The ability to selectively interfere with protein function not only is useful for studying cellular biology but also is an important therapeutic strategy. Hines et al. modified their previously developed protein degradation system called PROTAC (proteolysis targeting chimeras) to incorporate amino acid sequences that function as phosphorylation-dependent binding sites for specific proteins, along with an E3 ligase recognition sequence for degradation and a poly-D-arginine sequence to mediate cell permeability, and called this new system phosphoPROTAC. They created two such phosphoPROTACs: TrkAPPFRS2α contained a phosphorylation-dependent binding site for the adaptor FRS2α, an effector of the nerve growth factor (NGF) receptor TrkA, and ErbB2PPPI3K contained the p85 subunit binding site for phosphoinositide 3-kinase (PI3K), which is phosphorylated by the neuregulin receptor, the ErbB2-ErbB3 heterodimer. Treatment of PC12 cells, a rat pheochromocytoma cell line, with TrkAPPFRS2α resulted in a dose-dependent reduction in FRS2α and inhibition of activation of extracellular signal–regulated kinase (ERK) and neuronal differentiation in response to NGF. Application of ErbB2PPPI3K to various neuregulin-responsive cancer cell lines reduced the abundance of the p85 subunit, inhibited phosphorylation of the downstream kinase Akt, and reduced cell viability. Phosphorylation-deficient variants of these two phosphoPROTACs were not effective, and the stability of other phosphorylation-dependent binding proteins was not affected by the phosphoPROTACs, nor was the target protein affected by stimulation of phosphoPROTAC-treated cells with ligands for other receptors. Finally, mice injected subcutaneously with the ovarian cancer cell line OVCAR8 had smaller tumors when injected intraperitoneally with ErbB2PPPI3K compared with mice injected with either control solution or the nonphosphorylated variant ErbB2NPPI3K. Thus, phosphoPROTAC is a method of conditionally knocking down protein function and thus selectively disrupting phosphorylation-dependent signaling.
J. Hines, J. D. Gough, T. W. Corson, C. M. Crews, Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs. Proc. Natl. Acad. Sci. U.S.A. 110, 8942–8947 (2013). [Abstract] [Full Text]