SIAH ubiquitin ligases target the nonreceptor tyrosine kinase ACK1 for ubiquitinylation and proteasomal degradation

Abstract

Activated Cdc42-associated kinase 1 (ACK1) is a nonreceptor tyrosine kinase linked to cellular transformation. The aberrant regulation of ACK1 promotes tumor progression and metastasis. Therefore, ACK1 is regarded as a valid target in cancer therapy. Seven in absentia homolog (SIAH) ubiquitin ligases facilitate substrate ubiquitinylation that targets proteins to the proteasomal degradation pathway. Here we report that ACK1 and SIAH1 from Homo sapiens interact in a yeast two-hybrid screen. Protein–protein interaction studies and protein degradation analyses using deletion and point mutants of ACK1 verify that SIAH1 and the related SIAH2 interact with ACK1. The association between SIAHs and ACK1 depends on the integrity of a highly conserved SIAH-binding motif located in the far C-terminus of ACK1. Furthermore, we demonstrate that the interaction of ACK1 with SIAH1 and the induction of proteasomal degradation of ACK1 by SIAH1 are independent of ACK1’s kinase activity. Chemical inhibitors blocking proteasomal activity corroborate that SIAH1 and SIAH2 destabilize the ACK1 protein by inducing its proteasomal turnover. This mechanism apparently differs from the lysosomal pathway targeting ACK1 after stimulation with the epidermal growth factor. Our data also show that ACK1, but not ACK1 mutants lacking the SIAH binding motif, has a discernable negative effect on SIAH levels. Additionally, knockdown approaches targeting the SIAH2 mRNA uncover specifically that the induction of SIAH2 expression, by hormonally-induced estrogen receptor (ER) activation, decreases the levels of ACK1 in luminal human breast cancer cells. Collectively, our data provide novel insights into the molecular mechanisms modulating ACK1 and they position SIAH ubiquitin ligases as negative regulators of ACK1 in transformed cells.

Introduction

Seven in absentia homolog (SIAH) proteins are homologs of the Drosophila melanogaster Seven In Absentia (SINA) protein. All members of the SIAH family are evolutionally conserved across species.1, 2 In Homo sapiens, SIAH1 and SIAH2 are expressed in a broad range of tissues and share over 80% of amino acid homology and 69.8% sequence identity.1, 2, 3 Both of them contain a highly conserved Really Interesting New Gene (RING) domain, two zinc finger domains and a C-terminal substrate-binding domain. Sequence deviations mainly occur in the N-termini of SIAH1 and SIAH2 and both proteins function as ubiquitin ligases via their RING domains.1, 4

In eukaryotic cells, proteins are tightly regulated by the ubiquitin–proteasome system, in which polyubiquitinylated proteins are cleaved by a multiprotein complex termed the proteasome.5 Ubiquitinylation is usually carried out by three classes of enzymes. These enzymes act in a concerted manner to recognize and transfer ubiquitin molecules to substrate proteins. The three classes are a ubiquitin activating enzyme, ubiquitin conjugases and ubiquitin ligases (E3s).4 The selectivity of ubiquitinylation is primarily determined by ubiquitin ligases. E3s containing a RING domain, such as the SIAH proteins, possess no intrinsic catalytic activity. By binding ubiquitin conjugases, they coordinate the formation of polyubiquitin chains on their substrates.4

SIAH ubiquitin ligases facilitate the proteasomal degradation of diverse substrate proteins with multiple functions. Accordingly, SIAH-mediated degradation of targeted proteins is associated with severe consequences for the tumorigenicity of certain types of cancer.1, 2 The targets of SIAHs are, for example, the deacetylase HDAC3,6, 7 the polycomb protein HPH2,8 the corepressor N-CoR,7, 9 the alternative splicing factor T-STAR,10 the cell cycle regulator p27,11 the transcription factor β-catenin,12 the leukemia fusion protein AF4-MLL13 and the TNF signaling adapter molecule TRAF2.14 We recently demonstrated that SIAH1 and SIAH2 cooperate with the ubiquitin conjugase UBCH8 to induce proteasomal degradation of leukemia-associated oncoproteins, such as the mutated constitutively active receptor tyrosine kinase FLT3-ITD and the chimeric fusion proteins PML-RARα and AML1-ETO. Such processes promote the death of leukemic cells.15, 16, 17, 18 Remarkably, SIAH1 and SIAH2 are targets of the tumor suppressor p53.19, 20 Activation of p53 due to genotoxic stress, for example, induces SIAH1 expression and degradation of the transcriptional activator β-catenin, which leads to the suppression of cancer cell growth.1 Moreover, SIAH1 can restrict EGF-dependent cell growth by degradation of the phospholipase Cɛ,21 and SIAH1 suppresses the survival and motility of transformed breast cells by a JUN kinase-dependent induction of the proapoptotic protein BIM.22, 23

