Autophagy is a degradative process that recycles long-lived and faulty cellular components. It is linked to many diseases and is required for normal development. ULK1, a mammalian serine/threonine protein kinase, plays a key role in the initial stages of autophagy, though the exact molecular mechanism is unknown. Here we report identification of a novel protein complex containing ULK1 and two additional protein factors, FIP200 and ATG13, all of which are essential for starvationinduced autophagy. Both FIP200 and ATG13 are critical for correct localization of ULK1 to the pre-autophagosome and stability of ULK1 protein. Additionally, we demonstrate by using both cellular experiments and a de novo in vitro reconstituted reaction that FIP200 and ATG13 can enhance ULK1 kinase activity individually but both are required for maximal stimulation. Further, we show that ATG13 and ULK1 are phosphorylated by the mTOR pathway in a nutrient starvation-regulated manner, indicating that the ULK1⅐ATG13⅐FIP200 complex acts as a node for integrating incoming autophagy signals into autophagosome biogenesis.Macroautophagy (herein referred to as autophagy) is a catabolic process whereby long-lived proteins and damaged organelles are shuttled to lysosomes for degradation. This process is conserved in all eukaryotes. Under normal growth conditions a housekeeping level of autophagy exists. Under stress, such as nutrient starvation, autophagy is strongly induced resulting in the engulfment of cytosolic components and organelles in specialized double-membrane structures termed autophagosomes. Following fusion of the outer autophagosomal membrane with lysosomes, the inner membrane and its cytoplasmic cargo are degraded and recycled (1-3). Recent work has implicated autophagy in many disease pathologies, including cancer, neurodegeneration, as well as in eliminating intracellular pathogens (4 -8).The morphology of autophagy was first described in mammalian cells over 50 years ago (9). However, it is only recently through yeast genetic screens, that multiple autophagy-related (ATG) genes have been identified (10 -12). The yeast ATG proteins have been classified into four major groups: the Atg1 protein kinase complex, the Vps34 phosphatidylinositol 3-phosphate kinase complex, the Atg8/Atg12 conjugation systems, and the Atg9 recycling complex (13). Even though many ATG genes are now known, most of which have functional homologs in mammalian cells (14, 15), the molecular mechanism by which they sense the initial triggers and subsequently dictate autophagy-specific intracellular membrane events is far from understood.In yeast, one of the earliest autophagy-specific events is believed to involve the Atg1 protein kinase complex. Atg1 is a serine/threonine protein kinase and a key autophagy-regulator (16). Atg1 is complexed to at least two other proteins during autophagy, Atg13 and Atg17, both of which are required for normal Atg1 function and autophagosome generation (17-19). Classical signaling pathways such as the cAMP-dependent kinase (PKA) pat...

Autophagy is a cellular catabolic pathway by which long-lived proteins and damaged organelles are targeted for degradation. Activation of autophagy enhances cellular tolerance to various stresses. Recent studies indicate that a class of anticancer agents, histone deacetylase (HDAC) inhibitors, can induce autophagy. One of the HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA), is currently being used for treating cutaneous T-cell lymphoma and under clinical trials for multiple other cancer types, including glioblastoma. Here, we show that SAHA increases the expression of the autophagic factor LC3, and inhibits the nutrient-sensing kinase mammalian target of rapamycin (mTOR). The inactivation of mTOR results in the dephosphorylation, and thus activation, of the autophagic protein kinase ULK1, which is essential for autophagy activation during SAHA treatment. Furthermore, we show that the inhibition of autophagy by RNAi in glioblastoma cells results in an increase in SAHA-induced apoptosis. Importantly, when apoptosis is pharmacologically blocked, SAHA-induced nonapoptotic cell death can also be potentiated by autophagy inhibition. Overall, our findings indicate that SAHA activates autophagy via inhibiting mTOR and up-regulating LC3 expression; autophagy functions as a prosurvival mechanism to mitigate SAHA-induced apoptotic and nonapoptotic cell death, suggesting that targeting autophagy might improve the therapeutic effects of SAHA. transcription | ATG7 | necrosis H istone deacetylase (HDAC) inhibitors emerge as a new class of therapeutic agents with promising outcomes during the treatment of a wide range of cancer types (1). Hematological malignancies appear to be particularly sensitive to HDAC inhibitors; however, a number of additional cancer types are currently being tested for their response to HDAC inhibition therapy. For an example, suberoylanilide hydroxamic acid (SAHA, vorinostat), which inhibits HDACs 1, 2, 3, and 6, has been approved for treatment against cutaneous T-cell lymphoma and also has modest effects as a single agent on cancers of the prostate, ovaries, breast, colorectal, and glioblastoma (2, 3). Although their precise mode of action remains uncertain, a number of recent data suggest that HDAC inhibitors may induce apoptotic cell death through both chromatin-dependent and -independent mechanisms.Treatment with HDAC inhibitors most frequently induces apoptosis via the programmed activation of a series of proteases, called caspases (4-6). More recently, HDAC inhibition has been also shown to induce autophagy (7,8). Unlike apoptosis, the contribution of autophagy to cell death remains controversial and, most likely, context-dependent. Autophagy is a catabolic process by which cytosolic material is targeted for lysosomal degradation by means of double-membrane cytosolic vesicles, termed autophagosomes (9). The formation of autophagosomes is orchestrated by upstream signaling molecules, including the ULK1 and PI3K complexes, which signal to downstream complexes involved in the nucleation and...

