Cullin5 and SOCS Protein Regulation of Receptor Tyrosine Kinases

Abstract

Suppressors of cytokine signaling (SOCS) proteins are a family of eight proteins that can inhibit signal transduction through receptor tyrosine kinases (RTKs) by serving as substrate receptors in Cullin5-RING E3 ubiquitin ligase (CRL5) and through a variety of CRL5-independent mechanisms. We have shown that Cul5, SOCS2 and SOCS6 inhibit epithelial cell transformation. To identify heretofore-uncharacterized substrates of CRL5SOCS2 and CRL5SOCS6, we performed multiple mass spectrometry-based screens. First, a quantitative phosphoproteomic comparison of WT and Cul5-depleted cells identified increased abundance of phosphotyrosine (pY) peptides from the activation loop of the insulin receptor (INSR) and insulin-like growth factor receptor I (IGF-IR). Using western blotting we found pY1135 in the activation loop of INSR or IGF-IR was indeed increased in Cul5-depleted cells, suggesting that activation of either the INSR or IGF-IR or both are negatively regulated by CRL5SOCS1-depletion. Second, we identified proteins that interact with SOCS2 and SOCS6 using two parallel proteomics techniques: BioID and Flag affinity purification mass spectrometry. We found that the receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) interacted with SOCS2. Furthermore, SOCS2-EphA2 binding required the SOCS2 SH2 domain and autophosphorylation of a tyrosine in the activation loop of EphA2 induced by the ligand Ephrin A1 (EfnA1). SOCS2 interaction only occured with EphA2 that was internalized into endosomes following EfnA1 stimulation. Moreover, SOCS2 overexpression induces EfnA1 expression, and increased EfnA1 decreases the steady-state level of EphA2. Collectively, this suggests that SOCS2 may contribute to trafficking of EphA2. Third, we examined the relationship between Cul5 and the receptor tyrosine kinase epidermal growth factor receptor (EGFR) in mammary epithelial cells. Unlike control cells, Cul5-depleted cells grow independent of added epidermal growth factor (EGF) in the media. However, we found that the growth of Cul5-depleted cells in EGF-free media required EGFR ligand binding and EGFR kinase activity. Cul5-depleted cells showed increased expression of some EGFR ligands in EGF-free media and increased phosphorylation of EGFR, consistent with an autocrine requirement of EGFR signaling. Taken together, our results demonstrate new mechanisms by which Cul5-depletion or SOCS protein over-expression may regulate receptor tyrosine kinases in epithelial cells.