Fine-Tuning of Pten Localization and Phosphatase Activity Is Essential for Zebrafish Angiogenesis

Abstract

The lipid- and protein phosphatase PTEN is an essential tumor suppressor that is highly conserved among all higher eukaryotes. As an antagonist of the PI3K/Akt cell survival and proliferation pathway, it exerts its most prominent function at the cell membrane, but (PIP3-independent) functions of nuclear PTEN have been discovered as well. PTEN subcellular localization is tightly controlled by its protein conformation. In the closed conformation, PTEN localizes predominantly to the cytoplasm. Opening up of the conformation of PTEN exposes N-terminal and C-terminal regions of the protein that are required for both interaction with the cell membrane and translocation to the nucleus. Lack of Pten leads to hyperbranching of the intersegmental vessels during zebrafish embryogenesis, which is rescued by expression of exogenous Pten. Here, we observed that expression of mutant PTEN with an open conformation rescued the hyperbranching phenotype in pten double homozygous embryos and suppressed the increased p-Akt levels that are characteristic for embryos lacking Pten. In addition, in pten mutant and wild type embryos alike, open conformation PTEN induced stalled intersegmental vessels, which fail to connect with the dorsal longitudinal anastomotic vessel. Functional hyperactivity of open conformation PTEN in comparison to wild type PTEN seems to result predominantly from its enhanced recruitment to the cell membrane. Enhanced recruitment of phosphatase inactive mutants to the membrane did not induce the stalled vessel phenotype nor did it rescue the hyperbranching phenotype in pten double homozygous embryos, indicating that PTEN phosphatase activity is indispensable for its regulatory function during angiogenesis. Taken together, our data suggest that PTEN phosphatase activity needs to be carefully fine-tuned for normal embryogenesis and that the control of its subcellular localization is a key mechanism in this process.

PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer

“…Therefore, we isolated nuclear protein from the PTEN-WT and PTEN-4A expressing stable cell lines and performed immunofluorescence experiments in 293T cells. We found that PTEN-4A preferentially localized to the nucleus (Figure 2A-C), consistent with recent findings in various cell types [18,[44][45][46][47].…”

“…Further, several non-genomic mechanisms contribute to PTEN inactivation resulting in oncogenic transformation and progression. PTEN function and subcellular localization is frequently modulated by post-translational modifications, particularly phosphorylation and ubiquitination [16,18,47,61,62]. Indeed, PTEN phosphorylation has increasingly been observed to compromise PTEN function in several cancers [63][64][65][66] and other non-malignant diseases [67][68][69].…”

A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development

“…To confirm that Akt activation plays a role in zebrafish neural crest development, we expressed constitutively active human PTEN-mCherry fusion (PTEN S370A, S380A, T382A, T383A, S385A or PTEN QMA) in crestin:EGFP transgenic zebrafish embryos ( Gil et al, 2006 ; Stumpf et al, 2016 ). After sorting embryos based on mCherry fluorescence at 24 hpf, we found that PTEN QMA-mCherry decreases both phospho-Akt and crestin:EGFP expression ( Figure 7A–C ).…”

“…pCSDest was a gift from Nathan Lawson (Addgene plasmid # 22423). PTEN QMA-mCherry was from Stumpf et al, 2016 .…”

Establishment of pten knockout medaka with transcription activator–like effector nucleases (TALENs) as a model of PTEN deficiency disease

“…Furthermore, angiogenesis and expression of the vascular endothelial growth factor (VEGF) gene were also enhanced in ptena −/− ptenb −/− mutant embryos, and this hypervascularization phenotype was ameliorated by combined treatment with the VEGF receptor inhibitor sunitinib and the PI3K inhibitor LY294002 [ 13 ]. The hypervascularization phenotype was also applied to differentiate the role of the lipid and protein phosphatase activity of PTEN during embryonic development [ 14 ]. Database sequences suggest that medaka also possesses two PTEN genes ( ptena and ptenb ), with ptena being located on chromosome 8 but the chromosomal location of ptenb being unknown.…”