Conversation between engineered ubiquitin ligase and IGF-1R or IR was determined by co-immunoprecipitation assay. of the cancers with co-expressed IGF-1R/IR. and malignant actions of liver malignancy HepG2 and cervical cancer HeLa cells that over-express IGF-1R and IR. RESULTS The designed ubiquitin ligases specifically down-regulate IGF-1R and IR protein levels Upon activation by insulin and IGF-1, the -subunit tyrosine kinases of IR and IGF-1R mediate the phosphorylation IMD 0354 of additional tyrosine residues, which will serve as the docking sites for the adaptor proteins such as insulin receptor substrates (IRS) [9, 10] (Supplementary physique 1A). Therefore, we generated the designed ubiquitin ligases as shown in Fig.?Fig.1A.1A. PTB domain name, which is derived from IRS-1, a primary adaptor of IGF-1R/IR signaling [9, 10], is responsible for recognizing and interacting with specific phospho-tyrosine residues of active receptors [28]. U-box domain name from CHIP and RING finger domain name from Cbl confer E3 ubiquitin ligase activity [25, 29]. PTB-U-box and PTB-RING were supposed to be sufficient for the functional E3 ligase activity and IGF-1R/IR targeting. PTB was created as the control that has only the binding domain name. Additionally, PTB-U-box (HQ), which harbors a point mutation of H260Q that is known to disrupt the E3 activity of CHIP [30], was designed to serve as the counterpart of PTB-U-box without functional E3 activity. All of the constructs were cloned into pFLAG-CMV-4 to add the FLAG tag at the N-terminus. Open in a separate window Physique 1 Generation of the designed ubiquitin ligaseA, Schematic representation of the designed ubiquitin ligases. B, PTB-U-box promotes IGF-1R and IR down-regulation. HeLa and HepG2 cells were transiently transfected as indicated and analyzed by Western blotting. The bands intensity were quantified and normalized to the control. C, PTB-U-box does not change IGF-1R and IR transcription. Total RNA was isolated from the indicated transfectants and reverse-transcribed into cDNA. mRNA levels of IGF-1R (left) and IMD 0354 IR (right) were measured by quantitative Real-time PCR. To screen the effect of these recombinant IMD 0354 constructs on IGF-1R, IGF-1R-encoding plasmid was transiently transfected into HEK293 cells together SRSF2 with IMD 0354 vacant vector, PTB, PTB-U-box or PTB-RING. Compared with vacant vector and PTB, both PTB-U-box and PTB-RING are able to down-regulate IGF-1R protein in the presence of IGF-1, but PTB-U-box is usually more potent than PTB-RING (Supplementary physique 2). Thus, we mainly focused on PTB-U-box in this study. We examined several malignancy cell lines as for endogenous IGF-1R and IR levels, among which HepG2 and HeLa cells were chosen for the further study, because they express high levels of IGF-1R and IR and these receptors are constitutively activated when cultured in the serum-containing complete culture medium (Supplementary physique 1B). We found that IGF-1R and IR protein were significantly down-regulated in PTB-U-box transfected HepG2 cells and HeLa cells (Fig.?(Fig.1B).1B). However, the cells transfected with vector, PTB and PTB-U-box(HQ) did not show significant decrease in IGF-1R and IR levels. Similar results were also obtained in PTB-U-box-transfected pancreatic cancer cell line PANC-1 (Supplementary physique 3). Meanwhile, IGF-1R and IR mRNA levels, analyzed by quantitative real-time PCR, were not significantly changed (Fig.?(Fig.1C),1C), suggesting that their down-regulation occurred at post-transcriptional level. In addition, we examined the protein level of EGFR and Met, which were not designed to be targeted by our designed ubiquitin ligase, and found that PTB-U-box did not affect these receptors (Supplementary physique 4). Together, these data indicated that PTB-U-box specifically decreases IGF-1R and IR protein levels.