HER4 D-box sequences regulate mitotic progression and degradation of the nuclear HER4 cleavage product s80HER4

Abstract

Heregulin-mediated activation of HER4 initiates receptor cleavage (releasing an 80-kDa HER4 intracellular domain, s80(HER4), containing nuclear localization sequences) and results in G(2)-M delay by unknown signaling mechanisms. We report herein that s80(HER4) contains a functional cyclin B-like sequence known as a D-box, which targets proteins for degradation by anaphase-promoting complex (APC)/cyclosome, a multisubunit ubiquitin ligase. s80(HER4) ubiquitination and proteasomal degradation occurred during mitosis but not during S phase. Inhibition of an APC subunit (APC2) using short interfering RNA knockdown impaired s80(HER4) degradation. Mutation of the s80(HER4) D-box sequence stabilized s80(HER4) during mitosis, and s80(HER4)-dependent growth inhibition via G(2)-M delay was significantly greater with the D-box mutant. Polyomavirus middle T antigen-transformed HC11 cells expressing s80(HER4) resulted in smaller, less proliferative, more differentiated tumors in vivo than those expressing kinase-dead s80(HER4) or the empty vector. Cells expressing s80(HER4) with a disrupted D-box did not form tumors, instead forming differentiated ductal structures. These results suggest that cell cycle-dependent degradation of s80(HER4) limits its growth-inhibitory action, and stabilization of s80(HER4) enhances tumor suppression, thus providing a link between HER4-mediated growth inhibition and cell cycle control.

Figure 1. The intracellular domain of HER4, s80HER4, inhibits growth via G2/M delay in HaLa-rtTA cells A

Mutation of the HER4 γ-secretase cleavage site inhibits formation of s80^HER4 and interferes with HER4-mediated growth inhibition: A.1 Western analysis to detect phospho-tyrosine residues or total HER4 expression in HER4 immunoprecipitates (IPs) from membrane, cytoplasmic, or nuclear extracts from HeLa-rtTA cells expressing pLXSN, pLXSN-HER4, pLXSN-HER4^KD, or pLXSN-HER4^VA. Serum-starved cells were treated ± HRG (2h) before separation into cellular compartments using biochemical methods. Molecular weights are indicated at left. A.2: Cells grown in SF media ± HRG (48h) were stained with propidium iodide (PI) and analyzed by flow cytometry. Representative histograms are shown. B. Western analysis to detect GFP in HER4 IPs from HeLa-s80 and HeLa-s80^KD cells cultured +TET for 0-24 h. C. Growth inhibition and G2/M delay in cells expressing GFP-s80^HER4, but not in cells expressing GFP-s80^KD. C.1: Representative histograms of HeLa-pcDNA4 and HeLa-s80 cells grown in SF media ±TET (48h), stained with PI and analyzed by flow cytometry. *P < 0.002, Student's unpaired T-test. C.2 Equal numbers of HeLa-pcDNA4, HeLa-s80, and HeLa-s80^KD cells were plated, allowed to attach (24h), then treated for 0 or 96h ±TET in serum-free (SF) media. D. Equal numbers of HC11, MCF-10A, or MDA-453 cells expressing GFP-tagged s80 or GFP, were plated, allowed to attach (24h), then cultured for 0 or 96h in SF media. Each sample was counted in duplicate; each experiment was performed in triplicate. Values represent the average number of cells ± S.D. **P < 0.01; @P < 0.02; ˆP < 0.01; each calculated using Student's unpaired T-test Western analysis to detect s80^HER4 in cell lysates is shown in lower panel.

Figure 2. Cell-cycle-specific expression of s80^HER4. A

GFP imaging of HeLa-s80 cells cultured 24 h ± TET. B. HeLA-s80 cells were treated +TET for 24 h to induce expression of GFP-s80^HER4, followed by 16 h treatment +TET in SF media, or +TET in 10% serum +HU, or +OLO. Cytoplasmic and nuclear extracts were examined by western analysis, using HER4 IP followed by immunoblotting for GFP (upper panels) or by analysis of extracts by immunoblotting for SP-1 (lower panel). C. Immunocytofluorescence of HeLa-s80 cells cultured +TET (48 h). Upper: Cells were dual-stained for GFP and α-tubulin. DAPI images shown at right. Lower: Immunocytofluoresence HER4 and phospho-Ser10 histone H3. DAPI staining shown at right.

