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PubMed Central
Other literature type . 2019
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Affinity Purification of NF1 Protein–Protein Interactors Identifies Keratins and Neurofibromin Itself as Binding Partners

Authors: Rachel M. Carnes; Robert A. Kesterson; Bruce R. Korf; James A. Mobley; Deeann Wallis;

Affinity Purification of NF1 Protein–Protein Interactors Identifies Keratins and Neurofibromin Itself as Binding Partners

Abstract

Neurofibromatosis Type 1 (NF1) is caused by pathogenic variants in the NF1 gene encoding neurofibromin. Definition of NF1 protein–protein interactions (PPIs) has been difficult and lacks replication, making it challenging to define binding partners that modulate its function. We created a novel tandem affinity purification (TAP) tag cloned in frame to the 3’ end of the full-length murine Nf1 cDNA (mNf1). We show that this cDNA is functional and expresses neurofibromin, His-Tag, and can correct p-ERK/ERK ratios in NF1 null HEK293 cells. We used this affinity tag to purify binding partners with Strep-Tactin®XT beads and subsequently, identified them via mass spectrometry (MS). We found the tagged mNf1 can affinity purify human neurofibromin and vice versa, indicating that neurofibromin oligomerizes. We identify 21 additional proteins with high confidence of interaction with neurofibromin. After Metacore network analysis of these 21 proteins, eight appear within the same network, primarily keratins regulated by estrogen receptors. Previously, we have shown that neurofibromin levels negatively regulate keratin expression. Here, we show through pharmacological inhibition that this is independent of Ras signaling, as the inhibitors, selumetinib and rapamycin, do not alter keratin expression. Further characterization of neurofibromin oligomerization and binding partners could aid in discovering new neurofibromin functions outside of Ras regulation, leading to novel drug targets.

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Keywords

Neurofibromin 1, affinity purification, protein–protein interactors, neurofibromin, immunoprecipitation, binding partners, Article, neurofibromatosis Type I, HEK293 Cells, keratins, Humans, Keratins, Protein Multimerization, Ras, mass spectrometry, Protein Binding

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citations
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
16
Top 10%
Average
Top 10%
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gold