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

SUMO and ubiquitin : The Yin and Yang of IGF-1R function

Tumorigenesis is a multistep process involving genetic and epigenetic alterations that drive the progressive transformation of normal human cells into highly malignant derivatives. The insulin-like growth factor 1 receptor (IGF-1R) plays pivotal roles in cancer. Based on the traditional view, IGF-1R mediates its biological responses primarily through receptor phosphorylation and signaling. The subsequent termination of signaling occurs via receptor endocytosis and degradation. Over the last years we have begun to appreciate the biological roles of IGF-1R ubiquitination. In this thesis we established that IGF-1R ubiquitination is dependent on receptor phosphorylation and integrity of the C-terminal domain. We identified β-arrestin as an adapter protein essential for congregating IGF-1R and its ligase MDM2. The β-arrestin/MDM2 mediated IGF-1R ubiquitination was crucial for receptor degradation and also for IGF-1 mediated ERK activation and cell cycle progression. Further, Cbl was recognized as a new ligase for IGF-1R inducing Lys48 poly-ubiquitination of the receptor, whereas MDM2 modified the receptor with Lys63 ubiquitin chain. Cbl-mediated ubiquitination caused caveolin lipid-raft internalization of the receptor, while MDM2- mediated ubiquitination caused clathrin dependent internalization. Moreover, we identified IGF-1R as a novel target for SUMOylation. Ligand dependent SUMO-modification of IGF-1R promoted nuclear translocation of the receptor. Electrophoretic mobility shift assay and chromatin immunoprecipitation-based cloning studies demonstrated binding of IGF-1R to DNA and identified DNA binding regions. By cloning the obtained sequences upstream of a luciferase reporter, we found an IGF-1R induced enhancement of gene transcription. Altogether, Our data suggest that nuclear sequestered IGF-1R activates transcription by binding to enhancer regions. In conclusion, this thesis emphasizes the mechanisms by which ubiquitination of IGF-1R affects signaling and degradation. Furthermore, this thesis shows the importance of various ligases, orchestrating trafficking of the receptor. We also describe the novel involvement of IGF-1R SUMOylation in promoting the translocation of cell surface IGF-1R to the nucleus. Detailed understanding of how these processes are controlled under physiological and pathological conditions may be important for future therapeutic approaches

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

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

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

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

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