The aim of this study was to identify novel SIAH targets that might play a role in cancer development. By a systematic search, we identified the (proto) oncogenic nonreceptor tyrosine kinase activated Cdc42-associated kinase 1 (ACK1) as a novel interaction partner of SIAH1. We demonstrate that SIAH1 and SIAH2 facilitate the ubiquitinylation and degradation of ACK1 by the ubiquitin–proteasome system. Furthermore, we show that the association of SIAH1 and SIAH2 with ACK1 relies on a highly conserved binding site within ACK1. In addition, estrogen induces the expression of SIAH2, which we have identified here as a limiting factor for the degradation of ACK1 in transformed breast cancer cells. Our results suggest a new biological role for SIAH proteins as regulators of ACK1 expression in cancer cells.

Results

Identification of ACK1 as a binding partner of SIAH1

In order to find novel interaction partners of SIAH1, we performed yeast two-hybrid screening with full-length SIAH1 (as the bait) against human brain, testis and open reading frame cDNA libraries (as the prey). Under stringent screening conditions, 16 positive clones were identified to interact with SIAH1. Out of these clones, five have already been described as SIAH1 interaction partners (Figure 1a), for example, the SIAH1 interacting protein/CacyBP or SIAH1 itself.1 Two positive clones from the brain cDNA library encoded a large C-terminal fragment of human ACK1 (amino acids 819–1038). The association of ACK1 with SIAH1 has so far not been described.

Figure 1
figure1

Identification of ACK1 as SIAH1 interacting protein. (a) Yeast two-hybrid screens were performed with full-length SIAH1 (as bait) against human testis and brain cDNA libraries as well as a library of human full-length open reading frame cDNAs (as prey). The table displays a summary of the yeast two-hybrid results. Listed are prey proteins found in the screens that were already described as SIAH1-interacting proteins and the newly identified SIAH interaction partner ACK1. For each prey the numbers of isolated clones and numbers of different libraries in which this pair has been found are shown. (b) Detection of ACK1-Myc bound to GST-SIAH1 (left panel) or GST-SIAH2 (right panel) or GST (as control) in a GST pulldown assay. Bacterially expressed purified GST and GST-SIAH1/2 were incubated with total cell lysate protein of ACK1-Myc transfected HEK293T cells (IN, input representing 20% of cell lysate). Bound ACK1-Myc was detected by immunoblotting. Levels of GST fusion proteins were analyzed by membrane staining with Ponceau red (left panel) or by immunoblotting (right panel); kDa, relative molecular weight. (c) RING mutants of SIAH1 or SIAH2 were overexpressed in MCF-7 cells. SIAH1/SIAH2 was immunoprecipitated from cell lysates. The presence of endogenous ACK1 and SIAH1/SIAH2 was analyzed by immunoblotting (Input, 10% of cell lysate; pre, preimmune serum; IP, immunoprecipitation).

We performed protein–protein binding assays to test the initial result of an interaction between SIAH1 and ACK1. Since SIAH1 displays over 80% sequence similarity with the related protein SIAH22, 3 and as both share an overlapping spectrum of target proteins, we also tested for a putative interaction between SIAH2 and ACK1. We performed an in vitro GST pulldown assay with bacterially expressed GST-SIAH1 or GST-SIAH2 and full-length ACK1-Myc from HEK293T cells. ACK1 efficiently bound to the GST-SIAH fusion proteins but not to GST (Figure 1b), which corroborated the interaction of ACK1 with SIAH1 and SIAH2. Due to the strong self-ubiquitinylation and degradation of SIAH1, the interaction of SIAH1 its with binding partners is difficult to detect in coimmunoprecipitation assays.1, 6, 16, 24 Therefore, we used established RING mutants of SIAH1 and SIAH2.2 This approach is commonly used to test the interaction of SIAHs with their substrates, since these mutants still bind to their targets but fail to recruit ubiquitin conjugases.3, 14, 16, 17, 25 Indeed, we could coprecipitate SIAH1 as well as SIAH2 with endogenous ACK1 from MCF-7 cells (Figure 1c). Taken together, these data suggest that SIAH1 and SIAH2 occur in protein complexes containing ACK1.

