PROJECT SUMMARY Placental development is critical in the early stages of pregnancy, as dysregulation of this process can lead to intrauterine growth restriction of the fetus, preeclampsia, and miscarriage. A fundamental step in placental development is the invasion of extravillous trophoblast cells into the endometrium of the uterus. A key mediator of the invasive trophoblast phenotype is cytoskeletal remodeling driven by the activity of the small GTPase Rho. Dynamic changes in the cytoskeleton are indispensable for initiating cell polarity and forming cell protrusions and adhesions that drive cell migration, and the tightly coordinated regulation of these changes is critical for successful placental development. However, the specific signaling mechanisms that regulate Rho GTPase cytoskeletal remodeling during trophoblast invasion remain unclear. Previously published data have suggested that the c-Jun-N-terminal kinase (JNK) subfamily of mitogen-activated protein kinases (MAPKs) may be involved in trophoblast invasion, however the mechanism by which it accomplishes this is not well understood. Recent screens from our lab have identified the Rho GTPase activating proteins (Rho GAPs) SYDE1 and SYDE2 as novel JNK substrates based on kinase-substrate docking interactions. Existing literature indicate roles of SYDE1 and SYDE2 in migration in trophoblasts and cytoskeletal remodeling. It is therefore likely that SYDE1 and/or SYDE2 may be involved in a JNK signaling axis that regulates Rho GTPase cytoskeletal rearrangement and migration during trophoblast invasion. The objective of this proposal is to elucidate how JNK modulates SYDE1 and SYDE2 to control migration and invasion of trophoblasts. This proposal will determine the functional consequences of JNK-mediated regulation of SYDE1 and SYDE2 in trophoblasts by examining the effects on cell morphology, cytoskeletal organization, and migration in human trophoblast cells harboring SYDE1 or SYDE2 deletion or overexpression. Placental tissue samples will also be analyzed to examine clinical relevance of JNK signaling through these proteins. GTPase biosensor assays will be used to determine whether JNK phosphorylation of SYDE1 and SYDE2 positively regulates their activity as Rho GAPs. In vitro GTPase assays will determine whether this mechanism is accomplished by affecting GAP enzymatic activity. In order to establish whether any observed effects are dependent on JNK phosphorylation, JNK inhibitors and docking-site mutant SYDE1 and SYDE2 will be used to ablate JNK-specific phosphorylation. Ultimately, identifying JNK and small GTPase regulators as a key molecular players in trophoblast signaling, as well as understanding the mechanisms by which they act, will allow them to be explored as molecular targets to treat severe pregnancy complications related to aberrant trophoblast migration.