Tuberin and mTOR,a key apoptotic pathway in diabetes

Comment on: Velagapudi C, et al. J Am Soc Nephrol 2011; 22:262-73.     

Tuberous sclerosis complex gene products,Tuberin and Hamartin,control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb

BACKGROUND: Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs through the loss of heterozygosity of either TSC1 or TSC2, which encode Hamartin or Tuberin, respectively. Tuberin and Hamartin form a tumor suppressor heterodimer that inhibits the mammalian target of rapamycin (mTOR) nutrient signaling input, but how this occurs is unclear. RESULTS: We show that the small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, we show that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently. CONCLUSIONS: We show that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells.     

ErbB2/Neu-induced, cyclin D1-dependent transformation is accelerated in p27-haploinsufficient mammary epithelial cells but impaired in p27-null cells

ErbB2/Neu destabilizes the cyclin-dependent kinase (Cdk) inhibitor p27 and increases expression of cyclin D1. Therefore, we studied the roles of p27 and cyclin D1 in ErbB2-mediated mammary epithelial cell transformation. Overexpression of ErbB2 or cyclin D1 in p27(+/-) primary murine mammary epithelial cells resulted in increased proliferation, cyclin D1 nuclear localization, and colony formation in soft agar compared to those in p27(+/+) cells. In contrast, ErbB2- or cyclin D1-overexpressing p27(-/-) cells displayed reduced proliferation, anchorage-independent growth, Cdk4 activity, cyclin D1 expression, and cyclin D1 nuclear localization compared to wild-type cells. A cyclin D1 mutation in its nuclear export sequence (T286A) partially rescued nuclear localization of cyclin D1 in p27(-/-) cells but did not increase proliferation or Cdk4 kinase activity. Overexpression of E2F1, however, increased proliferation to the same degree in p27(+/+), p27(+/-), and p27(-/-) cells. Mammary glands from MMTV (mouse mammary tumor virus)-neu/p27(+/-) mice exhibited alveolar hyperplasia, enhanced proliferation, decreased apoptosis, and accelerated tumor formation compared to MMTV-neu/p27(+/+) glands. However, MMTV-neu/p27(-/-) glands showed decreased proliferation, cyclin D1 expression, and Cdk4 activity, as well as markedly prolonged tumor latency, compared to MMTV-neu/p27(+/+) glands. These results suggest that p27(+/-) mammary epithelium may be more susceptible to oncogene-induced tumorigenesis, whereas p27-null glands, due to severely impaired cyclin D1/Cdk4 function, are more resistant to transformation.     

Regulation of cell migration by mTOR is mediated through changes in p27Kip1 phosphorylation

Comment on: Moss SC, et al. J Biol Chem 2010; In press.     

PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest

Mechanisms linking mitogenic and growth inhibitory cytokine signaling and the cell cycle have not been fully elucidated in either cancer or in normal cells. Here we show that activation of protein kinase B (PKB)/Akt, contributes to resistance to antiproliferative signals and breast cancer progression in part by impairing the nuclear import and action of p27. Akt transfection caused cytoplasmic p27 accumulation and resistance to cytokine-mediated G1 arrest. The nuclear localization signal of p27 contains an Akt consensus site at threonine 157, and p27 phosphorylation by Akt impaired its nuclear import in vitro. Akt phosphorylated wild-type p27 but not p27T157A. In cells transfected with constitutively active Akt(T308DS473D)(PKB(DD)), p27WT mislocalized to the cytoplasm, but p27T157A was nuclear. In cells with activated Akt, p27WT failed to cause G1 arrest, while the antiproliferative effect of p27T157A was not impaired. Cytoplasmic p27 was seen in 41% (52 of 128) of primary human breast cancers in conjunction with Akt activation and was correlated with a poor patient prognosis. Thus, we show a novel mechanism whereby Akt impairs p27 function that is associated with an aggressive phenotype in human breast cancer.     

Cyclin D1 regulates p27 stability in B cells

p27^Kip1 is a cyclin-dependent kinase inhibitor that plays a critical role in regulating G₁/S transition, and whose activity is, in part, regulated through interactions with D-type cyclins. We have generated the BD1-9 cell line, a BaF3 pro-B cells derivative in which cyclin D1 can be induced rapidly and reversibly by ponasterone A. The induction of cyclin D1 expression leads to a targeted p27^Kip1 accumulation in both cytoplasmic and nuclear compartments. But, only the p27^Kip1 form phosphorylated on serine 10 (pSer10-p27^Kip1) accumulates in BD1-9 cells. We found that the binding of cyclin D1 and pSer10-p27^Kip1 prevents p27^Kip1 degradation by the cytoplasmic Kip1 ubiquitylation-promoting complex (KPC) proteosomic pathway. Importantly, the nuclear CDK2 activity which is crucial for G₁/S transition is not altered by p27^Kip1 increase. Using siRNA techniques, we revealed that p27^Kip1 inhibition does not affect the distribution of BD1-9 cells in the different phases of the cell cycle. Our study demonstrates that aberrant cyclin D1 expression acts as a p27^Kip1 trap in B lymphocytes but does not induce p27^Kip1 relocation from the nucleus to the cytoplasm and does not modulate the G₁/S transition. Since our cellular model mimics what observed in aggressive lymphomas, our data bring new insights into the understanding of their physiopathology.     

