Abstract: Jun N-terminal kinases or JNKs play a critical role in death receptor-initiated extrinsic as well as mitochondrial intrinsic apoptotic pathways. JNKs activate apoptotic signaling by the upregulation of pro-apoptotic genes through the transactivation of specific transcription factors or by directly modulating the activities of mitochondrial pro- and antiapoptotic proteins through distinct phosphorylation events. This review analyses our present understanding of the role of JNK in apoptotic signaling and the various mechanisms by which JNK promotes apoptosis.

Abstract: Changes in mitochondrial morphology that occur during cell cycle, differentiation, and death are tightly regulated by the balance between fusion and fission processes. Excessive fragmentation can be caused by inhibition of the fusion machinery and is a common consequence of dysfunction of the organelle. Here, we show a role for calcineurin-dependent translocation of the profission dynamin related protein 1 (Drp1) to mitochondria in dysfunction-induced fragmentation. When mitochondrial depolarization is associated with sustained cytosolic Ca(2+) rise, it activates the cytosolic phosphatase calcineurin that normally interacts with Drp1. Calcineurin-dependent dephosphorylation of Drp1, and in particular of its conserved serine 637, regulates its translocation to mitochondria as substantiated by site directed mutagenesis. Thus, fragmentation of depolarized mitochondria depends on a loop involving sustained Ca(2+) rise, activation of calcineurin, and dephosphorylation of Drp1 and its translocation to the organelle.

Abstract: Growth factors stimulate cells to take up excess nutrients and to use them for anabolic processes. The biochemical mechanism by which this is accomplished is not fully understood but it is initiated by phosphorylation of signalling proteins on tyrosine residues. Using a novel proteomic screen for phosphotyrosine-binding proteins, we have made the observation that an enzyme involved in glycolysis, the human M2 (fetal) isoform of pyruvate kinase (PKM2), binds directly and selectively to tyrosine-phosphorylated peptides. We show that binding of phosphotyrosine peptides to PKM2 results in release of the allosteric activator fructose-1,6-bisphosphate, leading to inhibition of PKM2 enzymatic activity. We also provide evidence that this regulation of PKM2 by phosphotyrosine signalling diverts glucose metabolites from energy production to anabolic processes when cells are stimulated by certain growth factors. Collectively, our results indicate that expression of this phosphotyrosine-binding form of pyruvate kinase is critical for rapid growth in cancer cells.

Abstract: PTEN/PI3K/AKT constitutes an important pathway regulating the signaling of multiple biological processes such as apoptosis, metabolism, cell proliferation and cell growth. PTEN is a dual protein/lipid phosphatase which main substrate is the phosphatidyl-inositol,3,4,5 triphosphate (PIP3), the product of PI3K. Increase in PIP3 recruits AKT to the membrane where it is activated by other kinases also dependent on PIP3. Many components of this pathway have been described as causal forces in cancer. PTEN activity is lost by mutations, deletions or promoter methylation silencing at high frequency in many primary and metastatic human cancers. Germ line mutations of PTEN are found in several familial cancer predisposition syndromes. Activating mutations which have been reported for PI3K and AKT, in tumours are able to confer tumourigenic properties in several cellular systems. Additionally, the binding of PI3K to oncogenic ras is essential for the transforming properties of ras. In summary, the data strongly support the view of the PTEN/PI3K/AKT pathway as an important target for drug discovery.

