Our research is organized into two inter-related subprojects. The first focuses on how 3'UTR sequences in mRNAs encoding substrates of oncogenic kinases such as ERK and CK2 regulate access of these proteins to their kinases in tumor cells. The 3'UTR mechanism involves subcellular mRNA localization, which can either inhibit or stimulate phosphorylation of the encoded protein by controlling access to kinases. The kinases in turn display different localization in normal and transformed cells. The second subproject involves investigating how oncogenic kinases partition to the perinuclear cytoplasm in tumor cells to form perinuclear signaling centers, or PSCs. PSCs serve as critical signaling engines that drive malignant transformation and cancer. 1. 3'UTR regulation of protein activity (UPA) and its underlying mechanisms. 3'UTR regulation of protein activity was discovered through our earlier observation that the CEBPB 3'UTR inhibits RAS-induced post-translational activation of the C/EBPb protein in tumor cells. Thus, the 3'UTR uncouples C/EBPb from RAS signaling, thereby constraining its pro-senescence activity. UPA also represses the ability of C/EBPb to activate transcription of pro-inflammatory senescence-associated secretory phenotype (SASP) genes. UPA requires a long G/U-rich element (GRE) motif in the 3'UTR and its cognate binding protein, HuR/ELAV1. These components exclude CEBPB mRNAs from a perinuclear cytoplasmic compartment where ERK1/2 and CK2 are present on signaling endosomes embedded within the ER network. Hence, newly translated C/EBPb is prevented from accessing its kinases. We later found that the RNA decay proteins UPF1 and Staufen (STAU1/2) are also essential UPA factors and are enriched within the perinuclear cytoplasm. Together with HuR, these proteins promote perinuclear mRNA decay (PMD) of CEBPB transcripts. Depletion of UPF1 or STAU in tumor cells increased the nuclear-proximal population of CEBPB transcripts, leading to C/EBPb phosphorylation on its CK2 site and senescence. High resolution imaging showed that the perinuclear CEBPB mRNAs frequently colocalize with CK2 foci. These observations suggest that when UPA is disabled, C/EBPb undergoes phosphorylation by CK2 which is contingent on close proximity of CEBPB transcripts (and thus newly translated CEBPb) with activating kinases. We identified a STAU binding site (SBS) adjacent to the GRE which, when deleted, activates the pro-senescence functions of C/EBPb and phosphorylation by CK2 but not its ability to induce SASP genes. Furthermore, deletion of the GRE alone also leads to C/EBPb-induced senescence but not phosphorylation by CK2. These and other observations imply that distinct 3'UTR sequences repress different C/EBPb functions, likely by differential inhibition of PTMs. We propose that various mRNA decay factors (e.g., STAU) which recognize discrete 3'UTR sequences are tethered to different types of signaling endosomes; e.g., those carrying ERK, CK2 or other kinases. Hence, individual 3'UTR elements may promote mRNA decay in the vicinity of particular kinases and thereby inhibit protein phosphorylation on specific sites. Future work will expand upon this novel relationship between modular 3'UTR motifs, localized mRNA decay and inhibition of specific PTMs on the encoded protein. To examine the in vivo relevance of UPA, we generated mice with a deletion that removes the Cebpb GRE and part of the adjacent SBS. This mutant strain was tested in a Kras model of lung tumorigenesis. Although overall lung tumor burdens in deltaGRE mice were similar to WT animals, regions of malignant adenocarcinoma were significantly reduced. Benign lesions such as adenomas were unaffected. These findings provide the first in vivo evidence that UPA constrains C/EBPb activity to facilitate tumor progression to carcinomatous lesions. We are currently performing senescent cell analysis and RNA-seq studies on tumors of different stages in the two genotypes to assess whether the delGRE mutation increases senescence and how this allele alters the C/EBPb transcriptome. A key goal of our work is to determine whether UPA is a general mechanism that regulates many proteins. We are using CRISPR-mediated deletion of 3'UTRs and 3'UTR swap strategies to identify other UPA-regulated genes. p53 is one such candidate. Its3'UTR suppresses the cytostatic activity of a p53 in RAS tumor cells, without affecting p53 protein levels, and excludes TP53 mRNAs from the kinase-rich perinuclear region, inhibiting phosphorylation on its CK2 site, Ser392. TP53 transcripts partition away from the nuclear-proximal region in tumor cells. However, TP53 mRNAs undergo perinuclear translocation in exposed to chemotherapeutic DNA damaging agents that induce p53-dependent senescence or apoptosis, coinciding with increased Ser392 phosphorylation. In summary, our findings demonstrate that 3'UTR-dependent changes in mRNA localization control the activity of p53, C/EBPb, and probably many other proteins. 2. Mechanisms and function of perinuclear signaling centers (PSCs) as key signaling engines in cancer cells. Oncogenic RAS induces perinuclear translocation of p-ERK and CK2 and the signaling scaffold KSR1. These proteins form signaling hubs on endosomes tethered to the ER network. These PSCs are critical to the 3'UTR (UPA) mechanism and are observed in all cancer cell lines and tumor tissues tested. PSCs are key signaling engines that drive cancer, allowing oncogenic kinases to access targets that are important for neoplastic transformation. We found that the endosomal adaptor TOLLIP is required for perinuclear localization of RAB11A+ endosomes harboring CK2 and KSR1, but not ERK. ERK resides on a different class of signaling endosomes. TOLLIP is perinuclear in human cancer cells and KRasG12D-driven mouse tumors but is pan-cytoplasmic in non-transformed cells and thus coincides with the presence of PSCs. A conserved "linker" region in TOLLIP mediates interactions with KSR1 pseudo-kinase domain. This association recruits CK2 signaling complexes to endosomes. A series of phosphoproteomic experiments showed that perinuclear CK2 phosphorylates selective substrates, including proteins involved in ribosome biogenesis and translation. One such target is the atypical kinase RIOK1, which regulates 18S rRNA processing and 40S subunit maturation. Mutant analysis suggests that phosphorylation on RIOK1 Ser22 by perinuclear CK2 is essential for RIOK1 function in tumor cells. KRasG12D-driven lung tumors in Tollip-/- mice progress less efficiently to the malignant adenocarcinoma stage. Furthermore, tumor cell lines carrying mutant KRAS or NRAS, but not HRAS or BRAF mutations, require TOLLIP for proliferation/survival. TOLLIP is therefore a key signaling adaptor in K/NRAS tumor cells whose inhibition is a potential vulnerability of these aggressive, treatment-resistant cancers. in collaboration with Dr. Nadya Tarasova (CIL), we are identifying small molecules that are projected to dock in TOLLIP pockets. One such candidate shows potent inhibition of proliferation/survival in KRAS mutant cancer cells. This and other compounds that that block PSC formation in tumor cells will be considered for further development as anti-cancer agents. As cancer cells driven by mutant HRAS, BRAF and other oncogenes display reduced dependence on TOLLIP but nevertheless exhibit perinuclear CK2, we believe that an alternative adaptor protein(s) provides a redundant perinuclear tethering function in these cells. Furthermore, ERK PSCs remain perinuclear in the absence of TOLLIP. Therefore, our future research will include identifying and characterizing additional endosomal adaptors involved in PSC formation by ERK, CK2 and other components of the RAS pathway.