The goal of the project is to understand the signaling pathways that control cytoskeletal changes during processes such as smooth muscle contraction and cell migration. In eukaryotes, cell migration in response to extracellular signals controls processes such as wound healing, embryogenesis, axonal guidance, metastasis of cancer cells, and aggregation of Dictyostelium cells to form a multicellular organism. In Dictyostelium, cGMP signaling plays a vital role in coordinating cytoskeletal rearrangements during chemotaxis, and is implicated in regulating myosin II and other unidentified targets. A bioinformatics approach was used to identify genes for cGMP-binding proteins, with the expectation that one or more of them would have a severe chemotaxis defect. One of these proteins - GbpC - is largely responsible for transmitting the downstream effects of cGMP during chemotaxis. GbpC is the Dictyostelium homolog of a novel family of protein kinases that is evolutionarily conserved throughout the metazoan lineage. These proteins contain leucine-rich repeats, a Ras-like domain, and a protein kinase domain in the MEKK family. GbpC additionally contains, at its C terminus, a RasGEF domain and two cGMP-binding binding sites, and therefore has properties of the cyclic nucleotide regulated GEFs found in higher eukaryotes. GbpC is required for chemoattractant-elicited recruitment of myosin to the cytoskeleton, a process regulated by threonine phosphorylation of the myosin heavy chain (MHC). The subcellular localization of myosin in GbpC-null cells will be examined, and localization of GbpC itself will be determined. Changes in MHC threonine phosphorylation and the regulation of MHC kinases and phosphatase during chemotaxis in wild-type and gbpC cells will be examined. These studies will lead to basic insight into how myosin dynamics in the cell are controlled during chemotaxis, and into GbpC's regulatory role during this process. The juxtaposition of cGMP, RasGEF, Ras and protein kinase domains on the GbpC protein suggests that GbpC may be activated by a novel intramolecular signaling mechanism. This model will be tested using in vivo complementation assays and in vitro protein kinase and GTPase assays. Since GbpC's protein kinase domain is in the MEKK family, it may play a role in the activation of MAP kinase pathways during chemotaxis, and activation of these pathways by GbpC will be tested using a gbpC- strain. These studies will reveal upstream signaling pathways for myosin II and ERK regulation, and will elucidate the role of the key regulatory protein GbpC in the complex network of signaling needed to achieve directional movement toward a chemoattractant.
