Synthetic Optimization of the MAPK and PI3K Pathways for Cell Proliferation and Survival

To fully characterize the effects of gene expression on cellular phenotypes, a range of expression levels needs to be explored.  Variable gene expression has typically been achieved at the level of transcription through the use of inducible transcription systems or varying strength synthetic promoters.  As an alternate approach, we employed a system that would control protein amount at the level of translation through the engineering of the Kozak sequence immediately preceding a gene of interest.  By using a GFP reporter, we established a translation initiation sequence (TIS) library exhibiting over a 200-fold range in GFP intensity with a high degree of consistency across five cell lines.  We then utilized the TIS library to determine the optimal contributions from the MAPK (mitogen-activated protein kinase) and PI3K (phosphatidylinositol 3-kinase) pathways towards survival through the overexpression of oncogenic H-Ras variants in heterogeneous culture.

Constitutively-active mutants of the GTPase H-Ras, are potent oncogenes that are involved in signal transduction down multiple cellular pathways.  Two of these pathways, the MAPK and PI3K pathways, are especially important in cellular transformation and have well defined downstream targets that allows for measurement of the signaling through that pathway.  We used our translation initiation sequence (TIS) library to translate (i) oncogenic H-RasG12V and (ii) mutant versions of H-RasG12V that are only able to signal down the MAPK branch (an H-RasG12V T35S mutant) or the PI3K branch (an H-RasG12V Y40C mutant).  Each transgene is fused to a fluorescent protein to enable quantitative analysis at the single-cell level by flow cytometry.  The TIS library allowed us to maintain a heterogeneous culture that contains cells translating our transgene over several orders of magnitude (i.e., inputs to the system).  Next we performed a population competition experiment.  By allowing this mixed culture to grow over time, optimal gene dosage levels become evident as cells expressing protein at an optimal level begin to outcompete the cells expressing sub-optimal levels (i.e., outputs from the system).  Here we report the optimization of the MAPK and PI3K signaling pathways in an antibody-producing murine plasmacytoma line, MPC-11.  We envision utilizing the TIS system in conjunction with engineered hetereogeneous cell culture to overexpress multiple genes in lymphocytes or CHO cells and to engineer pathways for optimal cell proliferation and/or antibody production.