Regulation of MLK3 by LATS

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ABSTRACT Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that regulates multiple MAPK signaling pathways. As a MAP3K, MLK3 phosphorylates MAP2Ks, which in turn activate c-Jun N-terminal Kinase (JNK) and p38 MAPKs to regulate stress-signaling responses. Through a kinase- independent scaffold function, MLK3 also activates B-Raf and extracellular signal-regulated kinase (ERK) signaling to promote cell proliferation. MLK3 is also critical for the migration and invasion of human ovarian, gastrointestinal, lung, and breast cancer cells. The LATS1 and 2 tumor suppressors are Ser/Thr kinases that control cellular responses including proliferation, migration, apoptosis and gene transcription. Loss of function of either LATS1 or LATS2 occurs in different types of tumors including ovarian, leukemia, prostate, breast, lung, and esophageal cancer. We identified that LATS1 phosphorylates MLK3 and promotes MLK3-14-3-3 binding, and we propose that this interaction results in retention of MLK3 in the cytoplasm. Accordingly, we observed that MLK3 is predominantly nuclear in ovarian cancer cells which have minimal functional LATS, and predominantly cytoplasmic in normal, ovarian epithelial cells that have functional LATS. Furthermore, our preliminary findings indicate that LATS together with β-TRCP regulate ubiquitination and turnover of MLK3. Our central hypothesis is that in ovarian epithelial cells, LATS phosphorylation of MLK3 promotes MLK3-14-3-3 binding and cytoplasmic retention, and also controls MLK3 protein turnover by mediating β-TRCP-dependent ubiquitination and degradation of MLK3. We postulate that in cells that lack functional LATS, MLK3 protein is aberrantly localized to the nucleus, and this results in inappropriate MLK3 interactions and signaling, which drives cellular transformation and tumorigenesis. The major focus of this research is to decipher the mechanism(s) by which LATS1 regulates MLK3 protein subcellular localization and turnover, and to investigate whether nuclear MLK3 drives cellular transformation of ovarian epithelial cells. Accordingly the specific aims of the proposal are: 1) To investigate how LATS and 14-3-3 regulate MLK3 localization 2) To analyze the regulation of MLK3 protein turnover by LATS, CK1ε and β-TrCP, and 3) To investigate whether nuclear MLK3 drives cellular transformation. These results will allow us to gain an understanding of how LATS regulates MLK3 in normal ovarian epithelial cells, and how loss of this regulation promotes aberrant MLK3 localization and heightened MLK3 protein, that drives cellular transformation.