Autophagy is an evolutionally conserved "self-eating" process. Although the genes essential for autophagy (termed Atg) have been identified in yeast, the molecular mechanism of how these Atg proteins control autophagosome formation in mammalian cells remains to be elucidated. Here, we demonstrate that Bif-1 (also known as Endophilin B1) interacts with Beclin 1 through UVRAG and acts as a positive mediator of the class III PI3-kinase (PI3KC3). In response to nutrition deprivation, Bif-1 localizes to autophagosomes where it colocalizes with Atg5, as well as LC3. Furthermore, loss of Bif-1 suppresses autophagosome formation. While the SH3 domain of Bif-1 is sufficient for binding to UVRAG, both the BAR and SH3 domains are required for Bif-1 to activate PI3KC3 and induce autophagosome formation. We also found that Bif-1 ablation prolongs cell survival under nutrient starvation. Moreover, knockout of Bif-1 significantly enhances the development of spontaneous tumors in mice. These findings suggest that Bif-1 joins the UVRAG-Beclin 1 complex as a potential activator of autophagy and tumor suppressor.Autophagy is a tightly orchestrated intracellular process for bulk degradation of cytoplasmic proteins or organelles that appears to be essential for many physiological processes such as cellular homeostasis, development, differentiation, tissue remodeling, cell survival and death, innate immunity, and pathogenesis in various organisms 1-4 . The process of autophagic degradation is initiated when a portion of the cytosolic components are sequestered in cupshaped membrane structures called isolation membranes 1, 2, 5, 6 . The isolation membranes are elongated and eventually sealed to become double-membrane vesicles called autophagosomes, which are then fused with lysosomes resulting in degradation of the enclosed components. Eighteen autophagy-related (Atg) genes have been characterized in S. cerevisiae and can be categorized into four functional groups: (1) the Atg1 protein kinase complex regulating the induction of autophagy, (2) the class III PI3-kinase (PI3KC3) lipid kinase complex controlling vesicle nucleation, (3) the Atg12-Atg5 and Atg8-phosphatidylethanolamine conjugation pathways for vesicle expansion and completion, and (4) the Atg protein retrieval system 2, 7 . Beclin 1, the mammalian homologue of yeast Atg6, is a key component of the PI3KC3 complex, which plays an essential role in autophagosome formation 8-11 . Although the phosphatidylinositol 3-phosphate (PtdIns-3-P) generated by PI3KC3 has been proposed to control membrane dynamics during autophagosome formation 3 , the molecular mechanism underlying this process remains unknown. Results Loss of Bif-1 suppresses caspase-independent cell deathWe have previously reported that Bif-1 localizes to mitochondria and regulates the activation of Bax and Bak during apoptosis induced by intrinsic death stimuli 21 . To examine Bif-1 localization in mouse embryonic fibroblast (MEF) cells during serum deprivation, we added a pancaspase inhibitor, z-VAD-fmk, to ...

Serum contains a growth factor derived from platelets and also growth factors derived from platelet-poor plasma. Extracts of heated (1000) human platelets function synergistically with platelet-poor plasma to induce DNA synthesis in quiescent, density-inhibited BALB/c 3T3 cells. Platelet-poor plasma alone did not induce DNA synthesis. Cells exposed to platelet extracts became competent to enter the cell cycle, but the rate of entry into the S phase depended upon the concentration of platelet-poor plasma. The time required for the induction of this competent state was a function of the concentration of the platelet extract. A 2-hr exposure to 100',g of the platelet extract at 370 caused the entire cell population to become competent to enter the S phase. At 40 or 250 the cells did not become competent to synthesize DNA. The platelet extract-induced competent state was stable for at least 13 hr after removal of the platelet extract; however, in the absence of platelet-poor plasma, these competent cells did not progress through the cell cycle. The addition of an optimal concentration of platelet-poor plasma (5%) to these competent cells initiated cell cycle traverse with a ra id, first-order entry of cells into the S phase beginning 12 hr after addition of the plasma. The addition of a suboptimal concentration of the plasma (0.25%) did not increase the rate of cell entry into the S phase. Thus, the induction of DNA synthesis in quiescent BALB/c 3T3 cells can be resolved into at least two phases, controlled by different serum components: (i) competence, induced by the plateletderived growth factor; and (ii) progression of competent cells into the cell cycle, mediated by factors in platelet-poor plasma.The growth of 3T3 cells (1), diploid fibroblasts (2), and smooth muscle cells (3) in vitro is controlled by the concentration of serum in the medium. Serum can be separated into two sets of components which control different cell functions. One set maintains cell viability (4), while the other stimulates replication (5). A heat-stable (1000) cationic growth factor (6) derived from. platelets (7) is released into serum during the clotting process (8, 9). Human serum contains about 770 pg of this polypeptide growth factor per mg of protein, as demonstrated by radioimmunoassay (7). Defibrinogenated platelet-poor plasma, a fraction prepared from unclotted blood, contains only low levels of the growth factor and does not stimulate the replication of diploid fibroblasts or BALB/c 3T3 cells (3, 7-9). Platelet-poor plasma does, however, contain the factors that maintain cell viability (3).The process by which resting cells become committed to enter the growth cycle remains unclear. Smith and Martin (10) have proposed that the commitment of quiescent cells to synthesize DNA is a random event characterized by a first-order rate constant, the transition probability. According to thisThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ...

