In the nucleus, PTEN has important tumor-suppressive functions. Given the absence of a classical nuclear localization signal, the mechanism of PTEN nuclear localization has yet to be fully elucidated. Numerous molecular mechanisms have been proposed, including simple diffusion [25] and active shuttling regulated by a putative nuclear localization signal NLS that mediate PTEN interaction with the major vault protein [26]. PTEN also has a cytoplasmic localization signal CLS in the N-terminal region amino acids 19—25 required for cytoplasmic localization that could acts as a non-canonical signal for nuclear export [27].
Nevertheless, the mechanisms of PTEN nucleus-cytoplasmic shuttling appear to involve also posttranslational modifications such as monoubiquitination or sumorylation. Recently also sumoylation-mediated PTEN nuclear retention has been reported [12]. Nuclear PTEN maintains chromosomal stability by interacting with the centromere proteins centromere protein-C CENP-C [30] , and participates in DNA-damage responses by up-regulating the transcription of Rad51 that leads to double-strand break repair [30].
In addition, nuclear PTEN controls cell-cycle progression by inducing G0—G1 arrest most likely as a result of cyclin D1 downregulation [31] , and regulates cellular senescence through anaphase promoting complex APC -CDH1-mediated protein degradation [32].
Cerebral ischemia was reported as the first physiological mechanism able to dynamically alter the subcellular localization of PTEN in vivo [33]. In this study, PTEN ubiquitination and translocation from the cytoplasm to the nucleus were shown to occur after cerebral ischemia in the brain, leading unexpectedly to neuron survival.
PTEN also localizes to the nucleolus, where it regulates nucleolar homeostasis and morphology [13]. Indeed, although nuclear pools of PIP3 have been reported, they belong to distinct, partially detergent-resistant proteolipid complexes that are not dynamically regulated and are therefore not likely PTEN substrates [34] , suggesting a potential role for nuclear PTEN beyond its lipid phosphatase activity.
Recently, several lines of evidence noted the ability of PTEN to also interact with intracellular membrane-containing organelles. This unique feature of PTEN was initially revealed by fluorescence recovery after photobleaching FRAP studies showing that that nuclear PTEN diffused very rapidly and appeared not to be tethered, while cytoplasmic PTEN diffused more slowly suggesting transient interactions with immobile cytoplasmic structures [25].
Therefore, it was important to understand if this tethering of PTEN to cytoplasmic structures prevents it from acting at the plasma membrane, or if it localizes PTEN to specific subcellular domains where it acts as a tumor suppressor through other mechanisms. Liang et al. The authors not only demonstrated that this longer form of PTEN is necessary for the maintenance of mitochondrial structure and function, potentially by maintaining cytochrome c oxidase COX in a hypo-phosphorylated state, but also suggested that it collaborates with canonical PTEN in regulating mitochondrial energy metabolism.
Putz et al. Hopkins et al. This longer form of PTEN is a membrane-permeable lipid phosphatase that is secreted from cells, and is capable of entering other cells where it antagonizes the PI3K signaling pathway. Indeed, in addition to mutation or deletion of the PTEN gene, other pathological mechanisms that result in the aberrant subcellular compartmentalization of the PTEN protein are associated with cancer.
A further investigation of the mechanisms underlying PTEN subcellular localization may provide a foundation for the development of novel therapies targeting PTEN. It is generally accepted that the subcellular localization of a protein is tied to its functions, and this has been widely proven, especially in the case of PTEN.
Therefore, it is important to be able to determine where PTEN resides and how its exact function relates to a specific subcellular localization. In the following paragraphs, we will describe different methods that have been used to highlight PTEN trafficking and subcellular localization.
Back in , Leslie et al. A remarkable number of studies that followed not only validated this puzzling observation, but also showed that dynamic membrane associations could be modulated temporally or spatially to alter PTEN activity, and demonstrated the functional role of PTEN in many other cellular compartments. Nuclear and cytosolic fractions were prepared from PC12 cells and CNS stem cells as described in [48]. In a recent study, PTEN was also found to be localized to the nucleolus [13].