Skp2 inversely correlates with p27 and tuberin in transformed cells

The cyclin-dependent kinase inhibitor p27^Kip1 (p27) is a major gatekeeper of the mammalian cell cycle progression known to be regulated by both, its subcellular localization and its degradation. To allow entrance into S phase and thereby mammalian cell cycle progression p27 must be degraded by a skp2-containing E3 ubiquitin ligase whose task is to target p27 for degradation by the proteasome. The tumor suppressor gene product tuberin directly binds to p27 and protects it from degradation via skp2. Whereas, p27 and tuberin are known to be localized to both, the cytoplasm and the nucleus, the localization of skp2 remained elusive. Here we demonstrate that skp2 is a cytoplasmic and nuclear protein. In addition we found an inverse correlation of the endogenous protein levels of skp2 with p27 and tuberin in different transformed cells and under different growth conditions. These data allow new important insights into this molecular network of cell cycle control.     

Polyamine depletion arrests cell cycle and induces inhibitors p21Waf1/Cip1, p27Kip1, and p53 in IEC-6 cells

The polyamines spermidine and spermine and their precursorputrescine are intimately involved in and are required for cell growthand proliferation. This study examines the mechanism by whichpolyamines modulate cell growth, cell cycle progression, and signaltransduction cascades. IEC-6 cells were grown in the presence orabsence ofDL--difluoromethylornithine(DFMO), a specific inhibitor of ornithine decarboxylase, which is thefirst rate-limiting enzyme for polyamine synthesis. Depletion ofpolyamines inhibited growth and arrested cells in theG₁ phase of the cell cycle. Cellcycle arrest was accompanied by an increase in the level of p53 proteinand other cell cycle inhibitors, including p21^Waf1/Cip1 andp27^Kip1. Induction of cell cycleinhibitors and p53 did not induce apoptosis in IEC-6 cells, unlike manyother cell lines. Although polyamine depletion decreased the expressionof extracellular signal-regulated kinase (ERK)-2 protein, a sustainedincrease in ERK-2 isoform activity was observed. The ERK-1 proteinlevel did not change, but ERK-1 activity was increased inpolyamine-depleted cells. In addition, polyamine depletion induced thestress-activated proteinkinase/c-JunNH₂-terminal kinase (JNK) type ofmitogen-activated protein kinase (MAPK). Activation of JNK-1 was theearliest event; within 5 h after DFMO treatment, JNK activity wasincreased by 150%. The above results indicate that polyamine depletioncauses cell cycle arrest and upregulates cell cycle inhibitors andsuggest that MAPK and JNK may be involved in the regulation of theactivity of these molecules.     

p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells

The F-box protein p45SKP2 is the substrate-targeting subunit of the ubiquitin-protein ligase SCFSKP2 and is frequently overexpressed in transformed cells. Here we report that expression of p45SKP2 in untransformed fibroblasts activates DNA synthesis in cells that would otherwise growth-arrest. Expression of p45SKP2 in quiescent fibroblasts promotes p27Kip1 degradation, allows the generation of cyclin-A-dependent kinase activity and induces S phase. Coexpression of a degradation-resistant p27Kip1 mutant suppresses p45SKP2-induced cyclin-A-kinase activation and S-phase entry. We propose that p45SKP2 is important in the progression from quiescence to S phase and that the ability of p45SKP2 to promote p27Kip1 degradation is a key aspect of its S-phase-inducing function. In transformed cells, p45SKP2 may contribute to deregulated initiation of DNA replication by interfering with p27Kip1 function.     

p27Xic1, a Cdk inhibitor, promotes the determination of glial cells in Xenopus retina

p27Xic1, a member of the Cip/Kip family of Cdk inhibitors, besides its known function of inhibiting cell division, induces Müller glia from retinoblasts. This novel gliogenic function of p27Xic1 is mediated by part of the N-terminal domain near but distinct from the region that inhibits cyclin-dependent kinases. Cotransfections with dominant-negative and constitutively active Delta and Notch constructs indicate that the gliogenic effects of p27Xic1 work within the context of an active Notch pathway. The gradual increase of p27Xic1 in the developing retina thus not only limits the number of retinal cells but also increasingly favors the fate of the last cell type to be born in the retina, the Müller glia.     