Quiescent BALB/c 3T3 cells exposed briefly to a platelet-derived growth factor (PDGF) become "competent" to replicate their DNA but do not "progress" into S phase unless incubated with growth factors contained in platelet-poor plasma. Plasma from hypophysectomized rats is deficient in progression activity; it does not stimulate PDGF-treated competent cells to synthesize DNA. Addition of somatomedin C to hypophysectomized rat plasma stimulates competent cells to synthesize DNA, demonstrating that somatomedin C is required for progression. Various growth factors were tested for progression activity and competence activity by using BALB/c 3T3 tissue culture assays. Multiplication stimulating activity and other members of the somatomedin family of growth factors are (like somatomedin C) potent mediators of progression. Other mitogenic agents, such as fibroblast growth factor, are (like PDGF) potent inducers of competence. Growth factors with potent progression activity have little or no competence activity and vice versa. In contrast, simian virus 40 provides both competence and progession activity. Coordinate control of BALB/c 3T3 cell growth in vitro by competence factors and somatomedins may be a specific example of a common pattern for growth regulation in animal tissues.

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Normal cellular functions of hamartin and tuberin, encoded by the TSC1 and TSC2 tumor suppressor genes, are closely related to their direct interactions. However, the regulation of the hamartin-tuberin complex in the context of the physiologic role as tumor suppressor genes has not been documented. Here we show that insulin or insulin growth factor (IGF) 1 stimulates phosphorylation of tuberin, which is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 but not by the mitogen-activated protein kinase inhibitor PD98059. Expression of constitutively active PI3K or active Akt, including Akt1 and Akt2, induces tuberin phosphorylation. We further demonstrate that Akt/PKB associates with hamartin-tuberin complexes, promoting phosphorylation of tuberin and increased degradation of hamartin-tuberin complexes. The ability to form complexes, however, is not blocked. Akt also inhibits tuberin-mediated degradation of p27 kip1 , thereby promoting CDK2 activity and cellular proliferation. Our results indicate that tuberin is a direct physiological substrate of Akt and that phosphorylation of tuberin by PI3K/Akt is a major mechanism controlling hamartin-tuberin function.Tuberous sclerosis complex (TSC) 1 is an autosomal dominant disorder and is characterized by the presence of hamartomas in many organs such as brain, skin, heart, lung, and kidney (1). It is caused by mutation of either the TSC1 or TSC2 tumor suppressor gene (2-5). TSC1 encodes a protein, hamartin, containing two coiled-coil domains that have been shown to mediate binding to hamartin (6). The TSC2 gene codes for tuberin, which contains a small region of homology to the rap1GTPase-activating protein, rap1GAP (7). These two proteins function within the same pathway(s) regulating cell cycle, cell growth, adhesion, and vesicular trafficking (4,5). However, the regulation of hamartin and tuberin in the context of physiologic role as tumor suppressor genes has not been documented.Among the various properties of these two proteins, the ability to interact and to form stable complex has been the most consistent finding. This led to the hypothesis that hamartin and tuberin function as a complex and that factors regulating their interaction are important in understanding physiologic roles. There is evidence to suggest that phosphorylation of tuberin may be a major mechanism of regulation of the hamartin-tuberin complex (8, 9). However, the kinases that are responsible for phosphorylation of this complex are currently unknown. Recent Drosophila genetic studies showed that dTsc1 and dTsc2 play an important role in the insulin/dPI3K/ dakt signal transduction pathway by demonstrating that reduced cell size and cell proliferation caused by either mutations in dINR and dakt or by overexpression of dPTEN are overridden by homozygous mutants of dTsc1 or dTsc2. This implies that dTsc1 and dTsc2 are either direct downstream targets of dakt or on a parallel pathway of the insulin cascade downstream from dakt (10 -13). Akt, also known as protein kinase B (PKB),...

