Role of Ubiquitination in IGF-1 Receptor Signaling and Degradation

BACKGROUND: The insulin-like growth factor 1 receptor (IGF-1R) plays numerous crucial roles in cancer biology. The majority of knowledge on IGF-1R signaling is concerned with its role in the activation of the canonical phosphatidyl inositol-3 kinase (PI3K)/Akt and MAPK/ERK pathways. However, the role of IGF-1R ubiquitination in modulating IGF-1R function is an area of current research. In light of this we sought to determine the relationship between IGF-1R phosphorylation, ubiquitination, and modulation of growth signals. METHODOLOGY: Wild type and mutant constructs of IGF-1R were transfected into IGF-1R null fibroblasts. IGF-1R autophosphorylation and ubiquitination were determined by immunoprecipitation and western blotting. IGF-1R degradation and stability was determined by cyclohexamide-chase assay in combination with lysosome and proteasome inhibitors. PRINCIPAL FINDINGS: IGF-1R autophosphorylation was found to be an absolute requirement for receptor ubiquitination. Deletion of C-terminal domain had minimal effect on IGF-1 induced receptor autophosphorylation, however, ubiquitination and ERK activation were completely abolished. Cells expressing kinase impaired IGF-1R, exhibited both receptor ubiquitination and ERK phosphorylation, however failed to activate Akt. While IGF-1R mutants with impaired PI3K/Akt signaling were degraded mainly by the proteasomes, the C-terminal truncated one was exclusively degraded through the lysosomal pathway. CONCLUSIONS: Our data suggest important roles of ubiquitination in mediating IGF-1R signaling and degradation. Ubiquitination of IGF-1R requires receptor tyrosine kinase activity, but is not involved in Akt activation. In addition we show that the C-terminal domain of IGF-1R is a necessary requisite for ubiquitination and ERK phosphorylation as well as for proteasomal degradation of the receptor

Regulation of insulin-like growth factor-1 receptor expression and signaling

Insulin-like growth factor-1 receptor (IGF-1R), a member of the insulin receptor tyrosine kinase family is a broadly expressed transmembrane receptor that plays a key role in malignant cell growth. IGF-1R transmits information provided by extracellular stimuli into intracellular signaling pathways resulting in the subsequent regulation of various effector systems. Under normal cellular conditions IGF-1R signaling network is tightly regulated. The most prominent regulator of IGF-1R signal termination is desensitization of receptors by the removal of activated receptors from the cell surface mediated by accelerated endocytosis. For some membrane receptors the signal mediating receptor internalization/downregulation is constituted by ubiquitination. Recently, we showed that IGF-1R undergoes ubiquitination following ligand stimulation. The proto-oncogene MDM2 was identified as an E3 ligase involved in IGF-1R ubiquitination. Studies on new events involved in IGF-1R downregulation and intracellular signaling constitute the subject of the present thesis. â-arrestins are ubiquitously expressed cytosolic proteins generally known to be involved in the regulation of endocytosis and signaling elicited by G protein-coupled receptors (GCPRs). We provide evidence that the two widely co-expressed isoforms of â-arrestin, bind to the IGF-1R and, by serving as adaptor proteins bring the oncoprotein E3 ligase MDM2 to the receptor. Thus, â-arrestins promote ubiquitination but also degradation of the receptor. In this respect, â-arrestin 1 is more potent then isoform 2. Actually, â-arrestins are an absolute requirement for interaction between MDM2 and IGF-1R, indicating their relevance for cell growth and cancer. We also investigated the role of â-arrestin 1 and MDM2 in intracellular signaling. We found that both MDM2 and â-arrestin 1 also are necessary for IGF-1 stimulated phosphorylation of ERK1/2 but not of Akt. In addition, the modulating effect of MDM2 and â-arrestin 1 on ERK activation has consequences on cell cycle progression. Thus, MDM2 and â-arrestin 1 do not only induce ubiquitination and degradation of IGF-1R but also influence cell growth by modulating the activity of ERKs. The cyclolignan PPP is an inhibitor of phosphorylation of IGF-1R and activation of downstream molecules, without interfering with the highly homologous insulin receptor (IR). Further, PPP has well established anti-tumor effects on several in vivo tumor models. We could demonstrate that PPP also causes downregulation of IGF-1R. Furthermore, the PPP-induced downregulation of IGF-1R required the expression of wild type MDM2 E3 ligase, indicating that MDM2-dependent ubiquitination and degradation of IGF-1R represents an important mechanism in this respect. Our data also suggest that this effect of PPP plays a role in induction of apoptosis. Finally, we demonstrated that PPP in fact induces IGF-1R ubiquitination, but also temporarily activates ERK1/2. This effect is IGF-1R-specific since PPP does not affect ERK phosphorylation in IGF-1R negative cells. Moreover, in the absence of MDM2, PPP-induced activation of ERK did not occur. The temporary MDM2-dependent ERK phosphorylation induced by PPP may contribute to the apoptotic effect of this compound