p27 and salivary cancer

This study examined p27 expression in a cohort of salivary malignancies (n = 74) for a prolonged period (20 years). Reduction of p27 expression was found to be a most powerful predictor for poor survival and more so when the tumor concurrently expressed high levels of p53, TUNEL and heparanase markers, dramatically dropping the patient survival probability to 0! While no patient whose tumor-staining profile included: p27 > 50%, p53 = 0, TUNEL = 0 and heparanase = 0, died of the disease during the 20-year follow up, the median of survival of the group with p27 ≤ 50%, p53 > 0, TUNEL > 0 and heparanase > 0 was only 39 months. The survival probabilities of these two groups at 5 years were 100 and 50%, respectively, and at 20 years they were 100 and 0%, respectively (P = 0.05). Significant p27 reduction also resulted in significantly larger tumor size (T value), higher spread of neck metastasis and extra capsular spread and in more advanced disease (higher stage). Significant correlation rates were found between age and poor survival, age and reduced p27 expression, and reduced p27 expression and other general co-existing malignancies, indicating p27 reduction as part of a general phenomenon—age related mutagenesis. Significantly more extensive therapy applied to patients with salivary reduced-p27 tumors could not prevent the rise in mortality rate, questioning the justification for extensive therapy which is naturally accompanied by higher morbidity. Additional therapeutic tools for fighting salivary cancer, possibly based on the new understanding of the p27, p53, TUNEL and heparanase carcinogenic network, are necessary.     

Role of Tuberin in Neuronal Degeneration

One of the tuberous sclerosis complex (TSC) gene products, tuberin is assumed to be the functional component, being involved in a wide variety of cellular processes. Here, we report for the first time that tuberin dysfunction may represent a mechanism for neuronal damage in Alzheimer’s disease (AD), Parkinson’s disease with dementia (PD/DLB), and a mouse model of PD. Tuberin was hyperphosphorylated at Thr1462 in post-mortem frontal cortex tissue of both AD and PD/DLB patients and in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP). Both PTEN and Akt phosphoactivation corresponded to the hyperphosphorylation patterns of tuberin suggesting that the PTEN–Akt pathway might be the mechanism of tuberin phosphorylation. Our data provide new information regarding the possible role of tuberin dysfunction in major neurodegenerative disorders, such as AD and PD, whereby inhibition of tuberin function may trigger an onset of neuronal cell death.     

CDK inhibitors,p21 and p27, participate in cell cycle exit of mammalian cardiomyocytes

Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21^Cip1 and p27^Kip1 but not p57^Kip2 showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21^Cip1 and p27^Kip1 bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21^Cip1 and p27^Kip1 knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21^Cip1 and p27^Kip play important roles in the cell cycle exit of postnatal cardiomyocytes.     

Constitutive mTOR activation in TSC mutants sensitizes cells to energy starvation and genomic damage via p53

Miscoordination of growth and proliferation with the cellular stress response can lead to tumorigenesis. Mammalian target of rapamycin (mTOR), a central cell growth controller, is highly activated in some malignant neoplasms, and its clinical implications are under extensive investigation. We show that constitutive mTOR activity amplifies p53 activation, in vitro and in vivo, by stimulating p53 translation. Thus, loss of TSC1 or TSC2, the negative regulators of mTOR, results in dramatic accumulation of p53 and apoptosis in response to stress conditions. In other words, the inactivation of mTOR prevents cell death by nutrient stress and genomic damage via p53. Consistently, we also show that p53 is elevated in TSC tumors, which rarely become malignant. The coordinated relationship between mTOR and p53 during cellular stress provides a possible explanation for the benign nature of hamartoma syndromes, including TSC. Clinically, this also suggests that the efficacy of mTOR inhibitors in anti-neoplastic therapy may also depend on p53 status, and mTOR inhibitors may antagonize the effects of genotoxic chemotherapeutics.     

MAP kinase-dependent degradation of p27Kip1 by calpains in choroidal melanoma cells. Requirement of p27Kip1 nuclear export

We investigated the status and the regulation of the cyclin-dependent kinases (CDK) inhibitor p27(Kip1) in a choroidal melanoma tumor-derived cell line (OCM-1). By contrast to normal choroidal melanocytes, the expression level of p27(Kip1) was low in these cells and the mitogen-activated protein (MAP) kinase pathway was constitutively activated. Genetic or chemical inhibition of this pathway induced p27(Kip1) accumulation, whereas MAP kinase reactivation triggered a down-regulation of p27(Kip1) that could be partially reversed by calpain inhibitors. In good accordance, ectopic expression of the cellular calpain inhibitor calpastatin led to an increase of endogenous p27(Kip1) expression. In vitro, p27(Kip1) was degraded by calpains, and OCM-1 cell extracts contained a calcium-dependent p27(Kip1) degradation activity. MAP kinase inhibition partially inhibited both calpain activity and calcium-dependent p27(Kip1) degradation by cellular extracts. Immunofluorescence labeling and subcellular fractionation revealed that p27(Kip1) was in part localized in the cytoplasmic compartment of OCM-1 cells but not of melanocytes, and accumulated into the nucleus upon MAP kinase inhibition. MAP kinase activation triggered a cytoplasmic translocation of the protein, as well as a change in its phosphorylation status. This CRM-1-dependent cytoplasmic translocation was necessary for MAP kinase- and calpain-dependent degradation. Taken together, these data suggest that in tumor-derived cells, p27(Kip1) could be degraded by calpains through a MAP kinase-dependent process, and that abnormal cytoplasmic localization of the protein, probably linked to modifications of its phosphorylation state, could be involved in this alternative mechanism of degradation.     

Correction for Volume 27, p. 159

JASTS Abstract: W1-1-3. MSG-induced response and