Glycogen Synthase Kinase (GSK) 3β phosphorylates and protects nuclear myosin 1c from proteasome-mediated degradation to activate rDNA transcription in early G1 cells

Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3β phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3β selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3β-/- mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3β directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3β-mediated phosphorylation of NM1 is required for pol I transcription activation

Two step activation of FOXO3 by AMPK generates a coherent feed-forward loop determining excitotoxic cell fate

Cerebral ischemia and excitotoxic injury induce transient or permanent bioenergetic failure, and may result in neuronal apoptosis or necrosis. We have previously shown that ATP depletion and activation of AMP-activated protein kinase (AMPK) during excitotoxic injury induces neuronal apoptosis by transcription of the proapoptotic BH3 only protein, Bim. AMPK, however, also exerts pro-survival functions in neurons. The molecular switches that determine these differential outcomes are not well understood. Using an approach combining biochemistry, single cell imaging and computational modeling, we here demonstrate that excitotoxic injury activated the bim promoter in a FOXO3-dependent manner. The activation of AMPK reduced AKT activation, and led to dephosphorylation and nuclear translocation of FOXO3. Subsequent mutation studies indicated that bim gene activation during excitotoxic injury required direct FOXO3 phosphorylation by AMPK in the nucleus as a second activation step. Inhibition of this phosphorylation prevented Bim expression and protected neurons against excitotoxic and oxygen/glucose deprivation-induced injury. Systems analysis and computational modeling revealed that these two activation steps defined a coherent feedforward loop; a network motif capable of filtering any effects of short-term AMPK activation on bim gene induction. This may prevent unwanted AMPK-mediated Bim expression and apoptosis during transient or physiological bioenergetic stress

Phosphatase and tensin homologue: a therapeutic target for SMA

Spinal muscular atrophy (SMA) is one of the most common juvenile neurodegenerative diseases, which can be associated with child mortality. SMA is caused by a mutation of ubiquitously expressed gene, Survival Motor Neuron1 (SMN1), leading to reduced SMN protein and the motor neuron death. The disease is incurable and the only therapeutic strategy to follow is to improve the expression of SMN protein levels in motor neurons. Significant numbers of motor neurons in SMA mice and SMA cultures are caspase positive with condensed nuclei, suggesting that these cells are prone to a process of cell death called apoptosis. Searching for other potential molecules or signaling pathways that are neuroprotective for central nervous system (CNS) insults is essential for widening the scope of developmental medicine. PTEN, a Phosphatase and Tensin homologue, is a tumor suppressor, which is widely expressed in CNS. PTEN depletion activates anti-apoptotic factors and it is evident that the pathway plays an important protective role in many neurodegenerative disorders. It functions as a negative regulator of PIP3/AKT pathway and thereby modulates its downstream cellular functions through lipid phosphatase activity. Moreover, previous reports from our group demonstrated that, PTEN depletion using viral vector delivery system in SMN delta7 mice reduces disease pathology, with significant rescue on survival rate and the body weight of the SMA mice. Thus knockdown/depletion/mutation of PTEN and manipulation of PTEN medicated Akt/PKB signaling pathway may represent an important therapeutic strategy to promote motor neuron survival in SMA

The ErbB signalling pathway: protein expression and prognostic value in epithelial ovarian cancer

Ovarian cancer is the most frequent cause of death from gynaecological cancer in the Western world. Current prognostic factors do not allow reliable prediction of response to chemotherapy and survival for individual ovarian cancer patients. Epidermal growth factor receptor (EGFR) and HER-2/neu are frequently expressed in ovarian cancer but their prognostic value remains unclear. In this study, we investigated the expression and prognostic value of EGFR, EGFR variant III (EGFRvIII), HER-2/neu and important downstream signalling components in a large series of epithelial ovarian cancer patients. Immunohistochemical staining of EGFR, pEGFR, EGFRvIII, Her-2/neu, PTEN (phosphatase and tensin homologue deleted on chromosome 10), total and phosphorylated AKT (pAKT) and phosphorylated ERK (pERK) was performed in 232 primary tumours using the tissue microarray platform and related to clinicopathological characteristics and survival. In addition, EGFRvIII expression was determined in 45 tumours by RT–PCR. Our results show that negative PTEN immunostaining was associated with stage I/II disease (P=0.006), non-serous tumour type (P=0.042) and in multivariate analysis with a longer progression-free survival (P=0.015). Negative PTEN staining also predicted improved progression-free survival in patients with grade III or undifferentiated serous carcinomas (P=0.011). Positive pAKT staining was associated with advanced-stage disease (P=0.006). Other proteins were expressed only at low levels, and were not associated with any clinicopathological parameter or survival. None of the tumours were positive for EGFRvIII. In conclusion, our results indicate that tumours showing negative PTEN staining could represent a subgroup of ovarian carcinomas with a relatively favourable prognosis

Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3β (GSK3β). KLC2 phosphorylation by GSK3β induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3β on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-β (TGFβ) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFβ-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling

Lithium chloride therapy fails to improve motor function in a transgenic mouse model of Machado-Joseph disease