A fractionation protocol modified from Busch et al. PTEN accumulation in the mitochondria was originally observed in mitochondria isolated from primary rat hippocampal neurons undergoing apoptosis [14]. The biological functions of PTEN in the subcellular space had never been studied before in the heart. To definitively characterize the localization of PTEN under basal conditions and in cells undergoing apoptosis, we utilized a cell fractionation protocol that we had previously established.
This comprehensive procedure is described in detail in [51]. A summary of the reagents is provided in Table 1 and a snapshot protocol is displayed in Fig. This protocol allows the isolation of a pure mitochondria fraction, wherein the ER, the nuclear and other non-mitochondrial markers become undetectable; moreover, it permits the isolation of the ER, as well as the MAMs fraction that contains unique regions of ER membranes attached to the outer mitochondrial membrane OMM.
Schematic steps of the subcellular fractionation protocol from cells. For a detailed protocol see [51]. Setup of subcellular fractionation stock solutions and buffers. Catalog numbers of the reagents are provided at the bottom of the table. Reagents Vendor, cat. P , sodium fluoride Sigma—Aldrich, cat. S , sodium orthovanadate Sigma—Aldrich, cat. S , Sucrose Merck, cat. Bold values were used to help the reader in the quick identification of volumes and weights required for the preparation of the different buffers when performing the protocol.
Italic values were used for a quick identification of the reagents to use to adjust the pH. To further discriminate whether PTEN associates with the OMM or resides within these organelles, purified mitochondria were treated with proteinase K PK , which can only digest those proteins that are not protected by closed phospholipidic bilayers. Using the method illustrated in Fig. The authors observed that the mitochondrial localization of the longer form of PTEN was much more prominent, compared to PTEN, and subfractionation of mitochondria isolated from the mouse brain cortex confirmed that PTEN is not able to enter the organelle.
However, the longer PTEN form was preferentially associated with the mitochondrial inner membrane and is less abundant in the outer membrane, suggesting that the extended N-terminal region endows a distinct cellular localization and function.
Schematic steps of the Mitochondria isolation for PK assay protocol. Setup of stock solutions and buffers to isolate mitochondria for PK assay. P , proteinase K from Tritirachium album Sigma—Aldrich, cat. P , sucrose Merck, cat. The subcellular fractionation protocol depicted in Fig. In cells undergoing apoptosis, the isolation of the pure mitochondrial fraction may be difficult because of the ongoing rupture of the organelles [44,53].
This is in agreement with previous studies demonstrating that under apoptosis-inducing conditions a narrowing of the ER-mitochondria associations occurs, and that this is an important step in the execution of some apoptotic mechanisms [54,55].
PTEN is regulated by multiple protein—protein interactions and the search for physiologically relevant protein substrates of PTEN continues. Thus, it is important to further investigate and understand the relevance of many of these interactions. To this end, we used the ER fraction isolated as shown in Fig. Given the difference in composition between the homogenization buffers employed during subcellular fractionation Table 1 and the common Co-IP-lysis buffers, we had to adjust the salt and detergent concentrations.
Using the procedure described in Fig. Recently, we also established a protocol that allows the isolation of plasma membrane-associated membranes PAMs , which are microdomains of the plasma membrane PM interacting with the ER and mitochondria for a detailed protocol, see [56].
This procedure is another useful addition to the methods that can be employed to study the localization of PTEN, and in particular how it changes in response to different stimuli. In addition to subcellular fractionation methods, there are a variety of cellular imaging methods that can be used to determine the subcellular localization of proteins. Immunostaining can be used to compare the localization of a protein of interest against known markers.
Another widespread approach that has been used to study PTEN localization is the generation of fusion proteins tagged with GFP or its derivatives. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparisons to that of various organelles or subcellular structures targeted by fluorescent fusion proteins with different spectral properties, or by direct comparison with fluorescent dyes.
Iijima et al. Later, Vazquez et al. Several other studies demonstrated that nuclear PTEN is essential for tumor suppression. In differentiating PC12 cells, PTEN was most evident as speckles in the nucleus, with only a faint diffuse staining in the cytoplasm.
More mature PC12 cells, however, showed strong staining in both the cytoplasm and nucleus. Image analysis using a laser-scanning confocal microscope revealed an overlapping spatial relationship between the PTEN signal in green and the centromere in red as a signal of discrete yellow speckles, indicating that a significant portion of nuclear PTEN colocalizes with centromeres in Pten -WT MEFs.
