Editors' ChoiceCell Biology

Discrete PTEN signaling

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Science Signaling  12 May 2015:
Vol. 8, Issue 376, pp. ec123
DOI: 10.1126/scisignal.aac4803

Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is a tumor suppressor protein. PTEN dephosphorylates the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3, also known as PIP3], which is the product of the phosphorylation of PI(4,5)P2 by phosphatidylinositol 3-kinase (PI3K). PTEN is thought (but rarely shown) to be localized and active at the plasma membrane together with the receptor tyrosine kinases (RTKs) and G protein–coupled receptors (GPCRs) that stimulate PI3K activity. Naguib et al. investigated the localization and function of PTEN in mouse embryonic fibroblasts (MEFs). Confocal immunofluorescence microscopy revealed that cytoplasmic PTEN was organized in discreet puncta, which were indistinguishable from those containing labeled transferrin, a marker for early endosomes. Super-resolution microscopy confirmed the colocalization of cytoplasmic PTEN and microtubules, and showed that PTEN was arranged along the microtubule network. The sizes of the PTEN puncta were unaffected by nocodazole, a drug that blocks microtubule polymerization, suggesting that this organization of PTEN was independent of microtubules. To investigate whether the PTEN puncta were the result of PTEN recruitment to vesicles, the authors performed experiments in which protein-lipid overlay was coupled with antibody detection. PTEN bound directly to phosphatidylinositol monophosphates (PIPs), and its highest affinity was for PI(3)P. Analysis of PTEN mutants demonstrated that the C2 domain of PTEN (C2PTEN) mediated its binding to PI(3)P. This association was confirmed by colocalization of a fluorescently tagged C2PTEN protein that was fused to the FYVE [PI(3)P-binding] domain of HRS (a protein that localizes to endosomes). Pharmacological inhibition or siRNA-mediated knockdown of Vps34, the kinase that produces PI(3)P, in MEFs suppressed the association of C2PTEN with vesicles. Further analysis of C2PTEN with synthetic liposomes that contained PIP3 alone or in combination with PI(3)P showed that there was increased dephosphorylation of PIP3 in the liposomes that contained PI(3)P, suggesting that the interaction between PTEN and PI(3)P was necessary for PTEN function. Mutational analysis of C2PTEN revealed that it bound to PI(3)P in vesicles through a membrane-interacting loop (mCBR3), and mutation of this region abolished its ability to bind to PI(3)P. When expressed in PTEN-deficient MEFs, the mCBR3 mutant PTEN fused to the FYVE-HRS domain restored PTEN function similarly to that in wild-type MEFs, even when it was low in abundance. In addition, pulse-chase experiments performed within minutes of the stimulation of the epidermal growth factor (EGF) receptor showed the colocalization of C2PTEN, labeled EGF, and FYVE-HRS in endosomes. Together, these data suggest the endosomal localization of PTEN is critical to its ability to inhibit PI3K-dependent signals.

A. Naguib, G. Bencze, H. Cho, W. Zheng, A. Tocilj, E. Elkayam, C. R. Faehnle, N. Jaber, C. P. Pratt, M. Chen, W. -X. Zong, M. S. Marks, L. Joshua-Tor, D. J. Pappin, L. C. Trottman, PTEN functions by recruitment to cytoplasmic vesicles Mol. Cell 58, 255–268 (2015). [PubMed]

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