In cerebellar granule neurons, a BH3-only Bcl-2 family member, death protein 5/harakiri, is up-regulated in a JNK-dependent manner during apoptosis induced by potassium deprivation. However, it is not clear whether c-Jun is directly involved in the induction of dp5. Here, we showed that the up-regulation of dp5, but not fas ligand and bim, after potassium deprivation was suppressed by the expression of a dominant negative form of c-Jun. Deletion analysis of the 5-flanking sequence of the dp5 gene revealed that a major responsive element responsible for the induction by potassium deprivation is an ATF binding site located at ؊116 to ؊109 relative to the transcriptional start site. Mutation of this site completely abolished promoter activation. Furthermore, a gel shift assay showed that a specific complex containing c-Jun and ATF2 recognized this site and increased in potassium-deprived cerebellar granule neurons. Chromatin immunoprecipitation demonstrated that c-Jun was able to bind to this site in vivo. Finally, we demonstrated that knockdown of Dp5 by small interfering RNA rescued neurons from potassium deprivation-induced apoptosis. Taken together, these results suggest that dp5 is a target gene of c-Jun and plays a critical role in potassium deprivation-induced apoptosis in cerebellar granule neurons.The Bcl-2 family proteins can be divided into three major subgroups (1). Antiapoptotic proteins, such as Bcl-2, Bcl-X L , and Mcl-1, typically share four conserved motifs termed Bcl-2 homology (BH) 3 domains and inhibit mitochondrial cytochrome c release and apoptosis. Multidomain proapoptotic proteins, the second subgroup, such as Bax, Bak, and Bok, typically have three BH domains but promote cytochrome c release and apoptosis. The third, and the most structurally diverse subgroup, is the BH3-only proteins, including Dp5/HRK (death protein 5/harakiri), Bim (Bcl2-interacting mediator of cell death), Bid, Bad, Puma, and Noxa, which share the BH3 domain. The BH3-only proteins are critical initiators of apoptosis. Upon challenge, BH3-only proteins translocate to mitochondria and promote the chromec release by neutralizing the antiapoptotic action of Bcl-2 family members. BH3-only proteins are stringently regulated at the transcriptional and post-translational levels during apoptosis, such as Dp5, Bim, and Puma, depending on the cell type and apoptotic stimulus (2-6). Among the BH3-only proteins, Dp5 is of particular interest to studies of apoptosis in the nervous system. In rodents, the expression of Dp5 is largely restricted to and is developmentally regulated in the nervous system (2, 7). Dp5 is the first found BH3-only protein to be induced by NGF deprivation in sympathetic neurons (2). dp5 is highly homologous to the human gene harakiri (HRK) cloned by a two-hybrid screen with Bcl-2 and Bcl-X L (3). As well as being induced in NGF-deprived sympathetic neurons, the induction of dp5 is also observed in cerebellar granule neurons (CGNs) deprived of potassium, cortical neurons exposed to toxic concentrations of amyl...
Glycogen synthase kinase-3 (GSK-3) plays a critical role in neuronal apoptosis. The two mammalian isoforms of the kinase, GSK-3␣ and GSK-3, are inhibited by phosphorylation at Ser-21 and Ser-9, respectively. Depolarization, which is vital for neuronal survival, causes both an increase in Ser-21/9 phosphorylation and an inhibition of GSK-3␣/. However, the role of GSK-3 phosphorylation in depolarization-dependent neuron survival and the signaling pathway contributing to GSK-3 phosphorylation during depolarization remain largely unknown. Using several approaches, we showed that both isoforms of GSK-3 are important for mediating neuronal apoptosis. Nonphosphorylatable GSK-3␣/ mutants (S21A/S9A) promoted apoptosis, whereas a peptide encompassing Ser-9 of GSK-3 protected neurons in a phosphorylation-dependent manner; these results indicate a critical role for Ser-21/9 phosphorylation on depolarization-dependent neuron survival. We found that Ser-21/9 phosphorylation of GSK-3 was mediated by Ca 2؉ / calmodulin-dependent protein kinase II (CaMKII) but not by Akt/PKB, PKA, or p90 RSK . CaMKII associated with and phosphorylated GSK-3␣/. Furthermore, the pro-survival effect of CaMKII was mediated by GSK-3 phosphorylation and inactivation. These findings identify a novel Ca 2؉ /calmodulin/CaMKII/ GSK-3 pathway that couples depolarization to neuronal survival.The survival or death of neurons is critical for the establishment of appropriate neural circuitry during brain development (1, 2). Considerable evidence supports that electrical activity plays a crucial role in neuronal survival (3, 4). For example, pharmacological blockade of electrical activity in rat brain induces extensive apoptotic neurodegeneration (5, 6). Deafferentiation of the cerebellar granule layer in adult rats resulted in massive and typical apoptosis of cerebellar granule neurons (CGNs), 3 suggesting the importance of afferent input-related factors for survival of CGNs in vivo (7). In culture, survival of rat CGNs can be maintained by electrical activity, which is effected by depolarizing concentrations of extracellular potassium [KCl] o ϭ 25 mM KCl ((25 K) or potassium depolarization) (8, 9). Lowering [KCl] o to 5 mM KCl ((5 K) or potassium deprivation) triggers typical apoptosis (10). Presumably, this recapitulates the naturally occurring neuronal death that takes place in the newborn rat cerebellum (11). These characteristics, along with an abundant neuronal population and up to 98% homogeneity, make cultured CGNs an excellent and extensively studied model for deciphering the signaling mechanisms that underlie depolarization-dependent neuron survival (4).It has been well documented that depolarizing conditions (such as elevated [KCl] o ) sustain neuronal survival by causing the influx of Ca 2ϩ through L-type Ca 2ϩ channels (8, 12, 13), implicating Ca 2ϩ as a necessary second messenger for survival signaling. When activated by elevated Ca 2ϩ , Ca 2ϩ /calmodulindependent protein kinase II (CaMKII) has been reported to mediate the depolarization-...
Background Triple negative breast cancer (TNBC) remains the most challenging breast cancer subtype so far. Specific therapeutic approaches have rarely achieved clinical improvements in treatment of TNBC patients and effective molecular biomarkers are largely unknown. Methods We used paired TNBC samples and high throughput RNA sequencing to identify differentially expressed circRNAs. Sucrose gradient polysome fractionation assay, antibody and Mass spectra were used to validate active circRNA translation. The novel protein function was validated in vitro and in vivo by gain or loss of function assays. Mechanistic results were concluded by immunoprecipitation analyses and kinase activity assay. Results Circular HER2 RNA (circ-HER2) encoded a novel protein, HER2–103. Unexpectedly, while HER2 mRNA and protein were barely detected, circ-HER2/HER2–103 was expressed in ~ 30% TNBC clinical samples. Circ-HER2/HER2–103 positive TNBC patients harbored worse overall prognosis than circ-HER2/HER2–103 negative patients. Knockdown circ-HER2 inhibited TNBC cells proliferation, invasion and tumorigenesis in vitro and in vivo, suggesting the critical role of circ-HER2/HER2–103 in TNBC tumorigenicity. Mechanistically, HER2–103 promoted homo/hetero dimerization of epidermal growth factor receptor (EGFR)/HER3, sustained AKT phosphorylation and downstream malignant phenotypes. Furthermore, HER2–103 shared most of the same amino acid sequences as HER2 CR1 domain which could be antagonized by Pertuzumab, a clinical used HER2 antibody. Pertuzumab markedly attenuated in vivo tumorigenicity of circ-HER2/HER2–103 expressing TNBC cells but showed no effects in circ-HER2/HER2–103 negative TNBC cells. Conclusion Our results not only demonstrated that certain TNBCs were not truly ‘HER2 negative’ but also highlighted the clinical implications of Pertuzumab in circ-HER2/HER2–103 expressing TNBC patients.
Background: GSK-3 is a key pro-apoptotic kinase, and its activity is strictly regulated. Results: GSK-3 is cleaved at both N and C termini by calpain. Conclusion: N-or C-terminal truncation activates GSK-3, and Ser-9/Ser-389 phosphorylation protects GSK-3 from calpain cleavage. Significance: The GSK-3 C terminus functions as an autoinhibitory domain, and Ser-9/Ser-389 phosphorylation and calpainmediated cleavage operate together in regulating GSK-3 activity.
Midkine (MDK) expression is associated with the proliferation of many cancers, including glioma. SP1 directly up-regulates the expression of MDK, and the SP1-MDK axis cooperates in glioma tumorigenesis.
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