Loss of PTEN promotes formation of signaling-capable clathrin-coated pits.
ABSTRACT: Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs) display highly heterogeneous dynamics on the plasma membrane where they can take from 20 s to over 1 min to form cytosolic coated vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], which is dephosphorylated by phosphatase and tensin homolog (PTEN), is a potent tumorigenic signaling lipid. By using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we found that EGF-bound EGFR and PTEN are enriched in a distinct subset of short-lived CCPs that correspond with clathrin-dependent EGF-induced signaling. We demonstrated that PTEN plays a role in the regulation of CCP dynamics. Furthermore, increased PI(3,4,5)P3 resulted in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Altogether, our findings provide evidence for the existence of short-lived 'signaling-capable' CCPs.
Project description:The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PI(3,4,5)P3. PI(3,4,5)P3 can be dephosphorylated by 3- or 5-phosphatases, the latter producing PI(3,4)P2. The PTEN tumor suppressor is thought to function primarily as a PI(3,4,5)P3 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PI(3,4)P2 3-phosphatase, both in vitro and in vivo. PTEN is a major PI(3,4)P2 phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PI(3,4)P2 4-phosphatase, leads to synergistic accumulation of PI(3,4)P2, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PI(3,4)P2 levels in a mouse model of prostate cancer, and it inversely correlated with PI(3,4)P2 levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PI(3,4)P2 in the phenotype caused by loss-of-function mutations or deletions in PTEN.
Project description:Phosphatidylinositides play important roles in cellular signaling and migration. Phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) is an important phosphatidylinositide because it acts as a secondary messenger to trigger cell movement and proliferation. A high level of PI(3,4,5)P3 at the plasma membrane is known to contribute to tumorigenesis. One key enzyme that regulates PI(3,4,5)P3 levels at the plasma membrane is phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which dephosphorylates PI(3,4,5)P3 through hydrolysis to form phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). It has been reported that PI(4,5)P2 is involved in positive feedback in the PI(3,4,5)P3 hydrolysis by PTEN. However, how PI(3,4,5)P3 dephosphorylation by PTEN is regulated, is still under debate. How other PI(3,4,5)P3-binding proteins affect the dephosphorylation kinetics catalyzed by PTEN also remains unclear. Here, we develop a fluorescent-protein biosensor approach to study how PI(3,4,5)P3 dephosphorylation is regulated by PTEN as well as its membrane-mediated feedback mechanisms. Our observation of sigmoidal kinetics of the PI(3,4,5)P3 hydrolysis reaction supports the notion of autocatalysis in PTEN function. We developed a kinetic model to describe the observed reaction kinetics, which allowed us to i) distinguish between membrane-recruitment and allosteric activation of PTEN by PI(4,5)P2, ii) account for the influence of the biosensor on the observed reaction kinetics, and iii) demonstrate that all of these mechanisms contribute to the kinetics of PTEN-mediated catalysis.
Project description:Phosphatase activities on PI(3,4,5)P3 and
This model describes the action of various phosphatases on
PI(3,4,5)P3 and PI(3,4)P2, in response to a stimulation by EGF. It
contains boolean switches to simulate knock-down and knock-out of
phosphatases as well as inhibition of PI3 kinase.
This model is described in the article:
PTEN Regulates PI(3,4)P2
Signaling Downstream of Class I PI3K
Mouhannad Malek, Anna Kielkowska,
Tamara Chessa, Karen E. Anderson, David Barneda, P?nar Pir,
Hiroki Nakanishi, Satoshi Eguchi, Atsushi Koizumi, Junko Sasaki,
Véronique Juvin, Vladimir Y. Kiselev, Izabella Niewczas,
Alexander Gray, Alexandre Valayer, Dominik Spensberger, Marine
Imbert, Sergio Felisbino, Tomonori Habuchi, Soren Beinke, Sabina
Cosulich, Nicolas Le Novère, Takehiko Sasaki, Jonathan
Clark, Phillip T. Hawkins and Len R. Stephens
The PI3K signaling pathway regulates cell growth and
movement and is heavily mutated in cancer. Class I PI3Ks
synthesize the lipid messenger PI(3,4,5)P3. PI(3,4,5)P3 can be
dephosphorylated by 3- or 5-phosphatases, the latter producing
PI(3,4)P2. The PTEN tumor suppressor is thought to function
primarily as a PI(3,4,5)P3 3-phosphatase, limiting activation
of this pathway. Here we show that PTEN also functions as a
PI(3,4)P2 3-phosphatase, both in vitro and in vivo. PTEN is a
major PI(3,4)P2 phosphatase in Mcf10a cytosol, and loss of PTEN
and INPP4B, a known PI(3,4)P2 4-phosphatase, leads to
synergistic accumulation of PI(3,4)P2, which correlated with
increased invadopodia in epidermal growth factor
(EGF)-stimulated cells. PTEN deletion increased PI(3,4)P2
levels in a mouse model of prostate cancer, and it inversely
correlated with PI(3,4)P2 levels across several EGF-stimulated
prostate and breast cancer lines. These results point to a role
for PI(3,4)P2 in the phenotype caused by loss-of-function
mutations or deletions in PTEN.