Abstract: The mammalian translational initiation machinery is a tightly controlled system that is composed of eukaryotic initiation factors, and which controls the recruitment of ribosomes to mediate cap-dependent translation. Accordingly, the mTORC1 complex functionally controls this cap-dependent translation machinery through the phosphorylation of its downstream substrates 4E-BPs and S6Ks. It is generally accepted that rapamycin, a specific inhibitor of mTORC1, is a potent translational repressor. Here we report the unexpected discovery that rapamycin's ability to regulate cap-dependent translation varies significantly among cell types. We show that this effect is mechanistically caused by rapamycin's differential effect on 4E-BP1 versus S6Ks. While rapamycin potently inhibits S6K activity throughout the duration of treatment, 4E-BP1 recovers in phosphorylation within 6 h despite initial inhibition (1-3 h). This reemerged 4E-BP1 phosphorylation is rapamycin-resistant but still requires mTOR, Raptor, and mTORC1's activity. Therefore, these results explain how cap-dependent translation can be maintained in the presence of rapamycin. In addition, we have also defined the condition by which rapamycin can control cap-dependent translation in various cell types. Finally, we show that mTOR catalytic inhibitors are effective inhibitors of the rapamycin-resistant phenotype.

Abstract: Objective TAR DNA-binding protein of 43kDa (TDP-43) is deposited as cytoplasmic and intranuclear inclusions in brains of patients with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Previous studies reported that abnormal phosphorylation takes place in deposited TDP-43. The aim of this study was to identify the phosphorylation sites and responsible kinases, and to clarify the pathological significance of phosphorylation of TDP-43. Methods We generated multiple antibodies specific to phosphorylated TDP-43 by immunizing phosphopeptides of TDP-43, and analyzed FTLD-U and ALS brains by immunohistochemistry, immunoelectron microscopy, and immunoblots. In addition, we performed investigations aimed at identifying the responsible kinases, and we assessed the effects of phosphorylation on TDP-43 oligomerization and fibrillization. Results We identified multiple phosphorylation sites in carboxyl-terminal regions of deposited TDP-43. Phosphorylation-specific antibodies stained more inclusions than antibodies to ubiquitin and, unlike existing commercially available anti–TDP-43 antibodies, did not stain normal nuclei. Ultrastructurally, these antibodies labeled abnormal fibers of 15nm diameter and on immunoblots recognized hyperphosphorylated TDP-43 at 45kDa, with additional 18 to 26kDa fragments in sarkosyl-insoluble fractions from FTLD-U and ALS brains. The phosphorylated epitopes were generated by casein kinase-1 and -2, and phosphorylation led to increased oligomerization and fibrillization of TDP-43. Interpretation These results suggest that phosphorylated TDP-43 is a major component of the inclusions, and that abnormal phosphorylation of TDP-43 is a critical step in the pathogenesis of FTLD-U and ALS. Phosphorylation-specific antibodies will be powerful tools for the investigation of these disorders. Ann Neurol 2008

Abstract: The Akt serine/threonine kinase (also called protein kinase B) has emerged as a critical signaling molecule within eukaryotic cells. Significant progress has been made in clarifying its regulation by upstream kinases and identifying downstream mechanisms that mediate its effects in cells and contribute to signaling specificity. Here, we provide an overview of present advances in the field regarding the function of Akt in physiological and pathological cell function within a more generalized framework of Akt signal transduction. An emphasis is placed on the involvement of Akt in human diseases ranging from cancer to metabolic dysfunction and mental disease.

Abstract: A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo

Abstract: On activation by receptors, the ubiquitously expressed class IA isoforms (p110alpha and p110beta) of phosphatidylinositol-3-OH kinase (PI(3)K) generate lipid second messengers, which initiate multiple signal transduction cascades. Recent studies have demonstrated specific functions for p110alpha in growth factor and insulin signalling. To probe for distinct functions of p110beta, we constructed conditional knockout mice. Here we show that ablation of p110beta in the livers of the resulting mice leads to impaired insulin sensitivity and glucose homeostasis, while having little effect on phosphorylation of Akt, suggesting the involvement of a kinase-independent role of p110beta in insulin metabolic action. Using established mouse embryonic fibroblasts, we found that removal of p110beta also had little effect on Akt phosphorylation in response to stimulation by insulin and epidermal growth factor, but resulted in retarded cell proliferation. Reconstitution of p110beta-null cells with a wild-type or kinase-dead allele of p110beta demonstrated that p110beta possesses kinase-independent functions in regulating cell proliferation and trafficking. However, the kinase activity of p110beta was required for G-protein-coupled receptor signalling triggered by lysophosphatidic acid and had a function in oncogenic transformation. Most strikingly, in an animal model of prostate tumour formation induced by Pten loss, ablation of p110beta (also known as Pik3cb), but not that of p110alpha (also known as Pik3ca), impeded tumorigenesis with a concomitant diminution of Akt phosphorylation. Taken together, our findings demonstrate both kinase-dependent and kinase-independent functions for p110beta, and strongly indicate the kinase-dependent functions of p110beta as a promising target in cancer therapy.

