Project Summary Many deadly cancers show mutations in a pathway driven by forms of cyclin D, Cdk4 or Cdk6, their inhibitory proteins, the Rb retinoblastoma protein, and the E2F transcription factors. These include breast cancer, melanoma, head and neck cancer, esophageal and pituitary cancers. We know that Rb suppresses E2F transcription factors to control genes critical for cell proliferation, but Rb also has transcription-independent activities that impact cell division and survival. Considerable interest is driven by the clinical efficacy of the cyclin D/Cdk4 inhibitor palbociclib, suggesting that additional components of this pathway may be targets of the inhibitor, or that new targets may reside within the pathway. Despite proteomic work identifying proteins that associate with pathway components, the key molecular effectors and regulators of the Rb pathway and protein post-translational modifications (PTMs) that regulate these factors remain unclear. Here we will focus first on systematically identifying (1) the proteins, PTMs, and signal dependencies observed during G1/S progression; (2) identifying which pathway components respond to Palbociclib by shotgun and phosphoproteomics; (3) defining nuclear and cytoplasmic regulated complexes for Cyclin D-Cdk4/6 and Rb/E2F components; (4) building protein interaction networks to link PTMs to protein interactions and activity; (5) determining the specificity of interactions among paralogs of the Cdk4/6, Cyclin D1/2/3, Rb and E2F components; and (6) identifying TCGA cancer lesions found within components of this pathway. These measurements and perturbations performed by the Proteomics Core will be assembled into a network of PTMs and protein-protein interactions (PPIs), driving new hypotheses for this critical cancer pathway.