SUMOylation regulates nuclear accumulation and signaling activity of the soluble intracellular domain of the ErbB4 receptor tyrosine kinase

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.

Keywords: ErbB4; HER4; RTK; breast cancer; nuclear transport; posttranslational modification (PTM); protein export; receptor tyrosine kinase; regulated intramembrane proteolysis (RIP); small ubiquitin-like modifier (SUMO).

Figure 1.

Lys-714 is the major SUMOylation site in ErbB4. A, schematic structure of ErbB4. Arrows indicate the mutated amino acids analyzed in B. Black color indicates transmembrane domain and TK indicates tyrosine kinase domain. B, COS-7 cells were transfected with wild-type or mutant HA-tagged ErbB4 ICD constructs and His₆-tagged SUMO1. Cells were lysed in denaturing buffer, and lysates were incubated with Ni²⁺-NTA agarose to pull down His₆-SUMO conjugates. Whole cell extracts or pulldown samples were analyzed by Western blotting with anti-ErbB4. Cons 4KR includes K1002R+K1143R+K1181R+K1202R; Cons 3KR includes K1143R+K1181R+K1202R. C, schematic structure of ErbB4 as in A. Blue color indicates the predicted NES and arrows indicate the mutated amino acids analyzed in D. D, COS-7 cells expressing wild-type or mutant HA-tagged ErbB4 ICD constructs and His₆-SUMO1 were analyzed as in B. 3KR includes K714R+K719R+K722R. E, COS-7 cells expressing wild-type or K714R HA-tagged ErbB4 ICD and His₆-SUMO1 or His₆-SUMO3 were analyzed as in B. F, COS-7 cells expressing wild-type or K714R HA-tagged ErbB4 ICD and His₆-SUMO1 with or without FLAG-tagged PIAS3 were analyzed as in B. G, crystal structure of the active ErbB4 kinase domain monomer (PDB ID: 3BCE). Lys-714 side chain is indicated in red, and side chains of the predicted NES amino acids are indicated in blue. H, sequence alignment of ErbB4 orthologues from the indicated species using Clustal Omega. The numbers refer to beginning and ending residues of each peptide. Red indicates Lys-714 and blue indicates the predicted NES. *, identical; :, conserved. ErbB4 ICD lysine-to-arginine mutant constructs were analyzed for SUMOylation in 4–11 independent experiments. The effect of PIAS3 on SUMOylation of wild-type and K714R ErbB4 ICD was analyzed in five independent experiments.

Figure 2.

ErbB4 ICD is deSUMOylated by SENP1, SENP2, and SENP5. A, COS-7 cells were transfected with HA-tagged ErbB4 ICD, His₆-tagged SUMO1, and FLAG-tagged wild-type or C603A mutant SENP1 as indicated. Cells were lysed in denaturing buffer, and lysates were incubated with Ni²⁺-NTA agarose to pull down His₆-SUMO conjugates. Whole cell extracts or pulldown samples were analyzed by Western blotting with anti-ErbB4. SENP1 expression was analyzed by Western blotting with anti-FLAG. B, COS-7 cells were transfected with HA-tagged ErbB4 ICD, His₆-SUMO1, or His₆-SUMO3 and FLAG-tagged SENP1 or SENP2 as indicated, and analyzed as in A. C, COS-7 cells were transfected as indicated with HA-tagged ErbB4 ICD, His₆-SUMO3, and FLAG-tagged SENP1, SENP2, SENP6, or SENP7 or GFP-tagged SENP3 or SENP5, and analyzed as in A. The effect of each SENP on deSUMOylation of ErbB4 ICD was analyzed in three to six independent experiments.

Figure 3.

