Abstract B16: The nuclear IGF-1R regulates DNA damage tolerance through tyrosine phosphorylation of PCNA in human embryonic stem cells

Abstract

Abstract Background: The type 1 insulin-like growth factor receptor (IGF-1R) and its signaling components promote cell proliferation, survival, and development of the malignant phenotype. Recently, the IGF-1R has been shown to translocate to the nucleus; however its nuclear functions are not fully elucidated. The proliferating cell nuclear antigen (PCNA) is a nuclear protein that coordinates DNA replication, and is involved in DNA damage repair and in the DNA damage tolerance (DDT) pathways. DDT allows the cell to replicate over polymerase-blocking lesions. DDT mechanisms include translesion DNA synthesis (TLS), and template switching, and the ubiquitination status of PCNA is crucial in these processes. Mono-ubiquitination of PCNA by Rad6 (E2)-Rad18 (E3) activates TLS. The mono-ubiquitin can be further extended though K63-linkages and the non degradative K63 polyubiquitination of PCNA by Ubc13 (E2)-SHPRH/HLTF (E3) leads to template switching. Purpose: The purpose of the present work was to study the functions of nIGF-1R in human embryonic stem cells (hESCs) and in the IGF-1R knockout (igf1r -/-) (R-) and the R- cells overexpressing human IGF1R (R+) mouse embryonic fibroblasts (MEFs), using a mass-spectrometry based approach. Methods and Results: Using subcellular protein fractionation (Qiagen Qproteome Cell Compartment Kit, Qiagen, Germany) and western blot (WB), as well as immunofluorescence, we detected the expression of the nIGF-1R in hESCs, human fibroblasts, MEFs, and in cancer cell lines. To investigate potential functions of nIGF-1R in hESCs, nIGF-1R binding partners were identified using co-immunoprecipitation (Co-IP) and mass spectrometry (MS) (hybrid LTQ-Orbitrap Velos mass spectrometer, Thermo Scientific). Multiple protein candidates were identified including PCNA. IGF-1R-PCNA association was confirmed by Co-IP/immunoblot and by in situ proximity ligation assay (PLA) (OLINK Bioscience, Uppsala, Sweden) to morphologically demonstrate the nuclear localization of the complex. The association of nIGF-1R with PCNA could only be detected in hESCs, MEFs and in human fibroblast cell lines, but not in any of the seven cancer cell lines used. Using a phospho-tyrosine antibody we detected the phosphorylation of PCNA immunoprecipitated from hESCs and R+ but not from R- MEFs or cancer cell lines. Using an in vitro kinase assay, recombinant IGF-1R kinase domain and His-tag recombinant PCNA, we showed that IGF-1R directly phosphorylates PCNA. Using LC-MS/MS and site directed mutagenesis, 3 tyrosine phosphorylation sites (Y60,133,250) were identified. Although PCNA ubiquitination was detected by WB of PCNA immunoprecipitated from R+ MEFs, PCNA protein stability was not affected. The ubiquitination pattern resembled the K63-linked polyubiquitination. Using Co-IP/WB, Ubc13 and SHPRH/HLTF were found to be associated with PCNA in R+ but not in R- cells. Similar results were also achieved for the mono-ubiquitin Rad18 E3 ligase and the main TLS polymerase DNA polymerase. Results were confirmed in hESCs but not in cancer cell lines. Further, PCNA ubiquitination was increased after DNA damage using UV irradiation in R+ MEFs and DNA replication was delayed after methyl methanesulfonate (MMS) treatment, while replication fork stalling in R- cells was highly significant (DNA-fiber labeling assay). Inhibition or knockout of IGF-1R abrogated PCNA phosphorylation, ubiquitination, progress through S-phase of cell cycle, or restart of DNA synthesis after inducing DNA damage. Conclusions: Our results demonstrate a novel role of nIGF-1R in regulating the DNA damage tolerance pathway through PCNA phosphorylation and ubiquitination, potentially maintaining genomic stability in in hESC and in normal fibroblasts. In contrast, nIGF-1R does not interact with PCNA in cancer cell lines. Citation Format: Ahmed Waraky, Yingbo Lin, Eiman Aleem, Olle Larsson. The nuclear IGF-1R regulates DNA damage tolerance through tyrosine phosphorylation of PCNA in human embryonic stem cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B16.