Ubiquitination of IGF-1R is crucial for ERK signaling.

<p>After 24 h serum starvation wt, Y1136F, K1003R, and Δ1245 cells were stimulated with IGF-1 for the indicated times and lyzed. The lysates were resolved by SDS-PAGE and analyzed by western blotting with anti-phospho-Akt, anti-phospho-ERK. The blots were then stripped and reprobed with anti-Akt, anti-Erk antibodies to demonstrate equal loading. The experiments were repeated with similar results.</p

C-terminal domain is essential for ubiquitination of IGF-1R.

<p>After 24-h serum starvation Y1136, Y950F, Δ1245 and Y950F-Δ1245 cells were stimulated for the indicated time points with IGF-1. Determinations of IGF-1R phosphorylation and ubiquitination were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000340#pone-0000340-g002" target="_blank">Fig. 2</a>. The investigated signals from three independent experiments were quantified (% of loading control) and presented in graphs above each blot. Means and SDs are shown.</p

Intact Y1136 and Y950 are important for lysosomal degradation of IGF-1R.

<p>Y1136F, Y959F, and Y950F-Δ1245 cells were pre-incubated without or with PI or LyI and treated with CHX as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000340#pone-0000340-g005" target="_blank">Fig. 5</a>. After western blotting quantification of IGF-1R, as normalized with GAPDH, was performed. Means and SDs of three different experiments are shown. <i>*P,<0.05</i>; ** <i>P,<0.005</i>.</p

Wild type and mutant IGF-1R constructs.

<p>A. IGF-1R is a heterodimeric receptor with two identical ß-subunit and two identical α-subunits. The α-subunit is completely extracellular, while the ß-subunit spans the membrane. The intracellular portion (residues 931–1337) has a TK domain with intrinsic kinase activity. Important tyrosine residues in the TK domain, know as activation loop, are (Y1131, Y1135 and Y1136) shown in green. Tyrosine residue Y950 (in blue) is, when phosphorylated, a docking site for binding of signaling molecules. Other important regions of the receptor are the ATP binding site (K1003) and the C-terminal domain important for phosphorylation and signaling, respectively. B. The expression of all IGF-1R variants were investigated on cells growing under basal conditions (complete medium supplemented with serum). Cell lysates were subjected to western blotting using antibodies to IGF-1R (β-subunit) and GAPDH (loading control).</p

Ubiquitination of IGF-1R is phosphorylation dependent.

<p>After 24-h serum starvation wt IGF-1R (A) and K1003R (B) cells were stimulated for indicated time points with 50 ng/ml IGF-1. Lysates were immunoprecipitated (IP) with anti-IGF-1Rß (H60), and blotted with either anti-phosphotyrosine (pY99) or anti-ubiquitin (p4D1). The graphs represent quantification (% of loading control) of three independent experiments. Means and SDs are shown. Equal loading was confirmed by stripping and re-immunoblotting with anti-IGF-1Rß (C20).</p

IGF-1R is degraded through both proteasomes and lysosomes.

<p>A. Wt, K1003R, and Δ1245 were either untreated (Unt) or pre-incubated with epoxomicin (PI) or chloroquine (LyI) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000340#s4" target="_blank">Materials and methods</a>. Cycloheximide (CHX) (50µg/ml) was added and the cells were further incubated for 6 or 12 h. The cells were lyzed and subjected to SDS-PAGE and western blotting with antibody to IGF-1Rß. The proreceptor and 95 kDa β-subunit of IGF-1R is indicated. B shows quantified data of IGF-1R, as normalized with GAPDH. Means and SDs of three separate experiments are shown. * <i>P,<0.05</i>; **<i>P,<0.005</i>.</p