|Ph.D Student||Volinsky Natalia|
|Subject||The Role of P21-Activated Kinase in SDF-1Alpha-induced T|
|Department||Department of Medicine||Supervisor||Dr. Deborah Yablonski|
|Full Thesis text|
Cell migration within a multicellular organism is indispensable for a variety of physiologic processes. In addition, during several pathologic conditions, such as invasion of tumor cells, enhanced cellular motility is observed. To investigate the signaling processes associated with migration, we employed as a model the Jurkat leukemic T cell line using a chemokine, SDF-1α, as a trigger. We made an attempt to discriminate signaling and cytoskeletal events specifically required for passing through restrictive gaps from those needed for migration per se, focusing on p21-activated kinase. We designed and characterized a potent and selective inhibitor of p21-activated kinase (Pak), named Paki. Paki consists of residues 83-265 of Pak1 and inhibits its catalytic activity by two mechanisms; the kinase inhibitory fragment enters the catalytic domain of endogenous Pak whereas the PIX-binding domain binds PIX proteins and prevents their interaction with Pak. A Pak-PIX signaling branch was found to be specifically required for passing through pores much smaller than the diameter of resting Jurkat cells. This feature distinguishes Pak and PIX from other molecules, such as Gi protein, Cdc42, Rac and PI3 Kinase, which contribute to cell migration regardless of the pore size. Interestingly, optimal activation of Pak and chemokine-induced cell migration depend on the Pak-interacting SH3 and Leucine-zipper domains of PIX, but not its small G protein exchange activity. The effects of Paki appear to be exerted in the cytoplasm; therefore we examined the role of two types of cytoskeletal structures, microtubules and vimentin, in mediating migration through small pores. Complete disruption of Vimentin didn't affect cell migration. Microtubule disruption abolished the ability of cells to distinguish different pore sizes, whereas the microtubule-stabilizing drug, Paclitaxel inhibited migration through small pores. Simultaneous usage of Paki and Paclitaxel led to a synergistic inhibitory effect when cells were allowed to migrate through large pores, suggesting that Pak may be required to promote microtubule dynamics, which in turn, are required for migration through small pores. Indeed, intact microtubules are absolutely required for Paki to exert its inhibitory effect on migration. Remarkably, Paki, actually helped migrating cells to overcome a lack of functional microtubules. Our results suggest that Pak may regulate microtubule dynamics and that this, previously unknown function of Pak is required for cell flexibility, allowing amoeboid migration through small pores. Determination of signaling pathways specifically required for this type of migration might further promote the development of potent drugs for cancer and autoimmune diseases therapy.