Figure 3. Degradation of s80^HER4 during mitosis directed by APC/C. A

HeLa-s80 cells were cultured +NOC/+TET in 10% serum for 16 h. Cells were released into 10% serum +TET at time (T) =0, and treated +CHX at T=0, 2, 4, 8, and 16h after NOC release. Expression of GFP-s80^HER4 was analyzed at 15 min intervals beginning at the time of CHX treatment, by HER4 IP followed by GFP immunoblot (IB). B. HeLa-s80 cells were synchronized +HU/+TET (upper panel) or +NOC/+TET (lower panel) in 10% serum for 16 h, then released into 10% serum +TET at T=0. Cells were treated +CHX at T=0 and 2h. Expression of GFP-s80^HER4 was analyzed as described above. C. Cells were cultured +NOC/+TET in 10% serum for 16 h, with PS-341 added for the final 4 h. Cells were released into 10% serum +TET/+PS-341 at T=0. CHX was added at T=0. Expression of GFP-s80^HER4 was analyzed as described above; blots were stripped then probed for ubiquitin (lower panel). D. HeLa-s80^HER4 cells transfected with 10 nmol siRNA sequences targeting luciferase or APC2 were treated +NOC/+TET +10% serum for 16 h. Cells were released into 10% serum +TET/+CHX. Expression of GFP-s80^HER4 and cyclin B were analyzed at 15 min intervals beginning at the time of CHX treatment, by IB of anti-HER4 or anti-cyclin B IP's using GFP or cyclin B antibodies, respectively. APC2 was analyzed by IB.

Figure 4. Mutation of the s80^HER4 D-box eliminates mitotic degradation of s80^HER4 and enhances s80^HER4-mediated growth inhibition. A

Mutation of the s80^HER4 D-box stabilized s80^HER4 during mitosis. A.1 Western analysis of HER4 IP's from HeLa-s80, -s80^db, and -s80^KD cells cultured 24 h +TET. IP's were analyzed for phospho-tyrosine or GFP. A.2: HeLa-s80^db cells were cultured +NOC/+TET in 10% serum (16 h). Cells were released into 10% serum +TET/+CHX at T=0, and extracts collected at 15 min intervals. Expression of s80^db and cyclin B was analyzed as described in Fig. 3. B. Equal numbers of cells were plated at day 0 ±TET. Cells were counted at 1-day intervals through 4 days. P= 0.011 comparing cell number of HeLa-s80^HER4 +TET at day 4 to cell number of HeLa-s80^db + TET at day 4, n=5, analyzed in triplicate. C. Cell cycle analysis of HeLa-s80^db cells cultured ±TET (48h). > 10,000 nuclei were analyzed per condition; n=3; representative histograms shown. D. Cells were in 10% serum +HU/+TET for 16 h to synchronize in S-phase. Cells were released into 10% serum +TET at T=0 h. Cells were collected at 0, 2, 4, 8, 12, and 24 h following HU release. Cells were stained with propidium iodide and analyzed by flow cytometry as described above.

Figure 5. Mutation of the D-box within full length HER4 does not reduce s80^HER4 production but does stabilize s80^HER4. A

HeLa cells stably transfected with pLXSN-HER4 or -HER4^db were serum-starved 16 h then treated +HRG (1h). HER4 IP's were analyzed for total HER4. B. Cells were serum-starved (16 h), treated +HRG (1h). Cells were analyzed for HER4 localization by immunocytofluorescence. DAPI staining is shown at bottom. N= nuclear localization of HER4. C= cytoplasmic localization of HER4. C. HeLa-HER4 and HeLa-HER4^db cells were cultured in 10% serum +NOC (16 h), then in SF media +NOC (16 h), adding HRG for the final hour. Cells were released into SF media +HRG/+CHX at T=0 and cells were harvested at T=0, 15, 30, and 45 min. HER4 IPs were analyzed as described above.

Figure 6. Decreased tumor formation in PyVmT-transformed HC11 cells expressing s80^db. A

HC11P cells expressing s80^HER4, s80^KD, s80^db, or the empty pcDNA4 vector were injected into the inguinal mammary fatpads of female BALB/c mice. Fifteen weeks post-implantation of cells, mice were euthanized and the mammary glands analyzed histologically; representative images are shown. B. Five tumor samples per group were analyzed by immunohistochemistry. PCNA positive (upper panel) or TUNEL positive nuclei (lower panel) were scored in 3 randomly chosen 400× fields per tumor section, for a total of 15 fields counted. HC11P-pcDNA4 and HC11P-s80^KD had equivalent percentages of PCNA-positive nuclei (29.4±2.2 and 34.1±2.9, respectively). HC11P-s80 had a statistically significantly lower percentage of PCNA-positive cells (12.5±1.1, p<0.0001) as compared to vector alone. While HC11P-s80^db percentage of PCNA-positive cells was the lowest of all groups (9.0±2.2), and was also significantly decreased when compared to HC11P-s80 (p<0.037).