UCLA

The leucine carboxyl methyltransferase and the protein L-isoaspartyl methyltransferase are two carboxyl methyltransferases responsible for mediating two protein modification chemistries. The leucine carboxyl methyltransferase, coded for by the Lcmt1 gene in mice, has only one known substrate, the C-terminal residue of protein phosphatase 2A (PP2A), a modification countered by protein phosphatase methylesterase 1. The protein L-isoaspartyl methyltransferase, coded for by the Pcmt1 gene in mice, is responsible for methylating a multitude of substrates, targeting isomerized aspartic acid residues for conversion, through methylation, to normal aspartic acid residues. Isoaspartyl residues are formed spontaneously in proteins from aspartyl and asparaginyl residues. Both of these enzymes have been implicated in the etiology of various disease states: PCMT1 in the onset of seizure disorders and spina bifida and LCMT1 as a tumor suppressor. Interestingly both enzymes have been implicated in the onset of Alzheimer's disease suggesting an important link to brain homeostasis.

Mice lacking the isoaspartyl methyltransferase demonstrate increased brain growth despite a decreased overall body size, hyperproliferation of T-cells and early death due to tonic clonic seizures. Although insulin signaling is constitutively activated in the brains of these animals, it is unclear whether this is involved in seizure onset. Additionally the convergence point between the isoaspartyl methyltransferase and the increased insulin signaling observed in Pcmt1-/- mice remains unknown. Recent work has implicated the isomerization/repair cycle mediated by the isoaspartyl methyltransferase in the regulation of a variety of proteins from cell surface receptors to p53, the "guardian of the genome". From my work, I hypothesize that an isoaspartyl-forming residue within a protein involved in the insulin signaling pathway may be responsible for the constitutive activation of this pathway seen in Pcmt1-/- mice. Through wortmannin-induced amelioration of the phosphatidylinositol 3-kinase-dependent insulin signaling pathway, I suggest the culpable residue or site of PCMT1 interaction may be the central effector of insulin signaling, the Akt kinase.

Expanding on the role of carboxyl methylation in the control of cell signaling, I characterized a mouse model hypomorphic for the leucine carboxyl methyltransferase, examining the tissue-specific decreases in PP2A methylation as well as a weak, but significant increase in insulin resistance in these animals. In an effort to discover as of yet unknown relationships between these two methyltransferases and the governance of cell signaling, I then attempted to define the LCMT1 and PCMT1 dependent "phosphome". Utilizing brain tissue from Pcmt1-/- animals as well as brain and muscle tissue from Lcmt1-/- animals, I employed a mass-spectrometry-based phosphoproteomic approach to identify tissues specific sites of phosphorylation sensitive to the levels of these two methyltransferases. These data will provide the foundation for future work examining the relationship between these two protein modification chemistries and their physiological role in mammalian cells.