Suppressors of T-cell Receptor Signaling Sts-1 and Sts-2 Bind to Cbl and Inhibit Endocytosis of Receptor Tyrosine Kinases

Abstract

The ubiquitin (Ub) ligase Cbl plays a critical role in attenuation of receptor tyrosine kinase (RTK) signaling by inducing ubiquitination of RTKs and promoting their sorting for endosomal degradation. Herein, we describe the identification of two novel Cbl-interacting proteins, p70 and Clip4 (recently assigned the names Sts-1 and Sts-2, respectively), that inhibit endocytosis of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor. Sts-1 and Sts-2 contain SH3 domains that interacted with Cbl, Ub-associated domains, which bound directly to mono-Ub or to the EGFR/Ub chimera as well as phosphoglycerate mutase domains that mediated oligomerization of Sts-1/2. Ligand-induced recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization, reduction in the number of EGFR-containing endocytic vesicles, and subsequent block of receptor degradation followed by prolonged activation of mitogenic signaling pathways. On the other hand, interference with Sts-1/ Sts-2 functions diminished ligand-induced receptor degradation, cell proliferation, and oncogenic transformation in cultured fibroblasts. We suggest that Sts-1 and Sts-2 represent a novel class of Ub-binding proteins that regulate RTK endocytosis and control growth factorinduced cellular functions.

Epidermal growth factor receptor double targeting by a tyrosine kinase inhibitor (Iressa) and a monoclonal antibody (Cetuximab). Impact on cell growth and molecular factors

“…There is thus a ligand-controlled turnover in the expression of EGFR, which could be deregulated in the combined presence of C225 and ZD1839. Recently, the identification of proteins p70 and Clip 4 was reported (Kowanetz et al, 2004); these proteins inhibit endocytosis of EGFR and interact with Cbl, a ubiquitin ligase which plays a critical role in EGFR endosomal degradation (Ettenberg et al, 2001). It is possible that the combined presence of C225 and ZD1839 may alter the interaction of Cbl with EGFR by a conformational change induced in EGFR.…”

Regulation of ubiquitin-binding proteins by monoubiquitination

“…HA-tagged hStam2 was kindly provided by S. Urbe (University of Liverpool, UK). The constructs for mammalian expression of Sts1 were all generated by polymerase chain reaction (PCR) using pcDNA3-FLAG (Invitrogen) and have been described recently 9 . The Sts1-ubiquitin and Sts2-ubiquitin chimerae were generated by subcloning the cDNAs for ubiquitin wild-type or I44A mutant, and then amplification by PCR in frame with the 3′ terminus of Sts1-2 or their deletions that had previously been subcloned into pcDNA3-FLAG.…”

“…For bacterial expression of Sts1, Sts1-ubiquitin, Flag-Sts1∆PGM and Flag-Sts1∆PGM-ubiquitin were cloned into the SalI and NotI sites of the pET24-SUMO vector and were expressed in BL21 cells according to the manufacturer's instructions (Lifesensor, Malvern, PA). GST Cbl-CT, containing the proline-rich sequences of Cbl (amino acids 450-860 of human c-Cbl), was expressed in BL21 cells and purified as described previously 9 . The constructs used in the FRET experiments were generated by subcloning citrine into the NheI and HindIII sites of pcDNA3-CFP and subsequent insertion of Sts2, Sts2 K202R , Sts2∆PGM and Sts2∆PGM K202R into the KpnI and BamHI sites of the same vector.…”

“…Given the fact that Eps15, Hrs and Sts proteins are able to dimerize or oligomerize, and are also found in multimeric protein complexes in cells 2,9 , it is possible that their monoubiquitinated forms engage in intramolecular (within the single molecule), intermolecular (between different molecules of the homo-oligomeric complex) or transmolecular (between different proteins in heterologous complexes) interactions. We therefore investigated whether the intramolecular binding between monoubiquitin and the UBA of Sts1 and 2 is sufficient for its auto-inhibition.…”

“…Sts1 and Sts2 have been shown to inhibit EGFR degradation by binding to the ubiquitin ligase Cbl and interacting with ubiquitinated receptor complexes via their UBA domains 9 . To reliably detect differences in the ability of Sts2, Sts2-ubiquitin and Sts2 K202R mutant to interfere with EGFR degradation even in the presence of endogenous Sts1, we overexpressed them along with EGFR.…”