SUMOylation regulates the nuclear accumulation of ErbB4 ICD. A, MCF-7 cells were transfected with HA-tagged wild-type or K714R ErbB4 ICD, treated with 100 μg/ml cycloheximide (CHX) for the indicated periods of time, and subjected to subcellular fractionation. Nuclear and cytoplasmic fractions were analyzed by Western blotting with anti-HA (H3663, Sigma-Aldrich). Equal loading of the nuclear fraction was controlled with anti-Sp1, and loading of the cytoplasmic fraction with anti-Mek1/2. Total protein loading in both fractions was controlled with Ponceau staining. Representative data of two independent experiments are shown. B, quantification of HA (ErbB4 ICD) signal intensities (shown in A) normalized to Sp1 or Mek1/2. Data are represented as mean ± range from two independent experiments. C, MCF-7 cells were transfected with HA-tagged ErbB4 ICD together with control or SENP2 siRNA and subjected to subcellular fractionation. Nuclear and cytoplasmic fractions were analyzed by Western blotting with anti-HA (H3663, Sigma-Aldrich). Fractionation was controlled with anti-Sp1 and anti-Mek1/2, and total protein loading with Ponceau staining. Efficiency of RNA interference analyzed by qRT-PCR is indicated as relative SENP2 mRNA levels in cells treated with SENP2-targeting siRNA compared with cells treated with control siRNA (SENP2 mRNA %). Representative data of two independent experiments are shown. D, quantification of HA (ErbB4 ICD) signal intensities (shown in C) normalized to Ponceau. The circles indicate individual data points from two independent experiments and the horizontal line indicates the mean.

Figure 4.

Lys-714 does not regulate the general stability of ErbB4. A, COS-7 cells expressing wild-type or K714R ErbB4 JM-a CYT-2 were starved without serum overnight and treated with 100 μg/ml cycloheximide (CHX) for the indicated periods of time. Lysates were analyzed by Western blotting with anti-ErbB4, and equal loading was controlled with anti-actin. B, quantification of 180 kDa or 75 kDa ErbB4 signal intensities (shown in A) normalized to actin. Data are represented as mean ± range from three independent experiments.

Figure 5.

Nuclear localization of ErbB4 ICD is regulated by CRM1-dependent nuclear export. A, MCF-7 cells were treated for 3 h without or with 5, 10, or 20 ng/ml leptomycin B (LMB) and subjected to subcellular fractionation. Nuclear and cytoplasmic fractions were analyzed by Western blotting with anti-ErbB4, anti-Lamin B, and anti-Mek1/2. Quantification of ErbB4 signal intensities normalized to Lamin B are presented as -fold change values relative to the nontreated control sample. The 75 kDa anti-ErbB4 reactive band represents the ICD. B, COS-7 cells were transfected with HA-tagged ErbB4 ICD2 with or without FLAG-tagged CRM1. Lysates were immunoprecipitated with anti-HA (2367, Cell Signaling Technology) followed by Western blotting with anti-FLAG. The membrane was reprobed with anti-HA (2367, Cell Signaling Technology). Representative data of four independent experiments are shown. C, schematic structure of ErbB4. Black color indicates the transmembrane domain and TK indicates tyrosine kinase domain. Blue color indicates the predicted NES amino acids and arrows indicate the mutated amino acids analyzed in D and E. Red color indicates the SUMOylation site analyzed in F and G. D, COS-7 cells expressing wild-type or NES-mutant (V721A+V723A+L724A) ErbB4 ICD2 were stained for anti-ErbB4 (red) and visualized by confocal microscopy using a 40× objective. Nuclei were stained with DAPI (blue). E, quantification of the nuclear staining intensity. Cells were scored for predominantly nuclear (equal signal in the nucleus and in the cytoplasm or more signal in the nucleus than in the cytoplasm; nuclear ≥ cytoplasmic) or cytoplasmic (more signal in the cytoplasmic than in the nucleus; nuclear < cytoplasmic) staining. Data are represented as a scatter plot, with circles indicating data points from four independent experiments and horizontal lines indicating the mean. 475 cells from four independent experiments were scored. F, MCF-7 cells were transfected with HA-tagged wild-type of K714R ErbB4, stimulated with 50 ng/ml NRG-1 for 15 min, and fixed. Complexes of ErbB4 and CRM1 were visualized with anti-HA and anti-CRM1 using in situ PLA. Red PLA foci represent ErbB4-CRM1 interactions. Nuclei were stained with DAPI (blue). G, quantification of PLA signals per cell. Signals were classified as nuclear, nuclear rim, or cytoplasmic using DAPI as a marker for cell nuclei. Data are presented as a box plot, with horizontal lines indicating the median, boxes indicating the second and third quartile, and error bars indicating the 1.5× interquartile range. Outliers are indicated as circles. n = 63 for cells expressing wild-type ErbB4; n = 53 for cells expressing ErbB4 K714R.