SIAH proteins mediate the degradation of ACK1 by the ubiquitin–proteasome system

SIAH proteins mediate the ubiquitinylation of substrate proteins to promote their proteasomal turnover. However, the interaction with SIAH1 and SIAH2 does not necessarily destabilize their binding partners.2, 3, 26 Therefore, we investigated whether SIAH proteins mediate the degradation of ACK1. HEK293T cells were transiently transfected with plasmids encoding ACK1-Myc and SIAH1 or SIAH2. Expression of the SIAHs resulted in a strong reduction of ACK1 protein levels and this was dependent on the concentration of SIAHs (Figure 2a). This inverse correlation between ACK1 and SIAH1 or SIAH2 identifies ACK1 as a new proteasomal target of SIAH1 and SIAH2. The protein levels of cotransfected green fluorescent protein as an internal control remained stable, which illustrates the specific effect of SIAHs on ACK1 (Figure 2a).

Figure 2
figure2

SIAH ubiquitin ligases trigger the proteasomal degradation of ACK1. (a) Myc-tagged ACK1 (0.5 μg) was transfected alone or together with different amounts of SIAH1 or SIAH2 (0.05–0.2 μg) into HEK293T cells. Green fluorescent protein was cotransfected to monitor equal transfection efficiency. Protein expression was analyzed by immunoblotting (SIAH1/2: polyclonal antibodies were used for the detection of SIAH1 and SIAH2). Detection of tubulin served as loading control. (b) ACK1-Myc (0.2 μg) was expressed in Hestrogen 93T cells and the knockdown of SIAH expression was achieved by transfection of shRNA plasmids (1 μg) directed against SIAH mRNA (shSIAH1/2) or unspecific shRNA as control. Immunoblotting was performed to detect protein expression. (c) ACK1-Myc and SIAH1 were expressed for 24 h in HEK293T cells. Where indicated, cells were incubated 6 h before cell lysis with the proteasome inhibitor MG132 (10 μM) to block the degradation of ubiquitinylated proteins. For detection of ubiquitinylated ACK1, ACK1-Myc was immunoprecipitated with an anti-Myc antibody and precipitates were immunoblotted for the presence of the ubiquitin (Ub) modification (IgG, immunoglobulin heavy chain). (d) ACK1-Myc was coexpressed with SIAH1 in HEK293T cells in the absence or presence of the very proteasome specific inhibitor lactacystin (Lact., 10 μM), added 8 h prior cell lysis. ACK1 and SIAH1 protein levels were analyzed by immunoblotting. (e) ACK1-Myc was coexpressed with SIAH2-Myc in HEK293T cells in the absence or presence of lactacystin and analyzed as described in (d). (f) To determine the specificity of the SIAH-mediated ACK1 turnover, ACK1 was coexpressed with SIAH1, SIAH2, RLIM-HA, TRIAD1-Myc and MDM2 ubiquitin ligases. Cell lysates were analyzed by immunoblotting. Green fluorescent protein and tubulin served as controls for equal transfection efficiency and equal loading, respectively (n.s., nonspecific band).

Since these data suggest that SIAH ubiquitin ligases accelerate the proteasomal degradation of ACK1, we also assessed whether the half-life of ACK1 is altered in the presence of SIAHs. We expressed ACK1 alone or with SIAH1, SIAH2 or an inactive RING mutant of SIAH2 in HEK 293T cells. Treatment with the ribosomal inhibitor cycloheximide stopped de novo protein synthesis and allowed the decay of ACK1 to be judged. The stability of ACK1 was drastically decreased when SIAH1 or SIAH2 were present. Expression of the inactive SIAH2 corroborated that SIAH2’s catalytic activity as ubiquitin ligase is a prerequisite for the accelerated turnover of ACK1 (Supplementary Figure S1).

