Abstract A35: Oncogenic Ras signaling involves formation of perinuclear CK2α nd p-ERK1/2 signaling complexes that require the KSR-1 scaffold protein and endosomal trafficking
Authors: Sandip K. Basu, Sook Lee, Krisada Sakchaisri, Vijay Walia, Christopher J. Westlake, Deborah K. Morrison, Peter F. Johnson
Journal: Cancer Research (2016)

Abstract Background: Ras GTPases play critical roles in transmitting signals from extracellular growth factors (GFs) to downstream effector pathways. Oncogenic Ras mutations occur frequently in human tumors and encode active Ras proteins that promote deregulated signaling in a GF-independent manner. Although the differences between oncogenic Ras and GF-driven signal transmission have been studied extensively, further insights into the features that define normal and tumorigenic Ras signaling may reveal important targets for development of novel anti-cancer therapies. Expression of oncogenic Ras in primary cells can provoke oncogene-induced senescence (OIS), an intrinsic tumor suppressor mechanism that is bypassed in cancer cells. The transcription factor C/EBPβ is post-translationally activated by Ras signaling, and activated C/EBPβ contributes to OIS in MEFs. We previously reported that C/EBPβ activation by H-RasV12 signaling is suppressed in transformed cells, but not in primary cells, by its 3'UTR. This novel RNA-mediated control mechanism is termed 3’UTR regulation of protein activity (UPA). A sequence in the 3’UTR regulates localization of Cebpb transcripts, directing them to a peripheral cytoplasmic region that is distinct from a perinuclear region containing the C/EBPβ activating kinase, p-ERK. Thus, UPA blocks C/EBPβ activation in tumor cells and suppresses its pro-senescence functions. These findings suggest an important role for subcellular localization of downstream kinases in Ras signal transmission. In the present study we have investigated the mechanism and functional consequences of perinuclear compartmentalization of the Ras effector kinases p-ERK and CK2α. Results: We identified CK2α as a Ras effector kinase that phosphorylates C/EBPβ on a Ser residue in its DNA-binding domain. This modification can be blocked by the Cebpb 3'UTR in transformed/immortalized cells. Like p-ERK, CK2α localizes to the perinuclear region in cells expressing oncogenic Ras or BRAFV600E. The MAPK signaling scaffold KSR-1 is essential for perinuclear targeting of p-ERK and CK2α, which can also be blocked by inhibitors of MEK or CK2. KSR-1 itself is also sequestered in the perinuclear region in response to oncogenic Ras-Raf signals. In addition, endocytic trafficking is required for perinuclear targeting of KSR-1, p-ERK and CK2α. CK2α co-localized with KSR-1 and Rab11 (a marker of recycling endosomes), while p-ERK was associated with a different KSR-1 positive endosomal population. Interestingly, we found that whereas oncogenic Ras induced constitutive perinuclear localization of p-ERK and CK2α in both transformed and senescing cells, GFs transiently elicited perinuclear compartmentalization of these kinases with delayed kinetics (4-6 hrs post-stimulation). Conclusions: We propose that oncogenic Ras signaling constitutively activates the delayed phase of GF signaling in which the effector kinases p-ERK and CK2α form KSR-1-dependent perinuclear signaling complexes (PSCs). In this subcellular location, ERK and CK2 and perhaps other kinases can access critical targets that promote oncogenic transformation or senescence. These distinct cellular responses may be controlled in part by differential localization of mRNAs encoding substrates such as C/EBPβ. PSCs were observed across a range of human cancer cell lines and in mouse lung tumor tissue, supporting a critical role for localized signaling in driving tumorigenesis. Citation Format: Sandip K. Basu, Sook Lee, Krisada Sakchaisri, Vijay Walia, Christopher J. Westlake, Deborah K. Morrison, Peter F. Johnson. Oncogenic Ras signaling involves formation of perinuclear CK2α nd p-ERK1/2 signaling complexes that require the KSR-1 scaffold protein and endosomal trafficking. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A35.