In order to survive and develop normally, eukaryotic cells must be able to respond to a variety of extracellular stimuli and in some cases adapt to adverse conditions. Exposure to certain extracellular stimuli can trigger cell growth and division, while some stimuli can induce other responses such as cell differentiation, changes in cell shape, or even programmed cell death. Small GTPases of the Ras superfamily serve as molecular switches which mediate responses to many types of extracellular stimuli. Our lab is interested in understanding how small GTPases integrate signals from extracellular stimuli and in turn induce various cellular changes in mammalian cells. A major focus is the Rho family of GTPases, which are a subset of the Ras superfamily. Members of the Rho family, including Cdc42, Rac, and Rho, were first identified as proteins that have key roles in regulating the organization of the actin cytoskeleton and thereby control cell shape, motility, and adhesion. Subsequently, the Rho GTPases were found to have other functions including the regulation of cell proliferation and activation of signal transduction pathways that lead to changes in gene expression patterns. When improperly regulated, the Rho GTPases also play key roles in oncogenic transformation and tumor invasiveness. This most likely reflects the fact that changes in cell shape, adhesion, and motility, all play an important part in the oncogenic process. Furthermore, the Rho GTPases have been found to regulate important cell cycle regulatory genes which may cause cells to enter into the cell cycle and proliferate.
One area of research in our laboratory has been the identification of new molecular targets for the Rho GTPases. We have cloned genes for two new protein kinases that bind to and get activated by Cdc42. Both of these protein kinases are members of the PAK family of serine/threonine kinases. We have found that one of these kinases, PAK4, mediates cytoskeletal and cell shape changes in response to Cdc42 activation. Importantly, we have also found that a constitutively active mutant of PAK4 can induce anchorage oncogenic independent growth in fibroblasts. This may be due in part to effects this protein has on cell adhesion. This is especially intriguing because anchorage independent growth is a key step in the oncogenic process, and activated Cdc42 has been shown to have a similar effect. Furthermore, inhibition of PAK4 expression blocks oncogenic transformation by oncogenic Dbl, which is a potent oncogene and an activator of Cdc42. We predict that PAK4 is a key player in the signaling pathway by which Rho GTPases regulate oncogenic transformation. Currently we are investigating the substrates for PAK4 that may be directly responsible for its functions.
The second gene encodes another member of the PAK family, PAK5, which is also a target for Cdc42. Unlike PAK4, however, which is ubiquitously expressed, PAK5 is expressed mostly in the brain. The regulation of cytoskeletal organization and cell shape are particularly important in neuronal cells, especially during development when neurons extend axons and dendrites and connections are made with target cells. Consistent with this, we have found that activated PAK5 promotes neurite outgrowth when it is overexpressed in a neuroblastoma cell line. Experiments are currently underway in our lab to determine whether PAK5 is indeed an important player in the process of neurite outgrowth. This involves further studies in cell lines as well as a mouse knockout project in which we are generating mice which are deficient in the PAK5 gene. A similar mouse knockout project is underway for PAK4.
Finally, an important function of the Rho GTPases is the regulation of cell proliferation and cell cycle entry in both normal and transformed cells. In fact, the Rho GTPases are essential for proliferation and transformation by oncogenes such as the Ras oncogene. This may be due in part to their ability to control the expression of cell cycle regulatory proteins such as p21 and Cyclin D1. Ongoing projects in the lab involve deciphering the molecular mechanism by which Rho GTPases may regulate the expression of cell cycle regulatory proteins and thereby control cell proliferation.
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