A previous study employed affinity purification and tandem mass spectroscopy to identify 142 SUMO-conjugation targets in the early Drosophila embryo. Multiple components of the Ras/MAPK signaling pathway are among these targets, and in accord with this discovery, SUMO was found to be required for activation of the Ras/MAPK pathway in Drosophila S2 cells. However, the question of what is SUMO’s role in the Ras/MAPK still remains. In this dissertation, I present experiments showing that SUMO is also required for Ras/MAPK signaling in the developing embryo. In addition, I explore the mechanism by which SUMO regulates Ras/MAPK signaling.
To determine if SUMO is required for Ras/MAPK signaling in developing embryos as it is in S2 cells, I investigated the role of SUMO in signaling through the Torso receptor tyrosine kinase, which signals through the Ras/MAPK pathway to specify the embryonic termini. I generated females containing SUMO mutant germline clones and performed immunoblotting and immunofluorescence on the resulting embryos to assess MAPK phosphorylation levels. In addition, I performed reverse transcriptase-qPCR to assess the expression of tailless, a target of the Torso pathway. The results show that reduction of the maternal SUMO contribution decreases both MAPK activation and tailless expression, consistent with a positive role for SUMO in Ras/MAPK signaling during embryogenesis.
Localization of Ras to the plasma membrane, which is required for Ras/MAPK signaling, depends on C-terminal prenylation as well as lysine residues in the Ras C-terminal hypervariable region. A published bacterial SUMOylation assay demonstrated that SUMO can be conjugated to the hypervariable region lysine residues suggesting a role for SUMO in membrane localization of Ras. However, my S2 cell SUMO knockdown studies revealed roles for SUMO in neither the lipid modification of Ras nor Ras trafficking. I saw no evidence for Ras SUMOylation in Drosophila cells, although I did detect a non-covalent complex that includes Ras and one or more SUMOylated proteins.
Included among the 142 SUMO-conjugated proteins detected in early embryos were protein phosphatase 2A (PP2A) and 14-3-3 family proteins, both of which modulate Ras/MAPK signaling through their interactions with Raf. These findings suggested that SUMO might modulate Ras/MAPK signaling via Raf, and I therefore determined how SUMO affects Raf phosphorylation and subcellular localization. An epitope-tagged form of Raf was expressed in wild-type and SUMO knockdown S2 cells followed by immunopurification and tandem mass spectroscopy to characterize the Raf phosphorylation state. Activation of the Ras/MAPK pathway promotes Raf dephosphorylation at S346, which is the counterpart of a residue in human C-Raf that needs to be dephosphorylated for efficient Ras/MAPK signaling. However, dephosphorylation of S346 did not occur upon activation of the Ras/MAPK pathway in SUMO knockdown cells. Furthermore, subcellular fractionation revealed that SUMO knockdown prevents Raf from localizing to the plasma membrane in insulin activated cells. By expressing a form of Raf that is constitutively targeted to the plasma membrane and that lacks S346, we were able to restore Ras/MAPK signaling in SUMO knockdown cells. These findings suggest that a primary role for SUMO in the Ras/MAPK pathway is to promote Raf dephosphorylation and targeting of Raf to the plasma membrane.
My studies have revealed a novel mechanism by which SUMO regulates Raf activity and therefore the Ras/MAPK pathway. Since mutations in the Ras/MAPK pathway are found in 30% of all cancers in humans, my findings may eventually lead to the development of novel therapies for cancer treatment that target the function of SUMO in Ras/MAPK signaling.