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To cite BioModels Database, please use:
Chelliah V et al. BioModels: ten-year
anniversary. Nucl. Acids Res. 2015, 43(Database
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Project description:The activation of phosphatidylinositol 3-kinase (PI 3-K) and subsequent production of PtdIns(3,4,5)P3 launches a signal transduction cascade that impinges on a plethora of downstream effects on cell physiology. Control of PI 3-K and PtdIns(3,4,5)P3 levels is an important therapeutic target in treatments for allergy, inflammation, cardiovascular, and malignant human diseases. We designed metabolically stabilized, that is, phosphatase resistant, analogues of PtdIns(3,4,5)P3 as probes for long-lived potential agonists or potential antagonists for cellular events mediated by PtdIns(3,4,5)P3. In particular, two types of analogues were prepared containing phosphomimetics that would be selectively resistant to the lipid 3-phosphatase PTEN. The total asymmetric synthesis of the 3-phosphorothioate-PtdIns(3,4,5)P3 and 3-methylenephosphonate-PtdIns(3,4,5)P3 analogues is described. These two analogues showed differential binding to PtdIns(3,4,5)P3 binding modules, and both were potential long-lived activators that mimicked insulin action in sodium transport in A6 cells.
Project description:In developing neurons, phosphoinositide 3-kinases (PI3Ks) control axon growth and branching by positively regulating PI3K/PI(3,4,5)P3, but how neurons are able to generate sufficient PI(3,4,5)P3 in the presence of high levels of the antagonizing phosphatase PTEN is difficult to reconcile. We find that normal axon morphogenesis involves homeostasis of elongation and branch growth controlled by accumulation of PI(3,4,5)P3 through PTEN inhibition. We identify a plasma membrane-localized protein-protein interaction of PTEN with plasticity-related gene 2 (PRG2). PRG2 stabilizes membrane PI(3,4,5)P3 by inhibiting PTEN and localizes in nanoclusters along axon membranes when neurons initiate their complex branching behavior. We demonstrate that PRG2 is both sufficient and necessary to account for the ability of neurons to generate axon filopodia and branches in dependence on PI3K/PI(3,4,5)P3 and PTEN. Our data indicate that PRG2 is part of a neuronal growth program that induces collateral branch growth in axons by conferring local inhibition of PTEN.
Project description:Clathrin-mediated endocytosis is a major regulator of cell-surface protein internalization. Clathrin and other proteins assemble into small invaginating structures at the plasma membrane termed clathrin-coated pits (CCPs) that mediate vesicle formation. In addition, epidermal growth factor receptor (EGFR) signaling is regulated by its accumulation within CCPs. Given the diversity of proteins regulated by clathrin-mediated endocytosis, how this process may distinctly regulate specific receptors is a key question. We examined the selective regulation of clathrin-dependent EGFR signaling and endocytosis. We find that perturbations of phospholipase C?1 (PLC?1), Ca2+, or protein kinase C (PKC) impair clathrin-mediated endocytosis of EGFR, the formation of CCPs harboring EGFR, and EGFR signaling. Each of these manipulations was without effect on the clathrin-mediated endocytosis of transferrin receptor (TfR). EGFR and TfR were recruited to largely distinct clathrin structures. In addition to control of initiation and assembly of CCPs, EGF stimulation also elicited a Ca2+- and PKC-dependent reduction in synaptojanin1 recruitment to clathrin structures, indicating broad control of CCP assembly by Ca2+ signals. Hence EGFR elicits PLC?1-calcium signals to facilitate formation of a subset of CCPs, thus modulating its own signaling and endocytosis. This provides evidence for the versatility of CCPs to control diverse cellular processes.
