Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs

Abstract

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 kinase-signaling 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.

Keywords: SH2 domain; protein degradation; signal transduction.

Fig. 1.

(A and B) Schematic diagram of conditional posttranslational target protein knockdown by a phosphoPROTAC. (A) In the absence of an activated RTK, the phosphoPROTAC is not phosphorylated and fails to bind to the target protein, sparing it from degradation. (B) Upon activation of the RTK, the phosphoPROTAC is phosphorylated, creating a binding site for the SH2- or PTB-domain–containing effector protein and its subsequent recruitment for ubiquitination by VHL and proteasomal degradation. (C) Schematic of phosphoPROTAC ^TrkAPP_FRS2α: the red peptide sequence corresponds to the TrkA autophosphorylation site; the blue peptide sequence enables binding to the E3 ubiquitin ligase VHL; and the green poly-d-arginine motif permits cell permeability of the peptide.

Fig. 2.

Function of ^TrkAPP_FRS2α, a phosphoPROTAC peptide that conditionally targets FRS2α for degradation upon TrkA activation. (A) Selective knockdown of FRS2α by ^TrkAPP_FRS2α and inhibition of NGF-dependent Erk1/2 activation in PC12 cells. Cells were incubated with the phosphoPROTAC for total of 7 h. (B) Knockdown of FRS2α by ^TrkAPP_FRS2α is time dependent. Cells were incubated with 60 µM ^TrkAPP_FRS2α for 30 min to 6 h. (C) Activated ^TrkAPP_FRS2α recruits FRS2α to be ubiquitinated. PC12 cells were pretreated with 100 nM epoxomicin for 1 h before incubation with 60 µM ^TrkAPP_FRS2α and with NGF for 7 h before lysis in boiling RIPA buffer. (D) Phosphorylation dependence of FRS2α knockdown by phosphoPROTAC. Phospho-null variant, ^TrkANP_FRS2α, is inactive in the presence of NGF. (E) Knockdown of FRS2α by ^TrkAPP_FRS2α is dependent on activation of TrkA but not on other receptor tyrosine kinases. NGF, EGF, and IGF1 were all used at 100 ng/mL. In lanes where NGF was not added, tonic activity of TrkA was blocked by supplementation with TrkA inhibitor (1 µM). (F) Knockdown of FRS2α by ^TrkAPP_FRS2α does not interfere with signaling of other RTKs through Erk1/2. NGF, EGF, and IGF1 were all used at 100 ng/mL.

Fig. 3.

^ErbB2PP_PI3K, a phosphoPROTAC that conditionally targets PI3K for degradation upon ErbB2 activation. (A) Streptavidin pulldowns from MCF-7 cells using biotinylated peptides demonstrate efficacy and phospho-dependence of a selected dual phosphotyrosine-containing ErbB3 sequence to bind PI3K p85. Pulldowns electrophoresed and immunoblotted with anti-p85 antibody as described in Materials and Methods. (B) ^ErbB2PP_PI3K-mediated degradation of PI3K is target specific and blocks NRG-dependent Akt activation in MCF-7 cells. (C) Null-phosphoPROTAC ^ErbB2NP_PI3K cannot cause degradation of PI3K p85 even in the presence of neuregulin-activated ErbB2/ErbB3 signaling.

Fig. 4.

Inhibition of PI3K signaling due to targeted knockdown by ^ErbB2PP_PI3K is phospho-dependent and reduces cell viability. MCF-7 cells were incubated with varying concentrations of ^ErbB2PP_PI3K or ^ErbB2NP_PI3K for 48 h, following which viability was assessed using MTS conversion. Bars represent the mean ± SD of three replicate experiments. Asterisk denotes significant difference in MTS conversion compared with control (untreated) cells determined by Student t test (P < 0.05).