The accumulation of misfolded proteins in neurons, leading to the formation of cytoplasmic and nuclear aggregates, is a common theme in age-related neurodegenerative diseases, possibly due to disturbances of the proteostasis and insufficient activity of cellular protein clearance pathways. Lithium is a well-known autophagy inducer that exerts neuroprotective effects in different conditions and has been proposed as a promising therapeutic agent for several neurodegenerative diseases. We tested the efficacy of chronic lithium 10.4 mg/kg) treatment in a transgenic mouse model of Machado-Joseph disease, an inherited neurodegenerative disease, caused by an expansion of a polyglutamine tract within the protein ataxin-3. A battery of behavioral tests was used to assess disease progression. In spite of activating autophagy, as suggested by the increased levels of Beclin-1, Atg7, and LC3II, and a reduction in the p62 protein levels, lithium administration showed no overall beneficial effects in this model concerning motor performance, showing a positive impact only in the reduction of tremors at 24 weeks of age. Our results do not support lithiumchronic treatment as a promising strategy for the treatment of Machado-Joseph disease (MJD).FCT -Fundação para a Ciência e a Tecnologia(SFRH/BD/51059/2010

Mechanisms of T cell organotropism

F.M.M.-B. is supported by the British Heart Foundation, the Medical Research Council of the UK and the Gates Foundation

Syk: a new player in the field of breast cancer

Breast tumor development and progression are thought to occur through a complex, multistep process, including oncogene activation (eg HER2/neu) and mutation or loss of tumor suppressor genes (eg p53). Determining the function of genetic alterations in breast carcinoma tumorigenesis and metastasis has been the focus of intensive research efforts for several decades. One group of proteins that play a critical role in breast cancer cell signaling pathways are tyrosine kinases. Overexpression of the tyrosine kinase HER2/neu is observed in many human breast cancers and is positively correlated with enhanced tumorigenesis [1]. Recently, another tyrosine kinase, Syk, has been implicated as an important inhibitor of breast cancer cell growth and metastasis [2]. This recent finding was unexpected, since Syk function has been predominantly linked to hematopoietic cell signaling, and is discussed further in this commentary

PTEN status in advanced colorectal cancer treated with cetuximab

BACKGROUND: Loss of phosphatase and tensin homologue deleted in chromosome 10 (PTEN) function in advanced colorectal cancer (CRC) may represent one of the resistance mechanisms to cetuximab by interfering with the epidermal growth factor receptor signal transduction pathway. METHODS: PTEN expression tested by indirect immunofluorescence was evaluated both on primary (n¼43) and on metastatic (n¼24) sites in CRC patients treated with cetuximab. RESULTS: The loss of PTEN expression tested on metastatic sites was negatively associated with response (100% progressive disease (PD) in PTEN-negative cases vs 30% PD in PTEN-positive cases; Po0.05), PFS (0.8 vs 8.2 months; Po0.001) and OS (2.9 vs 14.2 months; Po0.001). CONCLUSION: A potential role of PTEN in the anti-tumour activity of cetuximab could be hypothesised

Tumor Spectrum, Tumor Latency and Tumor Incidence of the Pten-Deficient Mice

BACKGROUND: Pten functionally acts as a tumor suppressor gene. Lately, tissue-specific ablation of Pten gene in mice has elucidated the role of Pten in different tumor progression models. However, a temporally controlled Pten loss in all adult tissues to examine susceptibility of various tissues to Pten-deficient tumorigenesis has not been addressed yet. Our goal was to explore the genesis of Pten-deficient malignancies in multiple tissue lineages of the adult mouse. METHODS AND FINDINGS: We utilized an inducible Cre/loxP system to delete Pten exon 5 in the systemic organs of ROSA26 (R26)-CreER(T);Pten(fx/fx) mice. On reaching 45 weeks 4OHT-induced Pten loss, we found that the R26-CreER(T);Pten(fx/fx) mice developed a variety of malignancies. Overall tumor mean latency was 17 weeks in the Pten-deficient mice. Interestingly, mutant females developed malignancies more quickly at 10 approximately 11 weeks compared with a tumor latency of 21 weeks for mutant males. Lymphoma incidence (76.9% in females; 40.0% in males) was higher than the other malignancies found in the mutant mice. Mutant males developed prostate (20.0%), intestinal cancer (35.0%) and squamous cell carcinoma (10.0%), whereas the mutant females developed squamous cell carcinoma (15.4%) and endometrial cancer (46.1%) in addition to lymphomas. Furthermore, we tested the pharmacological inhibition of the PTEN downstream effectors using LY294002 on Pten-deficient prostate hyperplasia. Our data revealed that, indeed, the prostate hyperplasia resulting from the induced Pten loss was significantly suppressed by LY294002 (p = 0.007). CONCLUSIONS: Through monitoring a variety of Pten-deficient tumor formation, our results revealed that the lymphoid lineages and the epithelium of the prostate, endometrium, intestine and epidermis are highly susceptible to tumorigenesis after the Pten gene is excised. Therefore, this R26-CreER(T); Pten(fx/fx) mouse model may provide an entry point for understanding the role of Pten in the tumorigenesis of different organs and extend the search for potential therapeutic approaches to prevent Pten-deficient malignancies