Coordinated activities of protein kinases and phosphatases ensure phosphorylation homeostasis, which, when perturbed, can instigate diseases, including cancer. Yet, in contrast to kinases, much less is known about protein phosphatase functions and their interactions and complexes. Here, we used quantitative affinity proteomics to assay protein-protein interactions for 54 phosphatases distributed across the three major protein phosphatase families, with additional analysis of their 12 co-factors. We identified 838 high-confidence interactions, of which 631, to our knowledge, have not been reported before. We show that inhibiting the activity of phosphatases PP1 and PP2A by okadaic acid disrupts their specific interactions, supporting the potential of therapeutics that target these proteins. Additional analyses revealed candidate physical and functional interaction links to phosphatase-based regulation of several signaling pathways and to human cancer. Our study provides an initial glimpse of the protein interaction landscape of phosphatases and their functions in cellular regulation.

Transcription factors (TFs) interact with several other proteins in the process of transcriptional regulation. Here, we identify 6703 and 1536 protein–protein interactions for 109 different human TFs through proximity-dependent biotinylation (BioID) and affinity purification mass spectrometry (AP-MS), respectively. The BioID analysis identifies more high-confidence interactions, highlighting the transient and dynamic nature of many of the TF interactions. By performing clustering and correlation analyses, we identify subgroups of TFs associated with specific biological functions, such as RNA splicing or chromatin remodeling. We also observe 202 TF-TF interactions, of which 118 are interactions with nuclear factor 1 (NFI) family members, indicating uncharacterized cross-talk between NFI signaling and other TF signaling pathways. Moreover, TF interactions with basal transcription machinery are mainly observed through TFIID and SAGA complexes. This study provides a rich resource of human TF interactions and also act as a starting point for future studies aimed at understanding TF-mediated transcription.

Ribosome biogenesis regulates animal growth and is controlled by nutrient-responsive mTOR signaling. How ribosome biogenesis is regulated during the developmental growth of animals and how nutrient-responsive signaling adjusts ribosome biogenesis in this setting have remained insufficiently understood. We uncover PWP1 as a chromatin-associated regulator of developmental growth with a conserved role in RNA polymerase I (Pol I)-mediated rRNA transcription. We further observed that PWP1 epigenetically maintains the rDNA loci in a transcription-competent state. PWP1 responds to nutrition in Drosophila larvae via mTOR signaling through gene expression and phosphorylation, which controls the nucleolar localization of dPWP1. Our data further imply that dPWP1 acts synergistically with mTOR signaling to regulate the nucleolar localization of TFIIH, a known elongation factor of Pol I. Ribosome biogenesis is often deregulated in cancer, and we demonstrate that high PWP1 levels in human head and neck squamous cell carcinoma tumors are associated with poor prognosis.

Engineering light-sensitive protein regulators has been a tremendous multidisciplinary challenge. Optogenetic regulators of MAPKs, central nodes of cellular regulation, have not previously been described. Here we present OptoJNKi, a light-regulated JNK inhibitor based on the AsLOV2 light-sensor domain using the ubiquitous FMN chromophore. OptoJNKi gene-transfer allows optogenetic applications, whereas protein delivery allows optopharmacology. Development of OptoJNKi suggests a design principle for other optically regulated inhibitors. From this, we generate Optop38i, which inhibits p38MAPK in intact illuminated cells. Neurons are known for interpreting temporally-encoded inputs via interplay between ion channels, membrane potential and intracellular calcium. However, the consequences of temporal variation of JNK-regulating trophic inputs, potentially resulting from synaptic activity and reversible cellular protrusions, on downstream targets are unknown. Using OptoJNKi, we reveal maximal regulation of c-Jun transactivation can occur at unexpectedly slow periodicities of inhibition depending on the inhibitor's subcellular location. This provides evidence for resonance in metazoan JNK-signalling circuits.