Structural basis for antigen recognition by methylated lysine-specific antibodies.

Proteins are modulated by a variety of posttranslational modifications including methylation. Despite its importance, the majority of protein methylation modifications discovered by mass spectrometric analyses are functionally uncharacterized, partly owing to the difficulty in obtaining reliable methylsite-specific antibodies. To elucidate how functional methylsite-specific antibodies recognize the antigens and lead to the development of a novel method to create such antibodies, we use an immunized library paired with phage display to create rabbit monoclonal antibodies recognizing trimethylated Lys260 of MAP3K2 as a representative substrate. We isolated several methylsite-specific antibodies that contained unique complementarity determining region sequence. We characterized the mode of antigen recognition by each of these antibodies using structural and biophysical analyses, revealing the molecular details, such as binding affinity toward methylated/nonmethylated antigens and structural motif that is responsible for recognition of the methylated lysine residue, by which each antibody recognized the target antigen. In addition, the comparison with the results of Western blotting analysis suggests a critical antigen recognition mode to generate cross-reactivity to protein and peptide antigen of the antibodies. Computational simulations effectively recapitulated our biophysical data, capturing the antibodies of differing affinity and specificity. Our exhaustive characterization provides molecular architectures of functional methylsite-specific antibodies and thus should contribute to the development of a general method to generate functional methylsite-specific antibodies by de novo design.

Interrogating PP1 Activity in the MAPK Pathway with Optimized PP1-Disrupting Peptides.

Protein phosphatase-1 (PP1)-disrupting peptides (PDPs) are selective chemical modulators of PP1 that liberate the active PP1 catalytic subunit from regulatory proteins; thus allowing the dephosphorylation of nearby substrates. We have optimized the original cell-active PDP3 for enhanced stability, and obtained insights into the chemical requirements for stabilizing this 23-mer peptide for cellular applications. The optimized PDP-Nal was used to dissect the involvement of PP1 in the MAPK signaling cascade. Specifically, we have demonstrated that, in human osteosarcoma (U2OS) cells, phosphoMEK1/2 is a direct substrate of PP1, whereas dephosphorylation of phosphoERK1/2 is indirect and likely mediated through enhanced tyrosine phosphatase activity after PDP-mediated PP1 activation. Thus, as liberators of PP1 activity, PDPs represent a valuable tool for identifying the substrates of PP1 and understanding its role in diverse signaling cascades.

SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer.

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.