In dysplastic intestinal polyps from Cowden syndrome patient colon sample slides, stained for PTEN using the monoclonal 6H2. PTEN was excluded from the nuclei of epithelial cells, but remarkably, interstitial lymphocytes which retain both WT and KE mutant proteins retained nuclear PTEN staining, serving as internal control for the staining procedure. Raw reads were de-multiplexed using a bcl2fastq conversion software v1.
Luciferase activities were normalized with the protein concentration. ChIP experiments were performed as previously described Beads were pelleted, washed, and eluted 2x by elution buffer. For mouse prostate analysis, protein extracts were prepared as previously described S1A — C. The high-grade PIN lesion displayed a highly dysplastic luminal epithelium and the presence of nuclear atypia. Kdm5b deficiency alone did not cause morphological changes to the prostates Fig.
S2A , as mice displayed the monolayer of luminal epithelial cells with normal recurrent mucosal folds projecting into the prostate gland.
These data support that Kdm5b deficiency resulted in suppression of the initiation and progression of PCa in vivo. B Quantification of AP weights from indicated genotypes of mice at 6 months of age. The averages of AP weight and numbers of mice for each cohort are indicated. S2C and D , indicating that Kdm5b ablation induces senescence. Two mouse prostate samples for each genotype. P protein acts as the catalytic subunit of the PI3K complex, while P85 is the regulatory subunit.
These findings indicated that Kdm5b plays an essential role in the regulation of PI3K levels as well as in the activation of AKT signaling in vivo. S3B , with no signal detected in nucleus and cytosol 34 — These results strongly support that Kdm5b ablation induces senescence in vitro. These findings from genetically-engineered mouse models in vivo and MEFs in vitro propelled us to investigate the impact of KDM5B loss on biological traits and molecular mechanisms in human PCa cells.
S4A — B. S5A — B , and Supplementary Fig. S6A — B. S5B and S6B. We previously demonstrated that KDM5B is noticeably increased in prostate tumors of Pten -null mice in which AKT signaling is hyper-activated due to Pten inactivation Meanwhile, a reduction of AKT signaling upon KDM5B knockdown was reported in gastric cancer cells and hepatocellular carcinoma 39 , Our data revealed that the expression level of SHIP1 protein in mouse prostates is very low as compared to that in mouse spleen Supplementary Fig.
AKT signaling pathway plays an important role on regulation of epigenetic events including posttranscriptional histone modifications 42 — S8B — E. KDM5B has mainly been studied as a transcriptional repressor. However, several studies reported that KDM5B could be a transcription activator, likely in a demethylase-independent manner.
For example, one study reported that KDM5B is a transcription activator of self-renewal-associated genes in embryonic stem cells ESCs , and KDM5B depletion decreased expression of genes associated with mitosis and nuclear metabolism However, most AKT inhibitors display a rather limited clinical activity as single agents. Our study suggests some options on the combinatorial treatments for better therapeutic perspective of PI3K or AKT inhibitors.
Importantly, the genetic evidence of this study highlights that KDM5B -deficient PCa cells decrease proliferation and are more vulnerable to another cancer inhibitor. Overall, our findings support the idea that targeting KDM5B can be a novel and effective therapeutic strategy of controlling PCa. We would like to thank Dr. Hui Yu for the assistance with bioinformatics analysis, and Dr. Simon W. Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.
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Find articles by Zhenbang Chen. In another trial, in which the researcher treated 19 AML patients with MK, only 1 response was observed, leading to early study termination The authors also showed that despite the use of MK at the maximum tolerated doses, only modest decreases in p-AKT Ser and limited inhibition of downstream targets were observed Reducing the cytotoxicity and increasing the treatment efficacy by a rational combination of different agents is a commonly used strategy for cancer therapies.
Thus, it would be interesting to investigate whether the combined administration of MK and a PDGFR inhibitor would achieve a better therapeutic effect than either agent alone in tumor treatment. Strikingly, we observed a synergistic anti-proliferative effect in vitro and in vivo after combined treatment with MK and CP As a mechanism, we found that CP enhanced MKinduced apoptosis.
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