Highlightsd 95 phosphosites on WC-1 and WC-2 were found and their roles in circadian feedback defined d Circadian negative feedback requires specific phosphorylations on both WC-1 and WC-2 d A complex including FRQ, FRH, and CK1 promotes essential phosphorylations on WC-1-WC-2 d Phosphorylations abolish WC-1-WC-2 DNA binding, impacting both core oscillator and output

PP2A is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B‐subunits. Only a limited number of PP2A‐regulated phosphorylation sites are known. This hampers our understanding of the mechanisms of site‐specific dephosphorylation and of its tumor suppressor functions. Here, we develop phosphoproteomic strategies for global substrate identification of PP2A‐B56 and PP2A‐B55 holoenzymes. Strikingly, we find that B‐subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit, resulting in unique patterns of kinase opposition. For PP2A‐B56, these patterns are further modulated by affinity and position of B56 binding motifs. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Binding of PP2A‐B56 to ADAM17 protease decreases growth factor signaling and tumor development in mice. This work provides a roadmap for the identification of phosphatase substrates and reveals unexpected mechanisms governing PP2A dephosphorylation site specificity and tumor suppressor function.

Highlights d R-DeeP implements the RNA dependence concept using density gradient centrifugation d Proteome-wide, specific, and quantitative analysis of 1,784 RNA-dependent proteins d Comprehensive online resource, including 537 proteins never before linked to RNA d Quantitative RNA dependence uncovers RNA effect on CTCF recruitment to chromatin

Highlights d The conserved PP4 holoenzyme binds to FxxP motifs that provide specificity d FxxP motifs bind to a conserved binding pocket on PP4 regulatory subunit d Binding to FxxP motifs can be regulated through phosphorylation d PP4 binding to an FxxP motif in WAPL regulates its cohesin release activity