Nectin-2 is a cell adhesion molecule encoded by a member of the poliovirus receptor gene family. This family consists of human, monkey, rat, and murine genes that are members of the immunoglobulin gene superfamily. Nectin-2 is a component of cell-cell adherens junctions and interacts with l-afadin, an F-actin-binding protein.Disruption of both alleles of the murine nectin-2 gene resulted in morphologically aberrant spermatozoa with defects in nuclear and cytoskeletal morphology and mitochondrial localization. Homozygous null males are sterile, while homozygous null females, as well as heterozygous males and females, are fertile. The production by nectin-2 ؊/؊ mice of normal numbers of spermatozoa containing wild-type levels of DNA suggests that Nectin-2 functions at a late stage of germ cell development. Consistent with such a role, Nectin-2 is expressed in the testes only during the later stages of spermatogenesis. The structural defects observed in spermatozoa of nectin-2 ؊/؊ mice suggest a role for this protein in organization and reorganization of the cytoskeleton during spermiogenesis.The human poliovirus receptor (Pvr) is a member of the immunoglobulin superfamily of proteins (38) [7]). Only pvr, agm1, and agm2 encode proteins that can function as cell receptors for poliovirus (18, 23; V. Racaniello, unpublished data, 1999). Pvr, Prr1, Prr2, and Mph/murine Prr2 are entry cofactors for alphaherpesviruses (9, 37).The cellular functions of members of the Pvr protein family are not known. Some members of the Ig superfamily are involved in cell-cell and cell-extracellular matrix interactions, and others are receptors for cytokines and growth factors. Expression of human Prr2 or Mph/murine Prr2 in cultured cells leads to aggregation, suggesting that these proteins are homophilic adhesion molecules (1, 20). The cytoplasmic domains of Prr1 and Prr2 proteins interact with l-afadin, an actin filament-binding protein (35). l-Afadin is ubiquitously expressed but is localized at specialized membrane structures, called adherens junctions, which are involved in cell-cell adhesion (22). l-Afadin contains one PDZ domain through which it interacts with a COOH-terminal amino acid motif of Prr1 and Prr2 as well as an actin filament-binding domain. Thus, Prr1 and Prr2 are linked to the cytoskeleton through l-afadin. The Prr1 and Prr2 proteins have been renamed Nectin-1 and Nectin-2 (35); the new terminology is used in this paper.To provide information on the function of Pvr family members, we disrupted the murine nectin-2 gene. Male mice lacking both alleles of nectin-2 are infertile and produce morphologically aberrant spermatozoa. Heterozygous males and females and homozygous null females are fertile and have no overt developmental defects. The heads of spermatozoa from nectin-2 Ϫ/Ϫ mice contain mitochondria, dense outer filaments, and misshapen nuclei, and the mitochondrial sheath of the middle piece is disorganized. These morphological defects may result from an overall disruption of cytoskeletal structure. In normal mice...

The gene most commonly activated by chromosomal rearrangements in patients with T-cell acute lymphoblastic leukemia (T-ALL) is SCL/tal. In collaboration with LMO1 or LMO2, the thymic expression of SCL/tal leads to T-ALL at a young age with a high degree of penetrance in transgenic mice. We now show that SCL LMO1 double-transgenic mice display thymocyte developmental abnormalities in terms of proliferation, apoptosis, clonality, and immunophenotype prior to the onset of a frank malignancy. At 4 weeks of age, thymocytes from SCL LMO1 mice show 70% fewer total thymocytes, with increased rates of both proliferation and apoptosis, than control thymocytes. At this age, a clonal population of thymocytes begins to populate the thymus, as evidenced by oligoclonal T-cell-receptor gene rearrangements. Also, there is a dramatic increase in immature CD44؉ CD25 ؊ cells, a decrease in the more mature CD4 ؉ CD8 ؉ cells, and development of an abnormal CD44 ؉ CD8 ؉ population. An identical pattern of premalignant changes is seen with either a full-length SCL protein or an amino-terminal truncated protein which lacks the SCL transactivation domain, demonstrating that the amino-terminal portion of SCL is not important for leukemogenesis. Lastly, we show that the T-ALL which develop in the SCL LMO1 mice are strikingly similar to those which develop in E2A null mice, supporting the hypothesis that SCL exerts its oncogenic action through a functional inactivation of E proteins.