Project description:The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5]P3). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)P3 in higher eukaryotes. Indeed, PI(3,4,5)P3 has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, PI(3,4,5)P3 became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)P3 levels. By examining various mutants, we show that synthesis of PI(3,4,5)P3 in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4-phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)P3 synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases.
Project description:PTEN, a 3-phosphatase of phosphoinositide, regulates asymmetric PI(3,4,5)P3 signaling for the anterior-posterior polarization and migration of motile cells. PTEN acts through posterior localization on the plasma membrane, but the mechanism for this accumulation is poorly understood. Here we developed an in vitro single-molecule imaging assay with various lipid compositions and use it to demonstrate that the enzymatic product, PI(4,5)P2, stabilizes PTEN's membrane-binding. The dissociation kinetics and lateral mobility of PTEN depended on the PI(4,5)P2 density on artificial lipid bilayers. The basic residues of PTEN were responsible for electrostatic interactions with anionic PI(4,5)P2 and thus the PI(4,5)P2-dependent stabilization. Single-molecule imaging in living Dictyostelium cells revealed that these interactions were indispensable for the stabilization in vivo, which enabled efficient cell migration by accumulating PTEN posteriorly to restrict PI(3,4,5)P3 distribution to the anterior. These results suggest that PI(4,5)P2-mediated positive feedback and PTEN-induced PI(4,5)P2 clustering may be important for anterior-posterior polarization.
Project description:In recent years, fluorescence microscopy has enabled researchers to observe the dynamics of clathrin-coated pit (CCP) assembly in real time. The assembly dynamics of CCPs shows striking heterogeneity. Some CCPs are long-lived (productive CCPs); they bind cargo and grow in size to form clathrin-coated vesicles. In contrast, other CCPs (abortive CCPs) are relatively short-lived and disassemble well before reaching vesicle size. Within both populations there is significant variance in CCP lifetime. We propose a stochastic biophysical model that links these observations with the energetics of CCPs and kinetics of their assembly. We show that without cargo, CCP assembly faces a high energy barrier that is difficult to overcome. As a consequence, CCPs without cargo are almost always abortive. We suggest a mechanism by which cargo binding stabilizes CCPs and facilitates their growth. The lifetime distribution of abortive pits calculated from our model agrees well with published experimental data. We also estimate the lifetimes of productive CCPs and show that the stochastic nature of CCP assembly plays a crucial role in causing their observed wide distribution.
Project description:PTEN prevents tumor genesis by antagonizing the PI3 kinase/Akt pathway through D3 site phosphatase activity toward PI(3,4)P2 and PI(3,4,5)P3. The structural determinants of this important specificity remain unknown. Interestingly, PTEN shares remarkable homology to voltage-sensitive phosphatases (VSPs) that dephosphorylate D5 and D3 sites of PI(4,5)P2, PI(3,4)P2, and PI(3,4,5)P3. Since the catalytic center of PTEN and VSPs differ markedly only in TI/gating loop and active site motif, we wondered whether these differences explained the variation of their substrate specificity. Therefore, we introduced mutations into PTEN to mimic corresponding sequences of VSPs and studied phosphatase activity in living cells utilizing engineered, voltage switchable PTENCiV, a Ci-VSP/PTEN chimera that retains D3 site activity of the native enzyme. Substrate specificity of this enzyme was analyzed with whole-cell patch clamp in combination with total internal reflection fluorescence microscopy and genetically encoded phosphoinositide sensors. In PTENCiV, mutating TI167/168 in the TI loop into the corresponding ET pair of VSPs induced VSP-like D5 phosphatase activity toward PI(3,4,5)P3, but not toward PI(4,5)P2. Combining TI/ET mutations with an A126G exchange in the active site removed major sequence variations between PTEN and VSPs and resulted in D5 activity toward PI(4,5)P2 and PI(3,4,5)P3 of PTENCiV. This PTEN mutant thus fully reproduced the substrate specificity of native VSPs. Importantly, the same combination of mutations also induced D5 activity toward PI(3,4,5)P3 in native PTEN demonstrating that the same residues determine the substrate specificity of the tumor suppressor in living cells. Reciprocal mutations in VSPs did not alter their substrate specificity, but reduced phosphatase activity. In summary, A126 in the active site and TI167/168 in the TI loop are essential determinants of PTEN's substrate specificity, whereas additional features might contribute to the enzymatic activity of VSPs.