LAUSR

The CDK4/6 inhibitor has been shown to increase recombinant protein productivity in Chinese hamster ovary cells (CHO). Therefore, we investigated the mechanism that couples cell cycle inhibitor (CCI) treatment with protein productivity utilizing proteomics and phosphoproteomics. We identified mTORC1 as a critical early signaling event that preceded boosted productivity. Following CCI treatment, mTOR exhibited a transient increase in phosphorylation at a novel site that is also conserved in human and mouse. Upstream of mTORC1, increased phosphorylation of AKT1S1 and decreased phosphorylation of RB1 may provide molecular links between CDK4/6 inhibition and mTORC1. Downstream, increased EIF4EBP phosphorylation was observed, which can mediate cap-dependent translation. In addition, the collective effect of increased phosphorylation of RPS6, increased phosphorylation of regulators of RNA polymerase I, and increased protein expression in tRNA-aminoacylation pathway may contribute to enhancing the translational apparatus for increased productivity. In concert, an elevated stress response via GCN2/EIF2AK4-ATF4 axis persisted over the treatment course, which may link mTOR to downstream responses including the unfolded protein response (UPR) and autophagy to enhance proper protein folding and secretion. Together, this comprehensive proteomics and phosphoproteomics characterization of CCI treated CHO cells offers insights into understanding multiple aspects of signaling events resulting from CDK4/CDK6 inhibition. This article is protected by copyright. All rights reserved.