vitro purposes, a GFP tag may allow for better imaging of low and medium expressing N- and K-Ras fusion proteins. This would allow for us to determine if there are differences in the localization of N- and K-Ras with different levels of induced expression. We are also interested in determining if there is an increase and a shift to PM localized N- and K-Ras when low and medium fluorescent cells are treated with TF. We predict there will be increased Ras signal at the PM when cells with lower levels of Ras expression are treated with TF.

This section was a prerequisite to creating mutant Ras proteins. These mutant Ras proteins will allow us determine the functional consequences of N- and K-Ras proteins which can only be GG’d. The work presented in this section confirms that N-terminally mKate-

tagged Ras proteins are functionally active to use as a tool for identifying transduced protein.

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B: Long term affects of N- and K-Ras mutants transduced into 293T cells

and K-CAIL associates with the PM.

Results

Point mutations in the CAAX motif of N-Ras and K-Ras were made in order for the Ras proteins to be WT (preferentially farnesylated (F’d)), CVLS (F’d only), cysteine mutated to serine (unprenylated), and CAIL (GG’d only) (Figure 14 A). To analyze the long term functional consequences of the constructs they were transduced into 293T cells and selected with puromycin for 3 days. These cells were then sorted in the FACScore facility based on mKate expression. To determine if increased mKate fluorescence correlated with increased active transduced Ras as in part A, CAIL and CVLS were sorted based on fluorescent intensity. We hypothesized CAIL to be toxic at high levels so sorting into these three expression levels may allow us to see toxicities associated with GG’d Ras. Stable transductions were plated in 10 cm dishes and lysates were collected once cells reached 80% confluence. As anticipated, increased intensities did correlate with increased Ras activation (A I 7 A). It was difficult to see separation of migration of unprenylated and prenylated proteins so remainders of the same lysates were ran on a 35 mm gel for 5 hours. The increased mobility of unprenylated protein from the C->S mutant protein runs at a higher mobility than prenylated proteins (A I 7 B).

293T TF mK at e W T H C A IL L C A IL M C A IL H C V L S M C V L S H C ->S N-Ras K-Ras W T H C A IL H C V L S H C ->S A. B. UP P