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Human VPS34 is required for internal vesicle formation within multivesicular endosomes.

Futter CE, Collinson LM, Backer JM, Hopkins CR - J. Cell Biol. (2001)

Bottom Line: In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation.Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs.Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Ophthalmology, University College London, London EC1V 9EL, United Kingdom.

ABSTRACT
After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3'-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3'-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3'-kinase activity, whereas vesicle formation requires PI 3'-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

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The effects of wortmannin on EGF-stimulated tyrosine phosphorylation in cells were the lysosomes have been cross-linked. HEp-2 cells were incubated with HRP for 30 min at 37°C, chased for 3 h at 37°C, and then incubated with DAB/H2O2 at 4°C to crosslink the lysosomes. Cells were then incubated in the presence or absence of EGF at 20°C for 1 h and then chased at 37°C in the presence or absence of wortmannin (wo). Cell lysates were analyzed by SDS-PAGE followed by Western blotting with antiphosphotyrosine antibody.
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fig8: The effects of wortmannin on EGF-stimulated tyrosine phosphorylation in cells were the lysosomes have been cross-linked. HEp-2 cells were incubated with HRP for 30 min at 37°C, chased for 3 h at 37°C, and then incubated with DAB/H2O2 at 4°C to crosslink the lysosomes. Cells were then incubated in the presence or absence of EGF at 20°C for 1 h and then chased at 37°C in the presence or absence of wortmannin (wo). Cell lysates were analyzed by SDS-PAGE followed by Western blotting with antiphosphotyrosine antibody.

Mentions: The above data suggests that EGFRs carry information that allows them to be retained on the perimeter membrane of MVB while TRs are being removed from this location to the recycling pathway, and that retention of EGFRs on the perimeter membrane is sufficient to target them for lysosomal delivery. This raises the question of the purpose of inward vesiculation. One possible purpose could be the removal of EGFRs from exposure to the cytoplasmic matrix and hence from substrates for their kinase activity. Therefore, we determined the effects of inhibition of inward vesiculation on the spectrum of proteins phosphorylated by the EGFR kinase. To enhance potential differences, lysosomes were cross-linked to prevent EGFR degradation, and then cells were incubated with EGF at 20°C followed by chase at 37°C in the presence or absence of wortmannin. MVBs with EGFRs on the perimeter membrane and exposed to the cytoplasm (in the presence of wortmannin) could be compared with MVBs with EGFRs on internal vesicles sequestered from the cytoplasm (in the absence of wortmannin). Tyrosine-phosphorylated proteins were detected by Western blotting cell lysates with antiphosphotyrosine antibody. As shown in Fig. 8, the pattern and strength of phosphorylation is increased significantly in wortmannin-treated cells. Since there is some loss of EGFRs from the perimeter membrane of MVBs in wortmannin-treated cells in which the lysosomes have been cross-linked, we cannot be sure that all the tyrosine-phosphorylated proteins are on the perimeter membrane of MVBs in wortmannin-treated cells. However, these data are consistent with the possibility that inward vesiculation within MVBs removes EGFRs from potential cytoplasmic substrates.


Human VPS34 is required for internal vesicle formation within multivesicular endosomes.

Futter CE, Collinson LM, Backer JM, Hopkins CR - J. Cell Biol. (2001)

The effects of wortmannin on EGF-stimulated tyrosine phosphorylation in cells were the lysosomes have been cross-linked. HEp-2 cells were incubated with HRP for 30 min at 37°C, chased for 3 h at 37°C, and then incubated with DAB/H2O2 at 4°C to crosslink the lysosomes. Cells were then incubated in the presence or absence of EGF at 20°C for 1 h and then chased at 37°C in the presence or absence of wortmannin (wo). Cell lysates were analyzed by SDS-PAGE followed by Western blotting with antiphosphotyrosine antibody.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199316&req=5

fig8: The effects of wortmannin on EGF-stimulated tyrosine phosphorylation in cells were the lysosomes have been cross-linked. HEp-2 cells were incubated with HRP for 30 min at 37°C, chased for 3 h at 37°C, and then incubated with DAB/H2O2 at 4°C to crosslink the lysosomes. Cells were then incubated in the presence or absence of EGF at 20°C for 1 h and then chased at 37°C in the presence or absence of wortmannin (wo). Cell lysates were analyzed by SDS-PAGE followed by Western blotting with antiphosphotyrosine antibody.
Mentions: The above data suggests that EGFRs carry information that allows them to be retained on the perimeter membrane of MVB while TRs are being removed from this location to the recycling pathway, and that retention of EGFRs on the perimeter membrane is sufficient to target them for lysosomal delivery. This raises the question of the purpose of inward vesiculation. One possible purpose could be the removal of EGFRs from exposure to the cytoplasmic matrix and hence from substrates for their kinase activity. Therefore, we determined the effects of inhibition of inward vesiculation on the spectrum of proteins phosphorylated by the EGFR kinase. To enhance potential differences, lysosomes were cross-linked to prevent EGFR degradation, and then cells were incubated with EGF at 20°C followed by chase at 37°C in the presence or absence of wortmannin. MVBs with EGFRs on the perimeter membrane and exposed to the cytoplasm (in the presence of wortmannin) could be compared with MVBs with EGFRs on internal vesicles sequestered from the cytoplasm (in the absence of wortmannin). Tyrosine-phosphorylated proteins were detected by Western blotting cell lysates with antiphosphotyrosine antibody. As shown in Fig. 8, the pattern and strength of phosphorylation is increased significantly in wortmannin-treated cells. Since there is some loss of EGFRs from the perimeter membrane of MVBs in wortmannin-treated cells in which the lysosomes have been cross-linked, we cannot be sure that all the tyrosine-phosphorylated proteins are on the perimeter membrane of MVBs in wortmannin-treated cells. However, these data are consistent with the possibility that inward vesiculation within MVBs removes EGFRs from potential cytoplasmic substrates.

Bottom Line: In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation.Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs.Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Ophthalmology, University College London, London EC1V 9EL, United Kingdom.

ABSTRACT
After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3'-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3'-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3'-kinase activity, whereas vesicle formation requires PI 3'-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

Show MeSH
Related in: MedlinePlus