Abstract: Altered degradation of α-synuclein (α-syn) has been implicated in the pathogenesis of Parkinson disease (PD). We have shown that α-syn can be degraded via chaperone-mediated autophagy (CMA), a selective lysosomal mechanism for degradation of cytosolic proteins. Pathogenic mutants of α-syn block lysosomal translocation, impairing their own degradation along with that of other CMA substrates. While pathogenic α-syn mutations are rare, α-syn undergoes posttranslational modifications, which may underlie its accumulation in cytosolic aggregates in most forms of PD. Using mouse ventral medial neuron cultures, SH-SY5Y cells in culture, and isolated mouse lysosomes, we have found that most of these posttranslational modifications of α-syn impair degradation of this protein by CMA but do not affect degradation of other substrates. Dopamine-modified α-syn, however, is not only poorly degraded by CMA but also blocks degradation of other substrates by this pathway. As blockage of CMA increases cellular vulnerability to stressors, we propose that dopamine-induced autophagic inhibition could explain the selective degeneration of PD dopaminergic neurons.

Abstract: Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc). This tandem system serves as a nutrient sensor to couple systemic metabolic status to cellular regulation of signal transduction, transcription, and protein degradation. Here we show that O-GlcNAc transferase (OGT) harbours a previously unrecognized type of phosphoinositide-binding domain. After induction with insulin, phosphatidylinositol 3,4,5-trisphosphate recruits OGT from the nucleus to the plasma membrane, where the enzyme catalyses dynamic modification of the insulin signalling pathway by O-GlcNAc. This results in the alteration in phosphorylation of key signalling molecules and the attenuation of insulin signal transduction. Hepatic overexpression of OGT impairs the expression of insulin-responsive genes and causes insulin resistance and dyslipidaemia. These findings identify a molecular mechanism by which nutritional cues regulate insulin signalling through O-GlcNAc, and underscore the contribution of this modification to the aetiology of insulin resistance and type 2 diabetes.

Abstract: Although some cancers are initially sensitive to EGFR tyrosine kinase inhibitors (TKIs), resistance invariably develops. We investigated mechanisms of acquired resistance to the EGFR TKI gefitinib by generating gefitinib-resistant (GR) A431 squamous cancer cells. In GR cells, gefitinib reduced phosphorylation of EGFR, ErbB-3, and Erk but not Akt. These cells also showed hyperphosphorylation of the IGFI receptor (IGFIR) and constitutive association of IRS-1 with PI3K. Inhibition of IGFIR signaling disrupted the association of IRS-1 with PI3K and restored the ability of gefitinib to downregulate PI3K/Akt signaling and to inhibit GR cell growth. Gene expression analyses revealed that GR cells exhibited markedly reduced IGF-binding protein 3 (IGFBP-3) and IGFBP-4 RNA. Addition of recombinant IGFBP-3 restored the ability of gefitinib to downregulate PI3K/Akt signaling and to inhibit cell growth. Finally, gefitinib treatment of mice with A431 xenografts in combination with an IGFIR-specific monoclonal antibody prevented tumor recurrence, whereas each drug given alone was unable to do so. These data suggest that loss of expression of IGFBPs in tumor cells treated with EGFR TKIs derepresses IGFIR signaling, which in turn mediates resistance to EGFR antagonists. Moreover, combined therapeutic inhibition of EGFR and IGFIR may abrogate this acquired mechanism of drug resistance and is thus worthy of prospective clinical investigation.

