Abstract
Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2, and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site compromised neither ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase.
Highlights
Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase–activated signaling cascades
Amino acid sequence analysis of the ErbB4 ICD revealed two ⌿KXE consensus motifs (PK1143QE and PK1181AE), a shorter KXE motif (GK1202AE) near the C terminus, and an inverted consensus motif DSK1002F adjacent to the kinase domain (Fig. 1A). To examine whether these lysine residues could serve as small ubiquitin-like modifier (SUMO) acceptor sites, we replaced them with arginines, cotransfected wild-type or lysine to arginine mutant soluble ErbB4 ICDs of CYT-2 type together with His6-tagged SUMO1 to COS-7 cells, and purified SUMOylated proteins using Ni2ϩNTA agarose under denaturing conditions to inactivate SUMO proteases [24]
The replacement of the three C-terminal lysines (K1143RϩK1181RϩK1202R; Cons 3KR) and the inverted motif lysine (K1002RϩK1143Rϩ K1181RϩK1202R; Cons 4KR) with arginines had no effect on content of SUMO-modified ErbB4 ICD (Fig. 1B), indicating that the lysine residue/residues targeted by SUMOylation are non-consensus sites
Summary
Regulated intramembrane proteolysis of cleavable ErbB4 isoforms releases a soluble intracellular domain that can localize in the cytoplasm, mitochondria, or nuclei, and that contains tyrosine kinase activity [18]. The K714R mutant ErbB4 ICD was ubiquitinated to a similar level as wild-type ICD when transfected to MCF-7 cells (supplemental Fig. S7), indicating similar susceptibility to ubiquitin-mediated targeting to proteasomes [34] Taken together, these data suggest that SUMOylation at Lys714 regulates accumulation of ErbB4 ICD in the nucleus, but not overall stability of the receptor in the cytoplasm or at the cell membrane. Similar results were obtained in coimmunoprecipitation experiments (supplemental Fig. S8) Taken together, these data indicate that nuclear localization of ErbB4 ICD is regulated by CRM1-dependent nuclear export, mediated by hydrophobic amino acids of the leucine-rich NES.
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