An ordered sequence of events must be completed before cells become committed to synthesize DNA. A platelet-derived growth factor (PDGF), present in heated (1000) extracts of human platelets, induces density-inhibited BALB/ c-3T3 cells to become competent to proliferate. Platelet-poor plasma induces these competent cells to leave the competence point, progress through Go/GI, and enter the S phase. Treatment of GO-arrested, incompetent cells with plasma, before the addition of PDGF, did not shorten the latent period for DNA synthesis or increase the rate of entry into the S phase. Growth arrest points in the plasma-dependent progression sequence were detected in Go/GI. PDGF-treated competent cells were exposed to an optimal concentration of plasma (5%) for various lengths of time and were then transferred to medium lacking plasma; the subsequent readdition of plasma stimulated the cells to enter the S phase. The lag period until DNA synthesis, in such experiments, was dictated by the length of the initial exposure to plasma. PDGF-treated competent cells that were incubated with plasma for 5 hr during the initial exposure did not leave the competence point; they began DNA synthesis 12 hr after the readdition of plasma. However, a population of cells treated with plasma for 10 hr became arrested at a point 6 hr before DNA synthesis, whereas a population treated with plasma for [12][13][14][15] hr became arrested at a point immediately before DNA synthesis. Cells remained arrested at this latter point for as long as 24 hr, and these arrested cells were not committed to DNA synthesis. The addition of plasma induced immediate entry into theS phase with an apparent first-order rate of entry being determined by the plasma concentration. This plasma-dependent commitment (transition) to DNA synthesis was blocked by cycloheximide but not by hydroxyurea. Removal ofthe hydroxyurea allowed cells to enter the S phase synchronously in the absence of plasma. Serum induces quiescent, density-inhibited 3T3 cells to leave Go, synthesize DNA, and replicate (1-3). There are two sets of growth factors in serum that control different phases of the cell cycle (4). One set is a heat-stable (100°) platelet-derived growth factor (PDGF) that is released into serum during the clotting process (5-9). PDGF induces BALB/c-3T3 cells to become competent to synthesize DNA. A second set of components, found in defibrinogenated platelet-poor plasma, allows competent cells to progress through GO/GI and synthesize DNA (4).

While the activated viral Src oncoprotein, v-Src, induces uncontrolled cell growth, the mechanisms underlying cell cycle deregulation by v-Src have not been fully de®ned. Previous studies demonstrated that v-Src induces constitutively active STAT3 signaling that is required for cell transformation and recent data have implicated STAT3 in the transcriptional control of critical cell cycle regulators. Here we show in mouse ®broblasts stably transformed by v-Src that mRNA and protein levels of p21 (WAF1/CIP1), cyclin D1, and cyclin E are elevated. Using reporter constructs in transient-transfection assays, the cyclin D1 and p21 promoters were both found to be transcriptionaly induced by v-Src in a STAT3-dependent manner. The kinase activities of cyclin D/ CDK4, 6 and cyclin E/CDK2 complexes were only slightly elevated, consistent with the ®ndings that coordinate increases in p21, cyclin D1 and cyclin E resulted in an increase in cyclin/CDK/p21 complexes. Similar results were obtained in NIH3T3 and BALB/c 3T3 cells stably transformed by v-Src, indicating that these regulatory events associated with STAT3 signaling represent common mechanisms independent of cell line or clonal variation. These ®ndings suggest that STAT3 has an essential role in the regulation of critical cell cycle components in v-Src transformed mouse ®broblasts.