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.

Keywords: Ras; affinity purification; binding partners; immunoprecipitation; keratins; mass spectrometry; neurofibromatosis Type I; neurofibromin; protein–protein interactors.

Figure 1

(A). Amino acid sequence of tandem affinity purification (TAP) tag cloned in frame with 3’ end of mNf1 cDNA and empty vector control. Blue letters represent the TEV cleavage site. Green letters indicate StrepII tag. Red letters indicate 6XHis tag. (B). Western blots showing neurofibromin protein levels in wildtype (WT, left) and NF1 null HEK293 cells (right). Cells were transfected with either the Empty Vector (EV) or the tagged mNf1 cDNA (WT) and probed with antibodies indicated. The first row of immunoblots shows the neurofibromin levels. The second row shows the functional His tag in only the lanes transfected with mNf1 (WT). The third row shows pERK. Total ERK levels are shown in the fourth row. Fifth row shows α-Tubulin for normalization of NF1 levels.

Figure 2

(A). The top row details the amino acid sequence of the fragment of human NF1 protein that affinity purified from WT HEK293 cells with the tagged mNf1 cDNA. The second row depicts the homologous mouse neurofibromin protein sequence and highlights the non-conserved amino acid residue T2489A. (B). Reciprocal immunoprecipitation of the tagged murine and human cDNAs. The left-hand side shows blots of input lysates after cell transfections with each indicated cDNA prior to affinity purification. The right-hand side shows blots after affinity purification. The left two columns show the Strep tag affinity purification and the right two columns show the Halo tag affinity purification. WT indicates the co-transfection of both cDNAs into HEK293 NF1 null cells; while EV indicates the empty vector transfection into the same cells. The first row of immunoblots is the visualization of neurofibromin. The second row shows the His tag from the mNf1 cDNA. The third row depicts the Halo tag from the hNF1 cDNA. The His tag is visible in the Halo immunoprecipitation WT lane and the Halo tag is visible in the Strep tag affinity purification WT lane, indicating that the two tagged neurofibromins are purifying the reciprocal neurofibromin when the individual affinity purification is completed.

Figure 3

Metacore network analysis identifies eight of the top 21 interactors within the same estrogen receptor protein network.

Figure 4

Keratin expression is unaffected by inhibition of Ras signaling pathways. NF1 null HEK293 cells were treated in dose response indicated with selumetinib or rapamycin and KRT8 levels (first row of bands) were evaluated by Western blot and normalized by α-tubulin (second row of bands). p-ERK/ERK ratios were also evaluated in response to selumetinib and pS6/S6 ratios in response to rapamycin to show drug efficacy (third and fourth rows of bands). Blots show an individual representative experiment. There is no statistical difference in KRT8 levels after N ≥ 3 represented in the histogram below the blots. p-ERK/ERK and pS6/S6 respond positively to selumetinib and rapamycin, respectively. Black bars indicate KRT8/tubulin ratios after treatment and white bars represent p-ERK/ERK or pS6/S6 ratios after treatment. (Rapamycin is toxic at doses higher than 100 nM). Error bars represent SEM.