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Download fileGlobal Snapshot of the Influence of Endocytosis upon EGF Receptor Signaling Output
journal contribution
posted on 2012-11-02, 00:00 authored by Jasminka Omerovic, Dean E. Hammond, Ian A. Prior, Michael J. ClagueTrafficking of activated receptors may dictate the signaling
output
through the exposure to a changing palette of substrates and effectors.
Here, we have used the acute application of a chemical inhibitor of
dynamin activity, Dynasore, to inhibit internalization of activated
EGF receptors together with quantitative mass spectrometry. This has
generated a global snapshot of phosphorylation associated changes,
which are contingent upon the endosomal trafficking of the activated
EGF receptor. Using a SILAC approach, we have been able to quantitate
>500 proteins in pTyr immunoprecipitation experiments and close
to
800 individual phosphopeptides through affinity based enrichment strategies.
This study provides >2 orders of magnitude increase in the coverage
of potential EGF effectors than hitherto assessed in the context of
endocytosis. There is a strong positive correlation between EGF responsiveness
and sensitivity to Dynasore, with ∼40% of EGF responses being
significantly changed by endocytic inhibition. Proteins which are
functionally linked to endosomal sorting are strongly influenced by
receptor entry, suggesting that the activated receptor can govern
its fate by influencing endosomal dynamics. However, the majority
of EGF-responsive enzymes which we quantify, do not exhibit this property.
Hence, our results provide many examples of key signaling proteins
that are impervious to EGFR receptor endocytosis but nevertheless
confirm the broad principle of endocytosis influence upon the network
response.
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Keywords
Global SnapshotEGF effectorsSILAC approachreceptor entrynetwork responseEGFR receptor endocytosismagnitude increasechemical inhibitormass spectrometryEGF receptorsEGF responsesendosomal dynamicsendosomal traffickingendocytosis influenceEGF responsivenessdynamin activitypTyr immunoprecipitation experimentsproteinEGF receptorenrichment strategiesEGF Receptor Signaling OutputTraffickingendocytic inhibition