Development of pan- and phospho-specific nanobodies for investigating MAPK and PP2A signaling

Vanderbilt University, Nashville TN

Investigators

Abstract

PROJECT SUMMARY The reversible phosphorylation of proteins is an essential process controlling cellular homeostasis. Protein kinases catalyze the transfer of phosphate from ATP to tyrosine (Tyr), serine (Ser), and threonine (Thr) residues in target proteins; protein phosphatases are responsible for removal of the phosphate group. Although Tyr phosphorylation is far less abundant than Ser and Thr phosphorylation (<0.1% of the total cellular phospho- amino acid content), it plays essential roles in many cellular signaling events. But Tyr phosphorylation is especially difficult to study, because it is low abundance and functions in rapid signaling events with a lifetime that is usually transient, increasing and then disappearing within minutes. Among the most useful tools for examining protein phosphorylation are phospho-specific antibodies (Abs), which can be used to readily quantify changes at phosphorylation sites and changes in their localization under various cell conditions. However, reliable phospho-specific Abs are difficult to obtain, due to poor specificity, limited availability of large-scale homogeneous preparations, and their inability to monitor protein phosphorylation in living cells. An attractive alternative to Abs is nanobodies (Nbs) – small (15 kDa), single domain, antigen binding fragments derived from camelid heavy chain-only Abs. Nbs targeting PTMs such as pTyr would be extremely valuable for the scientific community but reports describing such are very scant or non-existent. Although it has proven to be difficult developing Nbs targeting PTMs, our recent findings demonstrate remarkable success in developing Nbs targeting specific pTyr epitopes in two important families of signaling proteins – protein phosphatase 2A (PP2A) and the mitogen-activated protein kinase (MAPK) family member, ERK1/2. This proposal focuses on the development, characterization, and application of Nbs recognizing specific phosphosites in different subunits of PP2A, as well as the major MAPK family members, ERK1/2, JNK, and p38. We also will develop pan Nbs as probes for total protein abundances. Our approach will characterize the binding specificity, recognition, and affinity of each Nb for their targeted epitopes. We will determine the precise binding determinants by solving atomic resolution structures of Nb-peptide and Nb-protein complexes. We will systematically test our Nbs for their ability to recognize their respective phosphosite or protein target by Western analysis, immunoprecipitation, and immunohistochemistry, and determine effects of Nbs on MAPK and PP2A activity. Finally, we will develop Nb-based biosensors and PROTACs to respectively visualize phospho-epitope localization in cells and target them for degradation in living cells. The proposed work is responsive to PA-22-127, a technology development FOA requesting hypothesis-independent, broadly useful reagent and technology development. Completion of the proposed studies will not only yield novel tools for investigating signaling enzymes, but it will also open the door to a new technology that can be broadly applied to defining the phosphotyrosine proteome and expanding knowledge about the functions of this essential protein signaling event.