Jonathan M. Backer

Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation.

IRS‐1 undergoes rapid tyrosine phosphorylation during insulin stimulation and forms a stable complex containing the 85 kDa subunit (p85) of the phosphatidylinositol (PtdIns) 3′‐kinase, but p85 is not tyrosyl phosphorylated. IRS‐1 contains nine tyrosine phosphorylation sites in YXXM (Tyr‐Xxx‐Xxx‐Met) motifs. Formation of the IRS‐1‐PtdIns 3′‐kinase complex in vitro is inhibited by synthetic peptides containing phosphorylated YXXM motifs, suggesting that the binding of PtdIns 3′‐kinase to IRS‐1 is mediated through the SH2 (src homology‐2) domains of p85. Furthermore, overexpression of IRS‐1 potentiates the activation of PtdIns 3‐kinase in insulin‐stimulated cells, and tyrosyl phosphorylated IRS‐1 or peptides containing phosphorylated YXXM motifs activate PtdIns 3′‐kinase in vitro. We conclude that the binding of tyrosyl phosphorylated IRS‐1 to the SH2 domains of p85 is the critical step that activates PtdIns 3′‐kinase during insulin stimulation.

Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1- phosphatidylinositol 3-kinase complex

Beclin 1, a mammalian autophagy protein that has been implicated in development, tumour suppression, neurodegeneration and cell death, exists in a complex with Vps34, the class III phosphatidylinositol-3-kinase (PI(3)K) that mediates multiple vesicle-trafficking processes including endocytosis and autophagy. However, the precise role of the Beclin 1–Vps34 complex in autophagy regulation remains to be elucidated. Combining mouse genetics and biochemistry, we have identified a large in vivo Beclin 1 complex containing the known proteins Vps34, p150/Vps15 and UVRAG, as well as two newly identified proteins, Atg14L (yeast Atg14-like) and Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein). Characterization of the new proteins revealed that Atg14L enhances Vps34 lipid kinase activity and upregulates autophagy, whereas Rubicon reduces Vps34 activity and downregulates autophagy. We show that Beclin 1 and Atg14L synergistically promote the formation of double-membraned organelles that are associated with Atg5 and Atg12, whereas forced expression of Rubicon results in aberrant late endosomal/lysosomal structures and impaired autophagosome maturation. We hypothesize that by forming distinct protein complexes, Beclin 1 and its binding proteins orchestrate the precise function of the class III PI(3)K in regulating autophagy at multiple steps.

The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling.

GRB2, a small protein comprising one SH2 domain and two SH3 domains, represents the human homologue of the Caenorhabditis elegans protein, sem‐5. Both GRB2 and sem‐5 have been implicated in a highly conserved mechanism that regulates p21ras signalling by receptor tyrosine kinases. In this report we show that in response to insulin, GRB2 forms a stable complex with two tyrosine‐phosphorylated proteins. One protein is the major insulin receptor substrate IRS‐1 and the second is the SH2 domain‐containing oncogenic protein, Shc. The interactions between GRB2 and these two proteins require ligand activation of the insulin receptor and are mediated by the binding of the SH2 domain of GRB2 to phosphotyrosines on both IRS‐1 and Shc. Although GRB2 associates with IRS‐1 and Shc, it is not tyrosine‐phosphorylated after insulin stimulation, implying that GRB2 is not a substrate for the insulin receptor. Furthermore, we have identified a short sequence motif (YV/IN) present in IRS‐1, EGFR and Shc, which specifically binds the SH2 domain of GRB2 with high affinity. Interestingly, both GRB2 and phosphatidylinositol‐3 (PI‐3) kinase can simultaneously bind distinct tyrosine phosphorylated regions on the same IRS‐1 molecule, suggesting a mechanism whereby IRS‐1 could provide the core for a large signalling complex. We propose a model whereby insulin stimulation leads to formation of multiple protein‐‐protein interactions between GRB2 and the two targets IRS‐1 and Shc. These interactions may play a crucial role in activation of p21ras and the control of downstream effector molecules.

Phosphatidylinositol-3-OH kinases are Rab5 effectors

*Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse, 01307 Dresden, Germany †European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany ‡Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA §Ludwig Institute of Cancer Research, University College London, Riding House Street, London W1P 8BT, UK ¶e-mail: zerial@embl-heidelberg.de

hVps34 Is a Nutrient-regulated Lipid Kinase Required for Activation of p70 S6 Kinase

