Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage

We have previously shown that the insulin like growth factor 1 receptor (IGF1R) translocates to the cell nucleus, where it binds to enhancer like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF1R (nIGF1R) 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 the present study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF1R 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 IGF1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA coincubated with the IGF1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF1R targets, and PCNA phosphorylation was followed by mono and poly ubiquitination. Coimmunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF1R or mutation of Tyr60, Tyr133, or Tyr250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF1R, externally induced DNA damage in IGF1R negative cells caused G1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF1R in DDT.