We worked on finding a new kinase regulator to develop basic data to be used for cancer prevention. Our work found a link between three previously unrelated proteins involved in cancer, ERK2 and ERK6 and Scribble. The MAP kinase cascade is involved in cell proliferation, which is highly deregulated in cancer. Through the screening of ERK6 associating molecules, we found the cell polarity, and cell cycle related molecule Scribble. Furthermore, we found that Scribble was a dual-specific kinase regulator. We clearly demonstrated that these ERKs interact with Scribble through the LRR and the PDZ domains of Scribble. We hypothesize that Scribble may function as a scaffolding protein for ERK2 and ERK6, since Scribble has been found to down regulate the kinase activity of these ERKs.

Extracellular signal-regulated kinases (ERKs) modulate cellular activities in response to extracellular stimuli and play important biological roles. Thus, perturbed kinase pathways induce pathological conditions, such as tumor development. Rit, a novel member of the Ras family GTPases, activase ERK6, and its over-expression confers tumorigenicity. We hypothesized the presence of scaffolding molecules specific to ERK6, similar to other known MAP kinases. We performed yeast two-hybrid assays using ERK6 as bait, and Scribble was identified as a binding partner. Scribble contains 16 LRR domains and four PDZ domains. We performed immunoprecipitation (IP) assays and discovered ERK2 as another binding partner. Surprisingly, no interaction was observed with the highly homologous MAP kinase, ERK1. No other representative kinases showed binding capabilities with Scribble. IP data confirmed that both ERK2 and ERK6 bind to Scribble through its LRR and PDZ domains. Deletion of ten aminoi acids from the C-terminus of ERK2 and ERK6 abolished these interactions. In vitro kinase assays indicated the kinase suppressing ability of Scribble. Focus formation assays were performed with RitQ79L and H-RasV12 as constitutive activators of ERK6 and ERK2, respectively, in the presence of Scribble. Results confirmed the role of Scribble as a tumor suppressor.