Phosphatidylinositol 3-Kinase Encoded by Yeast
            <i>VPS</i>
            34 Gene Essential for Protein Sorting

Schu, Peter; Takegawa, Kaoru; Fry, Michael; Stack, Jeffrey H.; Waterfield, Michael D.; Emr, Scott D.

doi:10.1126/science.8385367

Cited by 906 publications

(764 citation statements)

References 32 publications

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“…Full length ATR, together with a mutant sequence which results in the introduction of the alteration D2494E in the kinase domain (Schu et al, 1993), were cloned into the mammalian expression vector pEBG-2T, which contains a GST tag (Sanchez et al, 1994). HEK293 cells were transfected by a calcium phosphate-mediated protocol and extracts subjected to glutathione-agarose puri®cation (see Materials and methods).…”

Section: Expression Of Atr In Hek293 Cells and Isolation By Glutathiomentioning

confidence: 99%

ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK

et al. 1999

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ATR is a large, 4300 kDa protein containing a carboxy-terminus kinase domain related to PI-3 kinase, and is homologous to the ATM gene product in human cells and the rad3/MEC1 proteins in yeast. These proteins, together with the DNA-PK, are part of a new family of PI-3 kinase related proteins. All members of this family play important roles in checkpoints which operate to permit cell survival following many forms of DNA damage. We have expressed ATR protein in HEK293 cells and puri®ed the protein to near-homogeneity. We show that pure ATR is a protein kinase which is activated by circular single-stranded, doublestranded or linear DNA. Thus ATR is a new member of a sub-family of PIK related kinases, founded by the DNA-PK, which are activated in the presence of DNA. Unlike DNA-PK, ATR does not appear to require Ku proteins for its activation by DNA. We show directly that, like ATM and DNA-PK, ATR phosphorylates the genome surveillance protein p53 on serine 15, a site which is up-regulated in response to DNA damage. In addition, we ®nd that ATR has a substrate speci®city similar to, but unique from, the DNA-PK in vitro, suggesting that these proteins have overlapping but distinct functions in vivo. Finally, we ®nd that the kinase activity of ATR in the presence and absence of DNA is suppressed by ca eine, a compound which is known to induce loss of checkpoint control. Our results are consistent with the notion that ATR plays a role in monitoring DNA structure and phosphorylation of proteins involved in the DNA damage response pathways.

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Section: Expression Of Atr In Hek293 Cells and Isolation By Glutathiomentioning

confidence: 99%

ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK

et al. 1999

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“…The yeast homologue of the p110 catalytic subunit of the mammalian PtdIns 3-kinase, VPS34p, is a lipid kinase with substrate specificity towards PtdIns. It cannot form PtdIns3,4P 2 or PtdIns3,4,5P 3 , and VPS34 mutants are disturbed in membrane flow (Schu et al, 1993;Stack & Emr, 1994). Therefore, the important factor here seems to be PtdIns3P, since the two other lipid molecules are absent in yeast cells.…”

Section: Autophagy and Signal Transductionmentioning

confidence: 99%

Untitled

Blommaart

1997

The Histochemical Journal

204

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SummaryThe rate of proteolysis is an important determinant of the intracellular protein content. Part of the degradation of intracellular proteins occurs in the lysosomes and is mediated by macroautophagy. In liver, macroautophagy is very active and almost completely accounts for starvation-induced proteolysis. Factors inhibiting this process include amino acids, cell swelling and insulin. In the mechanisms controlling macroautophagy, protein phosphorylation plays an important role. Activation of a signal transduction pathway, ultimately leading to phosphorylation of ribosomal protein S6, accompanies inhibition of macroautophagy. Components of this pathway may include a heterotrimeric G i3 -protein, phosphatidylinositol 3-kinase and p70S6 kinase. Recent evidence indicates that lysosomal protein degradation can be selective and occurs via ubiquitin-dependent and -independent pathways.

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“…An important advance in the study of protein sorting was the discovery that phosphatidylinositol 3-kinase (PI 3-kinase), which phosphorylates phosphatidylinositol to phosphatidylinositol 3-phosphate (PtdIns3P), is required for the transport of membrane proteins to the yeast vacuole [11]. Subsequent work with intact mammalian cells showed that pharmacologic inhibition of PI 3-kinase by the drugs LY294002 or wortmannin inhibits the intracellular trafficking of receptors, including the recycling of transferrin receptors [12][13][14][15] and the degradation of PDGF receptors [16,17].…”

Section: Introductionmentioning

confidence: 99%

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

Awwad

Iyer

Rosenfeld

et al. 2007

Experimental Cell Research

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Phosphatidylinositol 3-kinase inhibitors have been shown to affect the endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated β 2 -adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate β 2 -adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the β-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of β 2 -adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of β 2 -adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of β 2 -adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.

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Phosphatidylinositol 3-Kinase Encoded by Yeast VPS 34 Gene Essential for Protein Sorting

Cited by 906 publications

References 32 publications

ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK

ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK

Untitled

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

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