Jeannette Kunz

Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression

The yeast TOR2 gene encodes an essential 282 kd phosphatidylinositol (PI) 3-kinase homolog. TOR2 is related to the catalytic subunit of bovine PI 3-kinase and to yeast VPS34, a vacuolar sorting protein also shown to have PI 3-kinase activity. The immunosuppressant rapamycin most likely acts by inhibiting PI kinase activity because TOR2 mutations confer resistance to rapamycin and because a TOR1 TOR2 double disruption (TOR1 is a nonessential TOR2 homolog) confers G1 arrest, as does rapamycin. Our results further suggest that 3-phosphorylated phosphoinositides, whose physiological significance has not been determined, are an important signal in cell cycle activation. In yeast, this signal may act in a signal transduction pathway similar to the interleukin-2 signal transduction pathway in T cells.

Regulation of Dendritic Morphogenesis by Ras–PI3K–Akt–mTOR and Ras–MAPK Signaling Pathways

Dendritic arborization and spine formation are critical for the functioning of neurons. Although many proteins have been identified recently as regulators of dendritic morphogenesis, the intracellular signaling pathways that control these processes are not well understood. Here we report that the Ras–phosphatidylinositol 3-kinase (PI3K)–Akt–mammalian target of rapamycin (mTOR) signaling pathway plays pivotal roles in the regulation of many aspects of dendrite formation. Whereas the PI3K–Akt–mTOR pathway alone controlled soma and dendrite size, a coordinated activation together with the Ras-mitogen-activated protein kinase signaling pathway was required for increasing dendritic complexity. Chronic inhibition of PI3K or mTOR reduced soma and dendrite size and dendritic complexity, as well as density of dendritic filopodia and spines, whereas a short-term inhibition promoted the formation of mushroom-shaped spines on cells expressing constitutively active mutants of Ras, PI3K, or Akt, or treated with the upstream activator BDNF. Together, our data underscore the central role of a spatiotemporally regulated key cell survival and growth pathway on trophic regulation of the coordinated development of dendrite size and shape.

The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease.

The Saccharomyces cerevisiae targets of rapamycin, TOR1 and TOR2, signal activation of cell growth in response to nutrient availability. Loss of TOR or rapamycin treatment causes yeast cells to arrest growth in early G1 and to express several other physiological properties of starved (G0) cells. As part of this starvation response, high affinity amino acid permeases such as the tryptophan permease TAT2 are targeted to the vacuole and degraded. Here we show that the TOR signalling pathway phosphorylates the Ser/Thr kinase NPR1 and thereby inhibits the starvation‐induced turnover of TAT2. Overexpression of NPR1 inhibits growth and induces the degradation of TAT2, whereas loss of NPR1 confers resistance to rapamycin and to FK506, an inhibitor of amino acid import. NPR1 is controlled by TOR and the type 2A phosphatase‐associated protein TAP42. First, overexpression of NPR1 is toxic only when TOR function is reduced. Secondly, NPR1 is rapidly dephosphorylated in the absence of TOR. Thirdly, NPR1 dephosphorylation does not occur in a rapamycin‐resistant tap42 mutant. Thus, the TOR nutrient signalling pathway also controls growth by inhibiting a stationary phase (G0) programme. The control of NPR1 by TOR is analogous to the control of p70 s6 kinase and 4E‐BP1 by mTOR in mammalian cells.

Phosphatidylinositol phosphate kinases, a multifaceted family of signaling enzymes.

The importance of phosphoinositides as lipid signaling molecules in eucaryotic cells was first recognized by Lowell and Mabel Hokin in the 1950s (who also discovered the enzyme activities that phosphorylate phosphatidylinositol (PI)) (1–5). Since those early years, PI signaling pathways have expanded both in importance and complexity. The classical pathway transforms PI to PI-4,5-P2 by the successive actions of PI 4-kinases and PI-4-P 5-kinases. PI4,5-P2 is the precursor for second messengers and also acts directly to modify effectors, for example actin-binding proteins (6–9). Significant roles for other phosphoinositide lipid products in signaling, combined with recently identified lipid kinase activities, are illuminating the many mechanisms by which cells use lipid messengers (10–13). This review will focus on the phosphatidylinositolphosphate kinase (PIPK) family, which has the ability to synthesize all known PIP2 isomers and PIP3.

The v-ATPase V0 Subunit a1 Is Required for a Late Step in Synaptic Vesicle Exocytosis in Drosophila

The V(0) complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. We have isolated mutations in the largest V(0) component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is only required in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. Our data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion.

Cyclosporin A, FK506 and rapamycin: more than just immunosuppression.

