Quantitative Proteomics of the SMAD (Suppressor of Mothers against Decapentaplegic) Transcription Factor Family Identifies Importin 5 as a Bone Morphogenic Protein Receptor SMAD-specific Importin

Roy Baas,Ayestha Sijm,Hetty A.A.M Van Teeffelen,Robert Van Es,Harmjan R Vos,H Th Marc Timmers

doi:10.1074/jbc.m116.748582

Abstract

Gene-specific transcription factors (GSTFs) control gene transcription by DNA binding and specific protein complex recruitment, which regulates promoter accessibility for transcription initiation by RNA polymerase II. Mutations in the GSTFs Suppressor of Mothers Against Decapentaplegic 2 (SMAD2) and SMAD4 are frequently associated with colon and rectal carcinomas. These proteins play an important role in bone morphogenic protein (BMP) and transforming growth factor β (TGF-β) signaling pathways controlling cell fate and proliferation. To study the protein interactome of the SMAD protein family we generated a quantitative proteomics pipeline that allows for inducible expression of GFP-tagged SMAD proteins followed by affinity purification and quantitative mass spectrometry analysis. Data are available via ProteomeXchange with identifier PXD004529. The nuclear importin IPO5 was identified as a novel interacting protein of SMAD1. Overexpression of IPO5 in various cell lines specifically increases nuclear localization of BMP receptor-activated SMADs (R-SMADs) confirming a functional relationship between IPO5 and BMP but not TGF-β R-SMADs. Finally, we provide evidence that variation in length of the lysine stretch of the nuclear localization sequence is a determinant for importin specificity.

Highlights

Gene-specific transcription factors (GSTFs) control gene transcription by DNA binding and specific protein complex recruitment, which regulates promoter accessibility for transcription initiation by RNA polymerase II
Mutation experiments extending the lysine stretch of the nuclear localization signal (NLS) of SMAD3 shows that the length of the lysine stretch is involved in differentiating IPO5 and IPO7 as importins for the bone morphogenic protein (BMP) and TGF-␤ subgroup of SMAD transcription factors
Partially active TGF-␤ and BMP signaling pathways are consistent with detection of GFP-SMAD proteins both in the cytoplasm and nucleus (Fig. 2)

Summary

Results

GFP-SMAD Cell Line Generation—To study the interactome of the human SMAD proteins, stable doxycycline-inducible HeLa cell lines containing GFP-SMAD fusion proteins or GFP without fusion partner as control were generated using the Flp-In T-Rex system (supplemental Fig. S1). Phosphorylated GFP-SMAD2 and endogenous SMAD2 was detected in treated and untreated conditions suggesting the TGF-␤ pathway is (partially) active under normal HeLa cell culture conditions (Fig. 3A). Partially active TGF-␤ and BMP signaling pathways are consistent with detection of GFP-SMAD proteins both in the cytoplasm and nucleus (Fig. 2) These data show that we generated stable inducible HeLa cell lines expressing functional GFP-SMAD proteins. SMAD Interactome Identification Using Mass Spectrometry—Localization of GFP-SMAD proteins to both the cytoplasm and nucleus suggests different pools of proteins with different biological activity and/or interaction partners To systematically study these different SMAD pools we prepared cytoplasmic and nuclear extracts from the GFP-SMAD cell lines after 16 –20 h of doxycycline induction (Fig. 4A). 108 affinity purifications were performed and all data files were

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Discussion

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Experimental Procedures

Primers used for cloning