Abstract: The target of rapamycin (TOR), as part of the rapamycin-sensitive TOR complex 1 (TORC1), regulates various aspects of protein synthesis. Whether TOR functions in this process as part of TORC2 remains to be elucidated. Here, we demonstrate that mTOR, SIN1 and rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt and conventional protein kinase C (PKC) at the turn motif (TM) site. This TORC2 function is growth factor independent and conserved from yeast to mammals. TM site phosphorylation facilitates carboxyl-terminal folding and stabilizes newly synthesized Akt and PKC by interacting with conserved basic residues in the kinase domain. Without TM site phosphorylation, Akt becomes protected by the molecular chaperone Hsp90 from ubiquitination-mediated proteasome degradation. Finally, we demonstrate that mTORC2 independently controls the Akt TM and HM sites in vivo and can directly phosphorylate both sites in vitro. Our studies uncover a novel function of the TOR pathway in regulating protein folding and stability, processes that are most likely linked to the functions of TOR in protein synthesis.

Abstract: The SNF1/AMPK family of protein kinases is highly conserved in eukaryotes and is required for energy homeostasis in mammals, plants, and fungi. SNF1 protein kinase was initially identified by genetic analysis in the budding yeast Saccharomyces cerevisiae. SNF1 is required primarily for the adaptation of yeast cells to glucose limitation and for growth on carbon sources that are less preferred than glucose, but is also involved in responses to other environmental stresses. SNF1 regulates transcription of a large set of genes, modifies the activity of metabolic enzymes, and controls various nutrient-responsive cellular developmental processes. Like AMPK, SNF1 protein kinase is heterotrimeric. It is phosphorylated and activated by the upstream kinases Sak1, Tos3, and Elm1 and is inactivated by the Reg1-Glc7 protein phosphatase 1. Further regulation of SNF1 is achieved through autoinhibition and through control of its subcellular localization. Here we review the current understanding of SNF1 protein kinase pathways in Saccharomyces cerevisiae and other yeasts.

Abstract: Mitochondria are dynamic organelles that frequently move, divide, and fuse with one another to maintain their architecture and functions. However, the signaling mechanisms involved in these processes are still not well characterized. In this study, we analyze mitochondrial dynamics and morphology in neurons. Using time-lapse imaging, we find that Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) causes a rapid halt in mitochondrial movement and induces mitochondrial fission. VDCC-associated Ca2+ signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca2+/calmodulin-dependent protein kinase Iα (CaMKIα). In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 is associated with an increase in Drp1 translocation to mitochondria, whereas in vitro, phosphorylation of Drp1 results in an increase in its affinity for Fis1. CaMKIα is a widely expressed protein kinase, suggesting that Ca2+ is likely to be functionally important in the control of mitochondrial dynamics through regulation of Drp1 phosphorylation in neurons and other cell types.

Abstract: Although the best-defined function of type II major histocompatibility complex (MHC-II) is presentation of antigenic peptides to T lymphocytes, these molecules can also transduce signals leading alternatively to cell activation or apoptotic death. MHC-II is a heterodimer of two transmembrane proteins, each containing a short cytoplasmic tail that is dispensable for transduction of death signals. This suggests the function of an undefined MHC-II-associated transducer in signaling the death response. Here we describe a novel plasma membrane tetraspanner (MPYS) that is associated with MHC-II and mediates its transduction of death signals. MPYS is unusual among tetraspanners in containing an extended C-terminal cytoplasmic tail (140 amino acids) with multiple embedded signaling motifs. MPYS is tyrosine phosphorylated upon MHC-II aggregation and associates with inositol lipid and tyrosine phosphatases. Finally, MHC class II-mediated cell death signaling requires MPYS-dependent activation of the extracellular signal-regulated kinase signaling pathway.