Next, we addressed whether endogenously expressed SIAHs also regulate ACK1 levels. We expressed ACK1-Myc in HEK293T cells and lowered endogenous SIAH expression by transfecting these cells with small interfering RNAs (siRNAs) against SIAH1/SIAH2 or with small hairpin RNA (shRNAs) specifically targeting SIAH1 or SIAH2.27 The reduction of SIAH protein expression increased the levels of ACK1-Myc (Figure 2b and Supplementary Figure S2). These experiments show that endogenous as well as ectopically expressed SIAH proteins regulate ACK1 protein levels.

As SIAH ubiquitin ligases promote polyubiquitinylation targeting proteins for proteasomal degradation,1, 4 we analyzed if ACK1 undergoes SIAH1-mediated ubiquitinylation. We coexpressed ACK1-Myc with SIAH1 in HEK293T cells and added the proteasome inhibitor MG132 to block proteasomal degradation of the ubiquitinylated proteins. To detect ubiquitinylated ACK1, ACK1-Myc was immunoprecipitated from cell lysates with anti-Myc antibodies followed by immunoblotting with anti-ubiquitin antibodies. A slower migrating smear was detected in the ACK1 immunoprecipitates from lysates with SIAH1 and ACK1 expression (Figure 2c). The higher molecular weight of these ACK1 species indicates that SIAH1 mediates the ubiquitinylation of ACK1. Polyubiquitinylation of proteins is connected to their proteolytical cleavage by the proteasome. In order to establish whether ACK1 is degraded by the proteasome as a consequence of SIAH-dependent polyubiquitinylation, we tested if ACK1-Myc remains stable in the presence of increased levels of SIAH1 or SIAH2 in HEK293T cells with chemically inactivated proteasomes. Since ACK1 was found to be subjected to lysosomal degradation under specific cellular conditions28 and MG132 was also described to inhibit certain lysosome-associated proteases, we used the highly proteasome-specific inhibitor lactacystin.29 Coexpression of ACK1-Myc and SIAH1 or SIAH2 led to a robust degradation of the ACK1 protein that could be blocked by treatment with lactacystin (Figures 2d and e). These experiments demonstrate that SIAHs regulate the proteasomal degradation of ACK1.

Of note, ACK1-Myc protein was also stabilized by lactacystin in the absence of overexpressed SIAHs (Figure 2d). This finding suggests a basal proteasomal turnover of ACK1-Myc that is in agreement with the notion that endogenous SIAH1 and SIAH2 can control the protein levels of ectopically expressed ACK1 in HEK293T cells (Figure 2b and Supplementary Figure S2). Moreover, SIAH1 and SIAH2 were stabilized in the presence of lactacystin (Figures 2d and e), which is consistent with previous reports demonstrating their high proteasomal turnover.1, 16, 17, 25

To assess whether overexpression of other ubiquitin ligases can cause degradation of ACK1, we coexpressed ACK1-Myc together with HA-tagged RLIM, Myc-tagged TRIAD1 and MDM2 ubiquitin ligases in HEK293T cells. Coexpression of SIAH1 and SIAH2 resulted in a complete loss of ACK1, whereas ACK1 remained stable in the presence of coexpressed RLIM, TRIAD1 and MDM2 (Figure 2f). These findings suggest that SIAH proteins function as specific ubiquitin ligases for polyubiquitinylation and subsequent proteasomal degradation of ACK1.

SIAH1 binds and degrades ACK1 independent of its kinase activity

SIAH1 and SIAH2 have recently been reported to undergo phosphorylation at multiple tyrosine and serine residues and these modifications have been shown to regulate their substrate affinity and degradation.19, 20, 21, 26, 30 Furthermore, it is known that the specificity of SIAHs for certain targets can depend on the substrate phosphorylation status.15 ACK1 is a tyrosine kinase that catalyzes autophosphorylation and phosphorylation of interacting proteins.31, 32, 33, 34 To elucidate the relevance of ACK1 kinase activity for the degradation by SIAH1, we expressed SIAH1 together with wild-type ACK1, a kinase dead mutant (kdACK1) or a constitutively active mutant (caACK1) of ACK1 in HEK293T cells.31 ACK1 was immunoprecipitated and the phosphorylation status of ACK1 was verified by immunoblotting with antibodies recognizing phosphorylated tyrosine residues (Figure 3a, 2nd panel). Wild-type ACK1, caACK1 or kdACK1 exhibited no significant differences regarding their SIAH1-mediated degradation (Figure 3a, top panel). To investigate whether the interaction between SIAH1 and ACK1 depends on the kinase activity of ACK1, we performed a GST pulldown assay using GST-SIAH1 and wild-type ACK1, caACK1 or kdACK1 expressed in HEK293T cells. GST-SIAH1 associated well with all the three ACK1 variants in the pulldown assay (Figure 3b). These data coherently suggest that the interaction of these factors and the SIAH1-induced proteasomal degradation of ACK1 are independent of its kinase activity.

