Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of many diseases and in cancer therapy. Although
the unfolded protein response is known to alleviate ER stress by reducing ...the accumulation of misfolded proteins, the exact
survival elements and their downstream signaling pathways that directly counteract ER stress-stimulated apoptotic signaling
remain elusive. Here, we have shown that endogenous Akt and ERK are rapidly activated and act as downstream effectors of phosphatidylinositol
3-kinase in thapsigargin- or tunicamycin-induced ER stress. Introduction of either dominant-negative Akt or MEK1 or the inhibitors
LY294002 and U0126 sensitized cells to ER stress-induced cell death in different cell types. Reverse transcription-PCR analysis
of gene expression during ER stress revealed that cIAP-2 and XIAP, members of the IAP family of potent caspase suppressors,
were strongly induced. Transcription of cIAP-2 and XIAP was up-regulated by the phosphatidylinositol 3-kinase/Akt pathway
as shown by its reversal by dominant-negative Akt or LY294002. Ablation of these IAPs by RNA interference sensitized cells
to ER stress-induced death, which was reversed by the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone. The protective
role of IAPs in ER stress coincided with Smac release from mitochondria to the cytosol. Furthermore, it was shown that mTOR
was not required for Akt-mediated survival. These results represent the first demonstration that activation of endogenous
Akt/IAPs and MEK/ERK plays a critical role in controlling cell survival by resisting ER stress-induced cell death signaling.
A fundamental limitation in devising new therapeutic strategies for killing cancer cells with DNA damaging agents is the need to identify synthetic lethal interactions between tumor-specific ...mutations and components of the DNA damage response (DDR) in vivo. The stress-activated p38 mitogen-activated protein kinase (MAPK)/MAPKAP kinase-2 (MK2) pathway is a critical component of the DDR network in p53-deficient tumor cells in vitro. To explore the relevance of this pathway for cancer therapy in vivo, we developed a specific gene targeting strategy in which Cre-mediated recombination simultaneously creates isogenic MK2-proficient and MK2-deficient tumors within a single animal. This allows direct identification of MK2 synthetic lethality with mutations that promote tumor development or control response to genotoxic treatment. In an autochthonous model of non-small-cell lung cancer (NSCLC), we demonstrate that MK2 is responsible for resistance of p53-deficient tumors to cisplatin, indicating synthetic lethality between p53 and MK2 can successfully be exploited for enhanced sensitization of tumors to DNA-damaging chemotherapeutics in vivo.
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•A genetic switch maps synthetic lethality within tumors in single animals•MK2/p53 codeficiency enhances tumor growth in untreated NSCLCs•MK2/p53 codeficiency markedly sensitizes lung tumors to chemotherapy in vivo•This gene-switch approach can identify effective combination anticancer therapies
A fundamental limitation in devising new therapeutic strategies for killing cancer cells with genotoxic agents is the identification of synthetic lethal interactions between tumor-specific mutations and the DNA-damage response in vivo. In this study, Yaffe and colleagues use mouse genetics to model the therapeutic effect of MK2 inhibition in p53-deficient non-small-cell lung cancer, the leading cause of cancer-related deaths worldwide. This highly representative preclinical model validates the importance of MK2 as a critical target for synthetic lethal anticancer therapies.
Extracellular signal-regulated kinases (ERKs) play critical roles in numerous cellular processes, including proliferation and differentiation. ERK5 contains a kinase domain at the N-terminal, and the ...unique extended C-terminal includes multiple autophosphorylation sites that enhance ERK5-dependent transcription. However, the impact of phosphorylation at the various sites remain unclear. In this study, we examined the role of phosphorylation at the ERK5 C-terminal. We found that a constitutively active MEK5 mutant phosphorylated ERK5 at the TEY motif, resulting in the sequential autophosphorylation of multiple C-terminal residues, including Thr732 and Ser769/773/775. However, when ERK1/2 was selectively activated by an oncogenic RAS mutant, ERK5 phosphorylation at Thr732 was induced without affecting the phosphorylation status at TEY or Ser769/773/775. The Thr732 phosphorylation was U0126-sensitive and was observed in a kinase-dead mutant of ERK5 as well, suggesting that ERK1/2 can phosphorylate ERK5 at Thr732. This phosphorylation was also promoted by epidermal growth factor and nerve growth factor in HEK293 and PC12 cells, respectively. The ERK5-T732A mutant was localized in the cytosol under basal conditions. In contrast, ERK5 phosphorylated at Thr732 via the RAS-ERK1/2 pathway and ERK5-T732E, which mimics the phosphorylated form, were localized in both the nucleus and cytosol. Finally, ER-32A and U0126 blocked ERK5-dependent MEF2C transcriptional activity. Based on these findings, we propose a novel cross-talk mechanism in which ERK1/2, following activation by growth factor stimulation, phosphorylates ERK5 at Thr732. This phosphorylation event is responsible for ERK5 nuclear localization and ERK5-dependent transcription.