Mammalian cells respond to nutrient deprivation by inhibiting energy consuming processes, such as proliferation and protein synthesis, and by stimulating catabolic processes, such as autophagy. p70 S6 kinase (S6K1) plays a central role during nutritional regulation of translation. S6K1 is activated by growth factors such as insulin, and by mammalian target of rapamycin (mTOR), which is itself regulated by amino acids. The Class IA phosphatidylinositol (PI) 3-kinase plays a well recognized role in the regulation of S6K1. We now present evidence that the Class III PI 3-kinase, hVps34, also regulates S6K1, and is a critical component of the nutrient sensing apparatus. Overexpression of hVps34 or the associated hVps15 kinase activates S6K1, and insulin stimulation of S6K1 is blocked by microinjection of inhibitory anti-hVps34 antibodies, overexpression of a FYVE domain construct that sequesters the hVps34 product PI(3)P, or small interfering RNA-mediated knock-down of hVps34. hVps34 is not part of the insulin input to S6K1, as it is not stimulated by insulin, and inhibition of hVps34 has no effect on phosphorylation of Akt or TSC2 in insulin-stimulated cells. However, hVps34 is inhibited by amino acid or glucose starvation, suggesting that it lies on the nutrient-regulated pathway to S6K1. Consistent with this, hVps34 is also inhibited by activation of the AMP-activated kinase, which inhibits mTOR/S6K1 in glucose-starved cells. hVps34 appears to lie upstream of mTOR, as small interfering RNA knock-down of hVps34 inhibits the phosphorylation of another mTOR substrate, eIF4E-binding protein-1 (4EBP1). Our data suggest that hVps34 is a nutrient-regulated lipid kinase that integrates amino acid and glucose inputs to mTOR and S6K1.

The regulation and function of Class III PI3Ks: novel roles for Vps34.

The Class III PI3K (phosphoinositide 3-kinase), Vps34 (vacuolar protein sorting 34), was first described as a component of the vacuolar sorting system in Saccharomyces cerevisiae and is the sole PI3K in yeast. The homologue in mammalian cells, hVps34, has been studied extensively in the context of endocytic sorting. However, hVps34 also plays an important role in the ability of cells to respond to changes in nutrient conditions. Recent studies have shown that mammalian hVps34 is required for the activation of the mTOR (mammalian target of rapamycin)/S6K1 (S6 kinase 1) pathway, which regulates protein synthesis in response to nutrient availability. In both yeast and mammalian cells, Class III PI3Ks are also required for the induction of autophagy during nutrient deprivation. Finally, mammalian hVps34 is itself regulated by nutrients. Thus Class III PI3Ks are implicated in the regulation of both autophagy and, through the mTOR pathway, protein synthesis, and thus contribute to the integration of cellular responses to changing nutritional status.

Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation

Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3′-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.

Regulation of the p85/p110 Phosphatidylinositol 3′-Kinase: Stabilization and Inhibition of the p110α Catalytic Subunit by the p85 Regulatory Subunit

ABSTRACT We propose a novel model for the regulation of the p85/p110α phosphatidylinositol 3′-kinase. In insect cells, the p110α catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110α is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110α/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110α. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110α dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110α or by the growth of the HEK 293T cells at 30°C. These nonspecific interventions mimicked the effects of p85 on p110α, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110α in mammalian cells at 30°C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110α. Thus, we have experimentally distinguished two effects of p85 on p110α: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110α is both stabilized and inhibited by dimerization with p85.

Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity

Tyrosyl phosphorylation is implicated in the mechanism of insulin action. Mutation of the beta-subunit of the insulin receptor by substitution of tyrosyl residue 960 with phenylalanine had no effect on insulin-stimulated autophosphorylation or phosphotransferase activity of the purified receptor. However, unlike the normal receptor, this mutant was not biologically active in Chinese hamster ovary cells. Furthermore, insulin-stimulated tyrosyl phosphorylation of at least one endogenous substrate (pp185) was increased significantly in cells expressing the normal receptor but was barely detected in cells expressing the mutant. Therefore, beta-subunit autophosphorylation was not sufficient for the insulin response, and a region of the insulin receptor around Tyr-960 may facilitate phosphorylation of cellular substrates required for transmission of the insulin signal.

The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila

Degradation of cytoplasmic components by autophagy requires the class III phosphatidylinositol 3 (PI(3))–kinase Vps34, but the mechanisms by which this kinase and its lipid product PI(3) phosphate (PI(3)P) promote autophagy are unclear. In mammalian cells, Vps34, with the proautophagic tumor suppressors Beclin1/Atg6, Bif-1, and UVRAG, forms a multiprotein complex that initiates autophagosome formation. Distinct Vps34 complexes also regulate endocytic processes that are critical for late-stage autophagosome-lysosome fusion. In contrast, Vps34 may also transduce activating nutrient signals to mammalian target of rapamycin (TOR), a negative regulator of autophagy. To determine potential in vivo functions of Vps34, we generated mutations in the single Drosophila melanogaster Vps34 orthologue, causing cell-autonomous disruption of autophagosome/autolysosome formation in larval fat body cells. Endocytosis is also disrupted in Vps34−/− animals, but we demonstrate that this does not account for their autophagy defect. Unexpectedly, TOR signaling is unaffected in Vps34 mutants, indicating that Vps34 does not act upstream of TOR in this system. Instead, we show that TOR/Atg1 signaling regulates the starvation-induced recruitment of PI(3)P to nascent autophagosomes. Our results suggest that Vps34 is regulated by TOR-dependent nutrient signals directly at sites of autophagosome formation.