Interplay of PTEN subcellular localization and catalytic activities in vivo

Abstract

This thesis describes the use of mammalian cells, S. cerevisiae and D. rerio to unravel the complex interplay of PTEN subcellular localization and catalytic activities. In Chapter 1 we provide a general introduction to the PI3K/Akt(PKB)/PTEN axis, PTEN phosphatase-dependent and –independent functions and regulation of those functions through changes in protein conformation and subcellular localization. Last, we give a short résumé on the role of PTEN in human health and disease. In Chapter 2, we give an overview of developmental and pathological processes that have been successfully studied in zebrafish pten models. Further, we introduce techniques that we and others have developed for modulation of Pten expression in vivo and for the establishment of zebrafish cell lines from tumors. In Chapter 3 we performed a comprehensive mutational and functional analysis of the PTEN N-terminus. To analyze the contribution of this region to PTEN tumor suppressor function in vivo, we studied a panel of tumor-related mutations, employing S.cerevisiae and mammalian cells. We found that most tumor-related N-terminal PTEN mutations that lead to a loss of PIP3-phosphatase activity also showed impaired nuclear localization. This suggests that PIP3 catalytic activity and nuclear localization of PTEN are coordinated by the PTEN N-terminus in an overlapping manner. Our results from soft agar colony formation assays further indicated that both the PTEN PIP3 phosphatase activity and the PTEN capacity to accumulate in the nucleus were important for the full tumor suppression capacity of PTEN. In Chapter 4 we focused on the classical protein import pathway as a possible major nuclear transport mechanism for PTEN and, by immunofluorescence and confocal microscopy performed in mammalian cells, we identified importin alpha3 as a factor involved in PTEN nuclear translocation. Further, we found that the PTEN N-terminal region (residues 1-32) could mediate nuclear accumulation of PTEN through interactions with nuclear transporters. Systematic introduction of point mutations in the importin alpha 3 substrate binding pockets allowed us to further narrow down the region of importin alpha 3 that is important for nuclear accumulation of PTEN to the minor binding pocket. Using the zebrafish as a model organism, in Chapter 5 we unveil a differential requirement of Pten lipid and protein phosphatase activity during embryonic development. To this end, we performed rescue assays. We propose that the role of Pten during angiogenesis mainly consists of suppressing PI3K signaling via its lipid phosphatase activity, whereas the complex process of embryonic development requires lipid and protein phosphatase activity of Pten. In Chapter 6 we characterize the subcellular localization and the functional consquences of the expression of open conformation PTEN. The functional hyperactivity of open conformation PTEN in comparison to PTEN wild type in our model seemed to result predominantly from its enhanced recruitment to the cytoplasmic membrane. In conclusion, we show that enhanced membrane localization of phosphatase active PTEN dramatically increased its biological function in suppression of angiogenic sprouting. The findings of this chapter demonstrate the requirement of tightly regulated and equilibrated Pten activity during zebrafish development and angiogenesis. Finally, Chapter 7 provides a summarizing discussion of the work presented in each previous chapter.