Deborah N. Chadee - US grants

A grant has been awarded to the Department of Biological Sciences at The University of Toledo to support the acquisition of: 1) the Storm 860 Gel and Blot Imaging system; 2) the ImageQuant RT ECL Imaging system; and 3) a SpectraMax M5 Multi-detection plate reader. This instrumentation will support the research programs of the PI and Co-PIs and other members of the Department of Biological Sciences focused on cell signaling and regulation of gene expression in human cells and in model systems (nematodes and plants). In addition, these instruments will also be available to the broader research community in other departments at The University of Toledo. These instruments will provide investigators with more advanced and sensitive tools for conducting assays that they currently do not have the capabilities of performing.

The acquisition of these instruments will have a significant broader impact on the educational mission of the Department of Biological Sciences and The University of Toledo. The PI/Co-PIs as well as the department as a whole are strongly committed to the integration of research and education. The use of these instruments will be available to undergraduate students working on research projects under the direction of a faculty mentor. In addition, the use of these instruments will be incorporated into several upper division undergraduate laboratory courses, in particular the core Molecular Genetics and Cell Biology (taught by the PI) labs. Underrepresented groups, through the department?s commitment to the STARS and Glenn-Stokes programs will also benefit from the acquisition of these instruments.

[unreadable] DESCRIPTION (provided by applicant): Patients with NF2 exhibit loss of function mutations or deletions the NF2 tumor suppressor gene. The NF2 gene encodes a 595 amino acid tumor suppressor protein called Merlin. Merlin has homology to the Ezrin, Radixin and Moesin (ERM) group of cellular proteins that link integral membrane proteins to the actin cytoskeleton. Rac and Cdc42, small GTPases of the Rho family of proteins, are critical regulators of the cytoskeleton and Merlin has been implicated as an inhibitor of Rac-dependent signaling. Merlin also inhibits cell growth. The binding partners for Merlin may be critically important in its growth suppressive function. We have identified a novel interaction between Merlin and Mixed Lineage Kinase 3 (MLK3). MLK3 is a serine/threonine kinase that activates multiple MAP kinase signaling pathways. Cdc42 and Rac are upstream activators of MLK3. Our studies suggest that Merlin is a physiological inhibitor of MLK3. We postulate that Merlin blocks Rac- dependent signaling by inhibiting MLK3 activation of MAPK signaling. Furthermore, the lack of functional Merlin in NF2 tumor cells could cause an increase in the basal level of active MLK3 which may promote cell growth and cellular transformation. The aims of this proposal are to analyze the functional significance of the interaction between Merlin and MLK3, to investigate if loss of NF2 expression augments MLK3, ERK and JNK kinase activities, and to determine if Merlin inhibits cell proliferation and characteristics of malignant transformation of MLK3-transformed cells and human tumor cells. We will perform co- immunoprecipitations of full length and truncated, overexpressed MLK3 and Merlin proteins to identify the specific regions of Merlin and MLK3 that are required for their interaction. Indirect immunofluorescence studies will be employed to study the colocalization of MLK3 and Merlin in schwann cells. Binding of MLK3 to its upstream activators will be analyzed in the presence or absence of co-expressed Merlin. Normal cells, tumor cells lacking NF2 expression, cells that have regulated expression of NF2, and cells in which NF2 expression has been silenced, will be used to analyze the effect of Merlin on MLK3, ERK and JNK activation. The impact of Merlin on MLK3- mediated cellular transformation will be investigated in MLK3-transformed NIH-3T3 cells, SKOV3 ovarian tumor cells and HEI-193 tumor cells. Cells will be infected with a lentivirus that overexpresses Merlin, or Merlin lacking the MLK3 binding region, and cell proliferation, anchorage independent growth, and invasiveness of the cells will be analyzed. Collectively, the results of this study will allow us to define the biochemical significance of the Merlin-MLK3 interaction and the impact of Merlin on MLK3-dependent signaling and cellular transformation. We have identified a novel interaction between Merlin, the protein product of the NF2 tumor suppressor gene, and MLK3, a MAP3K that regulates multiple MAPK pathways. This proposal is focused on deciphering the function of Merlin in regulation of MLK3- dependent MAPK signaling, cell proliferation and cellular transformation. Since silencing mlk3 inhibits proliferation of NF2 human schwannoma and NF2 -/- mouse osteosarcoma cells, MLK3 could be a promising new target for the development of therapies to treat NF2 tumors. Collectively, the results of this study will be an important contribution to our current understanding of Merlin function, will give us valuable insight into MAPK signaling in NF2, and will expand our knowledge of potential signaling proteins that can be targeted for the development of treatments for patients with NF2. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Mixed Lineage Kinases (MLKs) are a family of mitogen-activated protein (MAP) kinase kinase kinases (MAP3Ks) that activate multiple MAP kinase (MAPK) signaling pathways to elicit specific cellular responses. The MLK subfamily consists of MLKs 1, 2, 3, 4 and 4. Of these, MLK3 has been the best characterized in terms of biological function and biochemical activity. MLK3 activates ERK, JNK and p38 MAPK pathways and has important functions in cell proliferation and tumor cell invasion. In contrast, very little is known about the biological functon and biochemical activity of MLK4, for which there are two alternatively spliced isoforms. Our preliminary results suggest that MLK4 does not function as a typical MAPK in promoting activation of MAPK signaling pathways. Instead MLK4 is a negative regulator of MAPK signaling and tumor cell invasion. Furthermore, we have identified a novel interaction between MLK4 and MLK3, and we propose that MLK4 suppresses MAPK signaling and invasion by directly inhibiting MLK3 activation. The experiments described in this proposal are designed to investigate the function of MLK4 in regulating MLK3 activation, MAPK signaling and tumor cell invasion. Accordingly, the specific aims of this proposal are as follows: (1) To analyze the role o MLK4 in regulating MAPK signaling. In vitro kinase assays will be performed to test if MLK4 has activity towards MAP2Ks, and if MLK4 inhibits the activity of other MAP3Ks in addition to MLK3. (2) To investigate the MLK3-MLK4 interaction and the mechanism by which MLK4 inhibits MLK3 activation. In vitro binding assays will be performed with MLK4 and MLK3 deletion mutants to identify the regions of MLK3 and MLK4 that interact. Co-immunoprecipitations will be performed in cells overexpressing MLK4 to determine if MLK4 inhibits the MLK3-TRAF, MLK3-Cdc42 or MLK3-B-Raf interactions. (3) To investigate the function of MLK4 in tumor cell proliferation and invasion. Cell proliferation assays and invasion assays (using a modified Boyden chamber) will be performed to test if MLK4 expression or knockdown affects cell proliferation or invasion. The results from the proposed studies will broaden our understanding of the different roles of MLKs in cell signaling and tumor cell invasion, which could provide insight into how altered MLK activity may contribute to establishment of the malignant phenotype. PUBLIC HEALTH RELEVANCE: MAPK signaling pathways have a central role in the regulation of numerous cellular responses such as proliferation, differentiation, migration and invasion. Since persistent activation of MAPK signaling is a major contributor to the establishment of a malignant phenotype, a thorough understanding of how MAPK pathways are tightly controlled by both positive and negative regulatory factors is essential to the development of novel cancer therapies.