The assembly of neuronal circuits during development requires the precise navigation of axons, which is controlled by attractive and repulsive guidance cues. In the developing spinal cord, ephrinB3 ...functions as a short-range repulsive cue that prevents EphA4 receptor-expressing corticospinal tract and spinal interneuron axons from crossing the midline, ensuring proper formation of locomotor circuits. Here we report that the small GTPase RhoA, a key regulator of cytoskeletal dynamics, is also required for ephrinB3/EphA4-dependent locomotor circuit formation. Deletion of RhoA from neural progenitor cells results in mice that exhibit a rabbit-like hopping gait, which phenocopies mice lacking ephrinB3 or EphA4. Consistent with this locomotor defect, we found that corticospinal tract axons and spinal interneuron projections from RhoA-deficient mice aberrantly cross the spinal cord midline. Furthermore, we determined that loss of RhoA blocks ephrinB3-induced growth cone collapse of cortical axons and disrupts ephrinB3 expression at the spinal cord midline. Collectively, our results demonstrate that RhoA is essential for the ephrinB3/EphA4-dependent assembly of cortical and spinal motor circuits that control normal locomotor behavior.
The small GTPases RhoA and Rac1 are key cytoskeletal regulators that function in a mutually antagonistic manner to control the migration and morphogenesis of a broad range of cell types. However, ...their role in shaping the cerebellum, a unique brain structure composed of an elaborate set of folia separated by fissures of different lengths, remains largely unexplored. Here we show that dysregulation of both RhoA and Rac1 signaling results in abnormal cerebellar ontogenesis. Ablation of RhoA from neuroprogenitor cells drastically alters the timing and placement of fissure formation, the migration and positioning of granule and Purkinje cells, the alignment of Bergmann glia, and the integrity of the basement membrane, primarily in the anterior lobules. Furthermore, in the absence of RhoA, granule cell precursors located at the base of fissures fail to undergo cell shape changes required for fissure initiation. Many of these abnormalities can be recapitulated by deleting RhoA specifically from granule cell precursors but not postnatal glia, indicating that RhoA functions in granule cell precursors to control cerebellar morphogenesis. Notably, mice with elevated Rac1 activity due to loss of the Rac1 inhibitors Bcr and Abr show similar anterior cerebellar deficits, including ectopic neurons and defects in fissure formation, Bergmann glia organization and basement membrane integrity. Together, our results suggest that RhoA and Rac1 play indispensable roles in patterning cerebellar morphology.
Protein histidine phosphorylation plays a crucial role in cell signaling and central metabolism. However, its detailed functions remain elusive due to technical challenges in detecting and isolating ...proteins bearing phosphohistidine (pHis), a labile posttranslational modification (PTM). To address this issue, we previously developed the first pHis-specific antibodies using stable, synthetic triazole-based pHis analogs. A second-generation, pyrazole-based pHis analog that enabled the development of a pan-pHis antibody with much improved pHis specificity is now reported.
