The role of protein methylation as a modifier of cellular pathways



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Present studies focus on the biological consequences of protein methylation of both arginine and lysine residues. We have previously identified in vitro substrates for the coactivator associated arginine methyltransferase 1 (CARM1) using highdensity protein arrays. These include TARPP (thymocyte cyclic AMP-regulated phosphoprotein) and CAS3 (CARM1 substrate 3). Here, we report that TARPP and CAS3 are arginine methylated by CARM1 in vivo. Using CARM1 knockout MEF cells, we were able to determine that no other enzymes methylate these substrates. In addition, we mapped the site of arginine methylation in TARPP and CAS3. Because TARPP expression is restricted to early thymocytes, we investigated the role of CARM1 in T cell development. CARM1 knockout embryos exhibited a partial developmental block in the earliest thymocyte progenitor subset. These findings implicate CARM1 in differentiation and/or survival of T cell precursors. To identify methyl-arginine binding proteins, we developed a protein domain microarray approach. Using these arrays we identified three Tudor domain proteins that specifically interact with methyl-arginine proteins. We further showed that CARM1 mediated methylation of CAS3 is critical for its interaction with these Tudor proteins, thereby identifying the first CARM1 dependent protein-protein interactions. Finally, we used this proteomic approach to identify novel methyl-lysine binding proteins that interact with histone tails. In addition to the chromo domain which has been previously identified as a methyl-lysine reader, our study identified Tudor and MBT domains as new classes of methyl-lysine-binding protein modules. These studies shed light on the biological roles of protein methylation, by identifying new methylated proteins and their methyl-dependent interacting proteins.