The mechanisms of action of the immunosuppressive drugs cyclosporin A (CsA), FK506 and rapamycin are strikingly conserved from yeast to human T cells. Recent results obtained with yeast corroborate calcineurin as the target of CsA-cyclophilin and FK506-FKBP complexes, and reveal a phosphatidylinositol 3-kinase homologue as the target of the rapamycin-FKBP complex.

The Activation Loop of Phosphatidylinositol Phosphate Kinases Determines Signaling Specificity

Phosphatidylinositol-4,5-bisphosphate plays a pivotal role in the regulation of cell proliferation and survival, cytoskeletal reorganization, and membrane trafficking. However, little is known about the temporal and spatial regulation of its synthesis. Higher eukaryotic cells have the potential to use two distinct pathways for the generation of phosphatidylinositol-4,5-bisphosphate. These pathways require two classes of phosphatidylinositol phosphate kinases, termed type I and type II PIP kinases. While highly related by sequence, these kinases localize to different subcellular compartments, phosphorylate distinct substrates, and are functionally nonredundant. Here, we show that a 20- to 25-amino acid loop spanning the catalytic site, termed the activation loop, determines both enzymatic specificity and subcellular targeting of PIP kinases. Therefore, the activation loop controls signaling specificity and PIP kinase function at multiple levels.

Focal adhesion disassembly requires clathrin-dependent endocytosis of integrins

Cell migration requires the controlled disassembly of focal adhesions, but the underlying mechanisms remain poorly understood. Here, we show that adhesion turnover is mediated through dynamin‐ and clathrin‐dependent endocytosis of activated β1 integrins. Consistent with this, clathrin and the clathrin adaptors AP‐2 and disabled‐2 (DAB2) distribute along with dynamin 2 to adhesion sites prior to adhesion disassembly. Moreover, knockdown of either dynamin 2 or both clathrin adaptors blocks β1 integrin internalization, leading to impaired focal adhesion disassembly and cell migration. Together, these results provide important insight into the mechanisms underlying adhesion disassembly and identify novel components of the disassembly pathway.

HEAT repeats mediate plasma membrane localization of Tor2p in yeast

The subcellular distribution of Tor1p and Tor2p, two phosphatidylinositol kinase homologs and targets of the immunosuppressive drug rapamycin in Saccharomyces cerevisiae, was analyzed. We found that Tor protein is peripherally associated with membranes. Subcellular fractionation and immunofluorescence studies showed that Tor1p and Tor2p associate with the plasma membrane and a second fraction that is distinct from Golgi, vacuoles, mitochondria, and nucleus and may represent vesicular structures. Pulse-chase experiments showed that association of Tor protein with plasma membrane and the second compartment is fast, does not appear to involve components of endocytic, secretory, or Golgi to vacuole transport pathways, and is not affected by the immunosuppressive drug rapamycin. Deletion analysis reveals that two domains within Tor2p independently mediate localization to both compartments. These domains are composed of HEAT repeats that are thought to act as protein-protein interaction surfaces. Our studies therefore place Tor proteins at the site of action of their known downstream effectors and suggest that they may be part of a multiprotein complex.

Coupled Inositide Phosphorylation and Phospholipase D Activation Initiates Clathrin-coat Assembly on Lysosomes*

Adaptors appear to control clathrin-coat assembly by determining the site of lattice polymerization but the nucleating events that target soluble adaptors to an appropriate membrane are poorly understood. Using an in vitro model system that allows AP-2-containing clathrin coats to assemble on lysosomes, we show that adaptor recruitment and coat initiation requires phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis. PtdIns(4,5)P2 is generated on lysosomes by the sequential action of a lysosome-associated type II phosphatidylinositol 4-kinase and a soluble type I phosphatidylinositol 4-phosphate 5-kinase. Phosphatidic acid, which potently stimulates type I phosphatidylinositol 4-phosphate 5-kinase activity, is generated on the bilayer by a phospholipase D1-like enzyme located on the lysosomal surface. Quenching phosphatidic acid function with primary alcohols prevents the synthesis of PtdIns(4,5)P2 and blocks coat assembly. Generating phosphatidic acid directly on lysosomes with exogenous bacterial phospholipase D in the absence of ATP still drives adaptor recruitment and limited coat assembly, indicating that PtdIns(4,5)P2functions, at least in part, to activate the PtdIns(4,5)P2-dependent phospholipase D1. These results provide the first direct evidence for the involvement of anionic phospholipids in clathrin-coat assembly on membranes and define the enzymes responsible for the production of these important lipid mediators.