Figure 6.

SUMOylation increases autophosphorylation of ErbB4 ICD. A, COS-7 cells were transfected with wild-type or kinase dead K751R ErbB4 ICD2 with or without His₆-tagged SUMO constructs as indicated. Cells were lysed in denaturing buffer, and lysates were incubated with Ni²⁺-NTA agarose to pull down His₆-SUMO conjugates. Whole cell extracts were analyzed by Western blotting with anti-ErbB4, and pulldown samples with anti-ErbB4 or anti-phosphotyrosine (4G10, Upstate). Representative data of two independent experiments are shown. B, structural model (displayed as secondary structures) of the covalent complex between the ErbB4 kinase asymmetric dimer X-ray structure (RCSB PDB ID: 3BCE) and an NMR structure for SUMO1 (in blue) (RCSB PDB ID: 2ASQ). Lys-714 (CPK representations) in both the activator and receiver kinase domains are indicated but SUMO1 is shown ligated only to Lys-714 of the receiver kinase via an isopeptide bond represented between the lysine side chain and the C-terminal Gly-Gly sequence of SUMO (CPK representations).

Figure 7.

Lys-714 is not required for signaling of full-length ErbB4 at the cell surface. A, COS-7 cells expressing wild-type or K714R ErbB4 ICD2 or JM-a CYT-2 were starved overnight without serum, lysed, and immunoprecipitated (IP) with anti-ErbB4. Phosphorylation was analyzed by Western blotting with anti-phosphotyrosine (4G10, Upstate), and the membrane was reprobed with anti-ErbB4. Representative data of five independent experiments are shown. *, IgG heavy chain. B, COS-7 cells expressing wild-type or K714R ErbB4 JM-a CYT-2 were starved without serum overnight, stimulated for 10 min with 50 ng/ml NRG-1, and lysed. Phosphorylation of Akt, Erk1/2, and ErbB4 was analyzed by Western blotting with phospho-specific antibodies. Loading was controlled using antibodies recognizing total Akt, Erk1/2, ErbB4, or actin. Representative data of three independent experiments are shown. C, quantification of pErk1/2 and pAkt signal intensities normalized to total Akt and Erk. Data are presented as scatter plots, with circles indicating data points from three independent experiments and horizontal lines indicating the mean. D, COS-7 cells expressing wild-type or K714R ErbB4 JM-a CYT-2 together with STAT5a were treated as in B. Phosphorylation of STAT5 and ErbB4 was analyzed by Western blotting with phospho-specific antibodies. Loading was controlled using antibodies recognizing total STAT5, ErbB4, or actin. Representative data of two independent experiments are shown. E, quantification of pSTAT5a signal intensity normalized to total STAT5a. Data are presented as a scatter plot, with circles indicating data points from two independent experiments and horizontal lines indicating the mean.

Figure 8.

Lys-714 is required for nuclear signaling of ErbB4 in mammary epithelial cells. A, HC11 cells stably expressing wild-type or K714R ErbB4 JM-a CYT-2 were analyzed for ErbB4 expression by Western blotting. B, representative images of control HC11 cells cultured for 15 days in growth factor reduced Matrigel in the presence of 50 ng/ml NRG-1. A photograph of a differentiated acinus (left) and an undifferentiated colony (right) are shown. Scale bar, 25 μm. C, quantification of the percentage of acini of all cell colonies of HC11 cells expressing the indicated constructs. Data of 12 wells from four independent experiments are presented as a box plot, with horizontal lines indicating the median, boxes indicating the second and third quartile, and error bars indicating the range of the data. n = 533 for vector cell colonies; n = 517 for ErbB4 wild-type colonies; n = 575 for ErbB4 K714R colonies.