Analysis of signalling pathways regulated by popx2 phosphatase.

Rho GTPases act as molecular switches to control a wide range of signal transduction pathways, which include actin cytoskeleton regulation, transcription regulation, cell motility, cell polarity and cell division in eukaryotic cells. There are more than twenty members in the Rho family of proteins, RhoA, CDC42 and RAC1 remain the most well studied. Many of the Rho effector proteins are kinases. The kinases in turn elicit their functions by phosphorylating other downstream proteins. One of the major effector proteins downstream of these Rho GTPases is the PAK (p21 (CDC42/RAC)-activated kinase) family of serine/threonine kinases. PAK is potently activated by auto-phosphorylation at multiple sites upon binding to active GTPases. Mammalian PAK phosphorylation state is highly regulated. Two PP2C-like serine/threonine phosphatases (POPX1 and POPX2) that inactivate PAK have been isolated. Phosphatases act in an antagonistic fashion to kinases by de-phosphorylating proteins. POPX (Partner of PIX) is a binding partner for the PAK interacting guanine nucleotide exchange factor βPIX. Both POPX and PAK are found in the same protein complex via their interaction with βPIX. This could facilitate the regulation of PAK activity in cells. Although PAK is identified as the major substrate of POPX2, many cellular functions and activities induced by POPX2 over-expression cannot be explained by the inhibition of PAK. Hence, it is highly possible that there are other POPX2 substrates, which might be regulated by the phosphatase leading to the observed phenotypes and functions of POPX2 expressing cells. The aim of this project is to elucidate the roles played by POPX phosphatases in cell signalling. So far, POPX2 has been implicated in the regulation of cell motility and maintenance of actin stress fibres. We also observed the exhibition or more dorsal ruffles. Dorsal ruffling, in turn, has been implicated in receptor internalization, cell motility and macropinocytosis. We have established a POPX2 stable expression cell line and have used this cell line to probe the functions of POPX2 through proteomics studies. Since our data suggest that POPX2 is involved in inducing dorsal ruffling of the cell membrane, we also studied if macropinocytosis is modulated accordingly. We showed that POPX2 phosphatase activity is necessary for an increase in macropinocytosis . We also showed that POPX2 levels are positively correlated to cell motility.Doctor of Philosophy (SBS