In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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Phosphoinositide 3-kinase (PI3K) activity is stimulated by diverse oncogenes and growth factor receptors, and elevated PI3K signaling is considered a hallmark of cancer. Many PI3K pathway-targeted therapies have been tested in oncology trials, resulting in regulatory approval of one isoform-selective inhibitor (idelalisib) for treatment of certain blood cancers, and a variety of other agents at different stages of development. In parallel to PI3K research by cancer biologists, investigations in other fields have uncovered exciting and often unpredicted roles for PI3K catalytic and regulatory subunits in normal cell function and in disease. Many of these functions impinge upon oncology by influencing the efficacy and toxicity of PI3K-targeted therapies. Here we provide a perspective on the roles of class I PI3Ks in the regulation of cellular metabolism and in immune system functions, two topics closely intertwined with cancer biology. We also discuss recent progress developing PI3K-targeted therapies for treatment of cancer and other diseases.

The multisubunit eukaryotic translation initiation factor (eIF) 4F recruits 40S ribosomal subunits to the 5 end of mRNA. The eIF4F subunit eIF4E interacts directly with the mRNA 5 cap structure. Assembly of the eIF4F complex is inhibited by a family of repressor polypeptides, the eIF4E-binding proteins (4E-BPs). Binding of the 4E-BPs to eIF4E is regulated by phosphorylation: Hypophosphorylated 4E-BP isoforms interact strongly with eIF4E, whereas hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quiescent cells, but is hyperphosphorylated on multiple sites following exposure to a variety of extracellular stimuli. The PI3-kinase/Akt pathway and the kinase FRAP/mTOR signal to 4E-BP1. FRAP/mTOR has been reported to phosphorylate 4E-BP1 directly in vitro. However, it is not known if FRAP/mTOR is responsible for the phosphorylation of all 4E-BP1 sites, nor which sites must be phosphorylated to release 4E-BP1 from eIF4E. To address these questions, a recombinant FRAP/mTOR protein and a FRAP/mTOR immunoprecipitate were utilized in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide mapping of the in vitro-labeled protein yielded two 4E-BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo. Mass spectrometry analysis indicated that these peptides contain phosphorylated Thr-37 and Thr-46. Thr-37 and Thr-46 are efficiently phosphorylated in vitro by FRAP/mTOR when 4E-BP1 is bound to eIF4E. However, phosphorylation at these sites was not associated with a loss of eIF4E binding. Phosphorylated Thr-37 and Thr-46 are detected in all phosphorylated in vivo 4E-BP1 isoforms, including those that interact with eIF4E. Finally, mutational analysis demonstrated that phosphorylation of Thr-37/Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. Taken together, our results suggest that 4E-BP1 phosphorylation by FRAP/mTOR on Thr-37 and Thr-46 is a priming event for subsequent phosphorylation of the carboxy-terminal serum-sensitive sites.

Tumor development is characterized by an initial phase of rapid expansion, followed by a period of slowed growth as the proliferating malignant cells outstrip the local supply of oxygen and nutrients. In the absence of a dedicated blood supply, early-stage tumors attain steady-state volumes of only a few cubic millimeters, at which time the rate of cell death, due to oxygen and nutrient depletion, equals the rate of cell division (19). To resume growth, these microtumors must adapt to hypoxic stress through alterations in cellular metabolism and the stimulation of neovascularization, which provides the additional blood needed to sustain cellular proliferation. Accordingly, cellular adaptation to growth during hypoxic stress contributes to malignant progression and is correlated with a poor clinical outcome in several types of cancer (3, 4, 18). Two hallmark features of hypoxic adaptation are increased rates of anaerobic glycolysis and the secretion of proangiogenic factors, such as vascular endothelial growth factors (VEGFs) (28, 39). The molecular mechanisms that underlie cellular responses to hypoxic stress are therefore of considerable relevance to cancer biology and therapy.A key regulator of the cellular response to oxygen deprivation is the transcription factor, hypoxia-inducible factor 1 (HIF-1). Originally identified as an oxygen-responsive activator of erythropoietin gene transcription, HIF-1 is now known to play a central role in the maintenance of oxygen homeostasis in virtually all bodily tissues (42, 43). The predominant form of HIF-1 is a heterodimer consisting of HIF-1␣ and HIF-1␤ subunits, both of which are members of the basic helix-loop-helix family of transcription factors. Although HIF-1␤ is a constitutively expressed nuclear protein, the expression of the HIF-1␣ subunit is tightly coupled to the ambient oxygen tension. Under normoxic conditions, the HIF-1␣ gene is continuously transcribed and translated; however, the HIF-1␣ protein is expressed at very low levels due to rapid destruction via the ubiquitin-proteasome pathway. In addition to its DNA-binding and transactivating motifs, HIF-1␣ contains a stretch of ca. 200 amino acids, termed the oxygen-dependent degradation (ODD) domain. As its name implies, the ODD domain mediates the interaction between HIF-1␣ and the E3 ubiquitin ligase complex that mediates continuous poly ubiquitination of HIF-1␣ in normoxic cells.The oxygen-dependent turnover of HIF-1␣ is governed by a novel family of prolyl 4-hydroxylases (PHDs) that specifically modify HIF-1␣ at two conserved proline residues (Pro-402 and Pro-564), both located in the ODD domain (5,15,27,41). Prolyl hydroxylation triggers the recognition of HIF-1␣ by the product of the VHL tumor suppressor gene, which serves as the targeting subunit of an E3 ubiquitin ligase complex (20). Although the exact mechanism remains unclear, a decrease in ambient oxygen tension leads to a correlative decrease in HIF-1␣ prolyl hydroxylation, which in turn leads to decreased rates of HIF-1␣ polyubiquitination and...