Protein histidine phosphorylation is increasingly recognized as a critical posttranslational modification (PTM) in central metabolism and cell signaling. Still, the detection of phosphohistidine ...(pHis) in the proteome has remained difficult due to the scarcity of tools to enrich and identify this labile PTM. To address this, we report the first global proteomic analysis of pHis proteins, combining selective immunoenrichment of pHis peptides and a bioinformatic strategy based on mechanistic insight into pHis peptide gas-phase fragmentation during LC–MS/MS. We show that collision-induced dissociation (CID) of pHis peptides produces prominent characteristic neutral losses of 98, 80, and 116 Da. Using isotopic labeling studies, we also demonstrate that the 98 Da neutral loss occurs via gas-phase phosphoryl transfer from pHis to the peptide C-terminal α-carboxylate or to Glu/Asp side chain residues if present. To exploit this property, we developed a software tool that screens LC–MS/MS spectra for potential matches to pHis-containing peptides based on their neutral loss pattern. This tool was integrated into a proteomics workflow for the identification of endogenous pHis-containing proteins in cellular lysates. As an illustration of this strategy, we analyzed pHis peptides from glycerol-fed and mannitol-fed Escherichia coli cells. We identified known and a number of previously speculative pHis sites inferred by homology, predominantly in the phosphoenolpyruvate:sugar transferase system (PTS). Furthermore, we identified two new sites of histidine phosphorylation on aldehyde-alcohol dehydrogenase (AdhE) and pyruvate kinase (PykF) enzymes, previously not known to bear this modification. This study lays the groundwork for future pHis proteomics studies in bacteria and other organisms.
Pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains are structurally related regulatory modules that are present in a variety of proteins involved in signal transduction, such as ...kinases, phospholipases, GTP exchange proteins, and adapter proteins. Initially these domains were shown to mediate protein-protein interactions, but more recently they were also found to bind phosphoinositides. Most studies to date have focused on binding of PH domains to phosphatidylinositol (PtdIns)-4-P and PtdIns-4,5-P2 and have not considered the lipid products of phosphoinositide 3-kinase: PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3. Here we have compared the phosphoinositide specificity of six different PH domains and the Shc PTB domain using all five phosphoinositides. We show that the Bruton's tyrosine kinase PH domain binds to PtdIns-3,4,5-P3 with higher affinity than to PtdIns-4,5-P2, PtdIns-3,4-P2 or inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4). This selectivity is decreased by the xid mutation (R28C). Selective binding of PtdIns-3,4,5-P3 over PtdIns-4,5-P2 or PtdIns-3,4-P2 was also observed for the amino-terminal PH domain of T lymphoma invasion and metastasis protein (Tiam-1), the PH domains of Son-of-sevenless (Sos) and, to a lesser extent, the PH domain of the β-adrenergic receptor kinase. The oxysterol binding protein and β-spectrin PH domains bound PtdIns-3,4,5-P3and PtdIns-4,5-P2 with similar affinities. PtdIns-3,4,5-P3 and PtdIns-4,5-P2 also bound to the PTB domain of Shc with similar affinities and lipid binding was competed with phosphotyrosine (Tyr(P)-containing peptides. These results indicate that distinct PH domains select for different phosphoinositides.
Arfophilin was first identified as a target protein for GTP-ARF5. The N-terminus of ARF5 (amino acids 2−17), which is distinct from that of class I or class III ARFs, is essential for binding to the ...C-terminus of arfophilin (amino acids 612−756). This study using GST fusion proteins in pulldown experiments in CHO-K1 cell lysates showed that, unexpectedly, ARF6 also bound to full-length arfophilin or the C-terminus of arfophilin (amino acids 612−756) in a GTP-dependent manner. Studies with ARF1/ARF6 chimeras further showed that the amino acid sequence of residues 37−80 of ARF6, which is different from the corresponding sequences in class I and class II ARFs, was essential for binding to arfophilin. Both GTP-ARF5 and GTP-ARF6 bound to arfophilin in CHO-K1 cell lysates, while GTP-ARF1 did not bind. In contrast, all three forms of ARF bound to arfaptin 2, with ARF1 showing the strongest binding. Yeast two-hybrid studies with wild-type, dominant negative, and constitutively active forms of ARF1, -5, and -6 and with ARF1/ARF6 chimeras confirmed these results, except that constitutively active ARF6 was autoactivating. Our findings suggest that both class II and III ARFs may influence the same cellular pathways through arfophilin as a common downstream effector.
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