Key role of Shc signaling in the transforming pathway triggered by Ret/ptc2 oncoprotein

Abstract

The RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of protoRET, a gene encoding two protein isoforms of a transmembrane tyrosine kinase receptor. By using Ret/ptc2 short isoform (iso9), we have previously demonstrated that Tyr586 (Tyr1062 of protoRet) is the docking site for both the PTB and the SH2 domains of Shc. To determine the relevance of this interaction for the transforming activity of Ret/ptc oncogenes, we have generated and characterized novel Ret/ptc mutants unable to activate Shc: Ret/ptc2 long isoform (iso51)-Y586F and both isoforms of Ret/ptc2-N583A. These mutants neither activate Shc nor transform NIH3T3 cells. Since Tyr1062 shows features of a multifunctional docking site, we have used a Shc mutant (Shc Y317F) to directly assess Shc role. We have demonstrated that in our cell system Shc Y317F behaves like a dominant interfering mutant on the activation of the Grb2-Sos pathway by endogenous Shc triggered by Ret/ptc2. A strong reduction of the transforming activity of Ret/ptc2 in presence of this mutant was also demonstrated. Our data suggest that Shc activation play a key role in the transforming pathways triggered by Ret/ptc oncoproteins. Moreover, we have shown that coexpression of the Shc-Y317F mutant with Ret/ptc2 speci®cally causes apoptosis, and that the surviving cells lose the long-term expression of one of the two genes. Oncogene (2001) 20, 3475 ± 3485.

Biological activity of the thyroid TRK-T3 oncogene requires signalling through Shc

“…The occurrence of apoptosis in our experimental system can be explained by several hypotheses: (1) Shc might transduce possible TRK-T3 anti-apoptotic signals; (2) signalling through tyrosine 317 of Shc could counteract a possible pro-apoptotic pathway induced by TRK-T3; (3) a pro-apoptotic signal might be the consequence of cell stress due to incorrectly transduced TRK-T3 signalling. Interestingly, results similar to ours have been obtained by co-expressing ShcY317F and the Ret/PTC2 oncogene (Mercalli et al, 2001), thus indicating the existence of a mechanism shared by other RTK oncogenes.…”

“…The ShcY317F mutant has been shown to exert a dominant-negative effect on the endogenous Shc activity in different contexts. In ErbB2-positive breast cancer cell lines, ShcY317F blocks growth by disrupting cell-cycle progression (Stevenson et al, 1999); in NIH3T3 cells it reduces the RET/PTC2 oncogene transforming activity (Mercalli et al, 2001); in adipocytes it suppresses the IGF-induced proliferation (Boney et al, 2000); moreover, a Shc mutant lacking the CH1 domain that includes Y317, suppresses NIH3T3 transformation induced by the neu oncogene (Li et al, 1999).…”

“…Tyr317 of Shc, in addition to Tyr239/240, recruits Grb2 upon phosphorylation; the ShcY317F mutant has been shown to exert a dominant-negative effect in different biological processes mediated by Shc (Li et al, 1999;Stevenson et al, 1999;Boney et al, 2000;Mercalli et al, 2001). The ShcY317F and the control ShcWT constructs (Figure 1) were tagged by the haemoagglutinin (HA) epitope, which confers a small electrophoretic shift with respect to the endogenous p52 Shc, and cloned into the pCGN expression vector harbouring the resistance to hygromycin.…”

Autocrine stimulation by osteopontin plays a pivotal role in the expression of the mitogenic and invasive phenotype of RET/PTC-transformed thyroid cells

“…This last residue is embedded in an NKLpY (single-letter aminoacid code) motif and mediates recruitment of various proteins to RET and RET/PTC (Hayashi et al, 2000;Manie' et al, 2001). Shc and FRS2 are recruited to pY1062 via their phosphotyrosine binding (PTB) domains (Melillo et al, 2001;Mercalli et al, 2001) and, by bridging RET/PTC to Grb2-Sos complexes, they play a key role in the activation of Ras small GTPases .…”

RET/PTC-induced dedifferentiation of thyroid cells is mediated through Y1062 signaling through SHC-RAS-MAP kinase

“…However, signal transduction pathways downstream of RET/PTC that are involved in its dedifferentiating effects have not been investigated. To study this we generated clonal cell lines derived from the well-differentiated rat thyroid cell line PCCL3, with doxycycline-inducible expression of RET/PTC mutants that are unable to interact with either PLCg or Shc, two downstream signaling intermediates important in mediating transformation of fibroblasts by RET/PTC (Borrello et al, 1996, Durick et al, 1998, Mercalli et al, 2001.…”