4E-BP1 is the primary gatekeeper for cancer cell, cap-dependent protein translation. It is directly targeted by the mTOR pathway that is frequently dysregulated in cancer. We have found that CDK1/CYCB1 substitutes for mTOR during mitosis to phosphorylate 4E-BP1 generating a novel phosphorylation mark at serine (S) 83 that is not present when mTOR phosphorylates 4E-BP1. A mutant form of 4E-BP1 unable to be phosphorylated at S83 partially reverses cell transformation caused by the Merkel cell polyomavirus (MCV) small T oncoprotein. This project is focused on investigating a novel CDK-1-dependent but mTOR-independent 4E-BP1 regulatory pathway. The central hypothesis for this proposal is that S83 phosphorylation modulates translation of a unique subset of mRNAs to facilitate mitosis-specific protein expression.
In Aim I, substitution of a mutant (S83A) and the phosphomimetic (S83D) 4EBP1 proteins into EIF4EBP1 null cells, as well as developing MEFs from the EIF4EBP1 S83A knock-in mice will be used to assay the effects of S83 phosphorylation on basic cell homeostasis, including cell cycle analysis, proliferation and protein synthesis.
In Aim II, we will identify differentially translated mRNAs as a result of mitotic 4E-BP1 phosphorylation through two complementary approaches: ribosomal profiling of mitosis-arrested cells, and RNA immunoprecipitation and sequencing (RIPseq).
In Aim III we will use live-cell imaging tools to track and quantify dynamics of translation in live cells. Finally in Aim IV, we will explore a unique knock-in mutant mouse model for 4E-BP1 dysregulation. Our studies will advance our fundamental understanding of how a mitosis-specific hyperphosphorylated form of 4E-BP1 functions in normally cycling cells and how its dysregulation in cancer cells may contribute to human malignancies.
Cancer cell proliferation is dependent on new protein synthesis to generate progeny cancer cells, and a critical enzyme regulating this protein synthesis is mTOR. We found that a second enzyme called cyclin-dependent kinase 1 (CDK1) substitutes for mTOR in some cancer cells. CDK1 is only active during cell mitosis and it may allow synthesis of a subset of proteins involved in the steps of cell division. The results of this proposal will shed light on a new pathway contributing to cancer cell proliferation.
