Journal of Biological Chemistry
Volume 292, Issue 44, 3 November 2017, Pages 18227-18239
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Cell Biology
Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage

https://doi.org/10.1074/jbc.M117.781492Get rights and content
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We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

insulin-like growth factor (IGF)
phosphorylation
post-transcriptional regulation
proliferating cell nuclear antigen (PCNA)
ubiquitin

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This work was supported by the Swedish Cancer Foundation, the Swedish Research Council, the Cancer Society in Stockholm, the Swedish Children Cancer Society, Stockholm County Council, and the Karolinska Institutet. The authors declare that they have no conflicts of interest with the contents of this article.

This article contains supplemental Figs. S1–S10 and Table S1.

1

Present address: Institute of Molecular Medicine at Phoenix Children's Hospital and Dept. of Child Health, University of Arizona College of Medicine-Phoenix, Phoenix, AZ 85004.