Abstract
The Hey protein family, comprising Hey1, Hey2 and HeyL in mammals, conveys Notch signals in many cell types. The helix-loop-helix (HLH) domain as well as the Orange domain, mediate homo- and heterodimerization of these transcription factors. Although distinct interaction partners have been identified so far, their physiological relevance for Hey functions is still largely unclear. Using a tandem affinity purification approach and mass spectrometry analysis we identified members of an ubiquitin E3-ligase complex consisting of FBXO45, PAM and SKP1 as novel Hey1 associated proteins. There is a direct interaction between Hey1 and FBXO45, whereas FBXO45 is needed to mediate indirect Hey1 binding to SKP1. Expression of Hey1 induces translocation of FBXO45 and PAM into the nucleus. Hey1 is a short-lived protein that is degraded by the proteasome, but there is no evidence for FBXO45-dependent ubiquitination of Hey1. On the contrary, Hey1 mediated nuclear translocation of FBXO45 and its associated ubiquitin ligase complex may extend its spectrum to additional nuclear targets triggering their ubiquitination. This suggests a novel mechanism of action for Hey bHLH factors.
Highlights
The mammalian Hey protein family comprises a small group of highly conserved basic helixloop-helix transcription factors with three members: Hey1, Hey2 and HeyL
Gel lanes were cut into 23 slices and after tryptic digest protein fragments were analyzed by mass spectrometry
We focused on proteins that were identified in both co-purification experiments (S1 Table)
Summary
The mammalian Hey protein family comprises a small group of highly conserved basic helixloop-helix (bHLH) transcription factors with three members: Hey, Hey and HeyL. They play essential roles in cardiovascular development [1] and during epithelial-to-mesenchymal transition [2], neural development [3, 4], myogenesis [5] and bone development [6]. Hey proteins are closely related to the D. melanogaster hairy and E(spl) proteins They show high structural similarity with the Hes protein family, especially in the DNA binding basic domain as well as the HLH and Orange domains that mediate homo- and heterodimerization. Hey proteins are further characterized by two conserved peptide motifs—YRPW and PLOS ONE | DOI:10.1371/journal.pone.0130288 June 12, 2015
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