? DESCRIPTION (provided by applicant): Mixed lineage kinase 3 (MLK3) is a serine/threonine mitogen-activated protein kinase (MAP3K) that regulates multiple MAPK signaling pathways in response to mitogenic and stress stimuli. Depending on the cellular context, MLK3 can phosphorylate MKK4 or MKK7 MAPK kinases (MAP2Ks), which activate c-Jun N-terminal Kinase (JNK) to trigger cell death. Alternatively, MLK3, through a kinase-independent scaffold function, can activate B-Raf and extracellular signal-regulated kinase (ERK) signaling to promote cell proliferation. MLK3 is required for the migration and invasion of human ovarian, gastrointestinal, lung, and breast cancer cells. A requirement for MLK3 in mitogen-dependent ERK activation and colon cancer cell proliferation has been demonstrated; however its function in colon cancer cell invasion is not known. Many cancer cells have persistent, intrinsic oxidative stress and higher levels of reactive oxygen species (ROS) than normal cells. Stimuli such as pro-inflammatory cytokines, irradiation, toxins and chemotherapeutic drugs can trigger ROS production. At very high levels ROS can elicit activation of signal transduction pathways and induce apoptosis or necrotic cell death, and at low levels ROS can stimulate cell proliferation. Our preliminary findings indicate that oxidative stress strongly activates ERK1/2 signaling which promotes the phosphorylation of MLK3 in colon cancer cells. Oxidative stress also promotes an ERK-dependent increase in MLK3 protein level and a decrease in MLK3 kinase activity. Furthermore, we demonstrated that ERK could phosphorylate MLK3 in vitro. Thus, we have identified ERK as a novel, positive regulator of MLK3 protein level and a negative regulator of MLK3 kinase activity in response to oxidative stress. We postulate that the enhancement of inactive MLK3 protein in response to oxidative stress could cause persistent MLK3-dependent stimulation of ERK signaling and thereby promote cellular transformation and proliferation of colon cancer cells. The overall goal of this proposal is to define the mechanism by which oxidative stress induces ERK-dependent regulation of MLK3, and elucidate the impact of this regulation on MLK3-dependent stimulation of cellular transformation, proliferation and apoptosis in colon cancer cells. Accordingly the specific aims of the proposal are: 1) To investigate the role of ERK signaling in H202-mediated upregulation of MLK3 in colon cancer cells. 2) To determine the impact of ROS and ERK activation on MLK3 kinase activity and function, 3) To elucidate the function of MLK3 in colon cancer cell proliferation and invasion. The experiments described in this proposal will allow us to gain a thorough understanding of how oxidative stress modulates MLK3 protein level and kinase activity and how this affects MLK3-dependent activation of MAPK signaling, proliferation and invasion of colon cancer cells. Furthermore, we will examine novel strategies to deplete cellular MLK3 protein levels to inhibit colon cancer cell proliferation and invasion.

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 ?-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 ?-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, CK1? and ?-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.