Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinasesignaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2α or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short-and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects.A ntagonizing the function of a protein is an effective strategy for determining its role within a cellular context. For some enzymes and/or receptor proteins, this can be accomplished by incubation with a small molecule inhibitor or antagonist. However, specific small molecule inhibitors have not been discovered or developed for many proteins. As alternative strategies, gene deletion and RNAi approaches function at the DNA and mRNA levels to reduce protein expression, thus offering the ability to control function even for those proteins lacking a specific inhibitor (1, 2). However, such nucleic acid-based approaches do not afford the same rapid, direct assessment of protein function as posttranslational intervention; furthermore, in vivo application of these strategies is even today still complicated and relatively cumbersome. Combining the attractive qualities of both RNAi and small molecule inhibitor approaches, we developed a strategy, proteolysis targeting chimeras (PROTACs) for targeted posttranslational knockdown of proteins. Each PROTAC is heterodimeric, consisting of an E3 ubiquitin ligase-binding moiety linked to a ligand that binds to the target protein (3). As such, each PROTAC recruits i...