Figure 3
figure3

SIAH1 facilitates ACK1 degradation independent of ACK1 activity. (a) Cells were transfected with SIAH1 and wild-type (WT), constitutively active (ca) or kinase dead (kd) ACK1-Myc followed by analysis of cell lysates by immunoblotting. Kinase activity of ACK1 was determined by immunoprecipitation (IP) of ACK1-Myc and immunoblotting (IB) with a phospho-tyrosine (pY) specific antibody for detection of phosphorylated ACK1. (b) HEK293T cells were transfected with ACK1-Myc expression vectors as described in (a). Pulldown experiment was performed by incubating HEK293T cell lysates with bacterially expressed GST or GST-SIAH1. Interaction of GST-SIAH1 with ACK1-Myc was assessed by immunoblotting against precipitated ACK1-Myc (IN, input representing 10% of cell lysate). GST fusion proteins were detected by Ponceau-staining of the membrane.

Identification of a SIAH binding site in the far C-terminus of ACK1

The majority of SIAH interacting proteins have a SIAH-binding site also named degron motif (abbreviation for ‘degradation on’).2, 35, 36 The consensus peptide sequence for a degron motif comprises PxAxVxP, with the core sequence VxP (x, random amino acid; V, valine; P, proline). Although the degron motif is not a prerequisite for the association with SIAHs, all of the SIAH1-interacting proteins that we identified in our yeast two-hybrid assay possess conserved degrons (Figure 4a). In silico analysis of the ACK1 peptide sequence revealed an amino acid stretch, PTATVRP, which matched perfectly with the degron consensus sequence (Figure 4a). This sequence is located in the C-terminal proline rich region of ACK1 (Figure 4b, 1.). The ACK1 clone that we identified in the yeast two-hybrid assay encodes the C-terminal fragment of ACK1 from amino acid 819–1038 (Figure 4b, 3.), which also comprises the putative SIAH degron. Collectively, these data suggest that SIAH1 bound to ACK1 within this region. To further analyze the putative role of the SIAH degron in ACK1, we performed a pulldown binding assay with a C-terminally truncated mutant of ACK1 lacking the degron sequence (wACK1, Figure 4b, 2.). GST-SIAH1 bound to full-length ACK1 but not to C-terminally truncated wACK1 (Figure 4c). In agreement with this finding, ACK1 was efficiently degraded by SIAH1 whereas wACK1 was unaffected (Figure 4d). Already in the absence of overexpressed SIAH proteins, we detected a much higher expression of wACK1 compared to the basal expression of full-length ACK1. This is congruent with previous publications reporting that full-length ACK1 is expressed poorly compared to wACK1.31, 37

Figure 4
figure4

SIAH proteins bind to a distinct C-terminal binding site in ACK1. (a) Alignment of the consensus sequence of the SIAH binding motif (degron) to the peptide sequences of ACK1 and already known SIAH-interacting proteins found in the yeast two-hybrid screens. (b) Schematic representation of ACK1 variants with domain structure: (I) full-length ACK1 (ACK1), (II) C-terminal truncated ACK1 (wACK1); (III) prey fragment of ACK1 identified in the yeast two-hybrid screen (Y2H-ACK1) (kinase domain, SH3 domain, Cdc42/Rac binding domain (C), proline-rich domain (PR), ubiquitin binding domain (U), SIAH degron). (c) Cell extracts of Myc-tagged ACK1 or wACK1 transfected HEK293T cells were used for pulldown experiments with recombinant GST-SIAH1 or GST (IN, Input representing 10% of cell lysate; equal input levels of ACK1 WT and wACK1 were obtained by adjusted lysate volumes). ACK1-Myc protein was detected by immunoblotting. Levels of GST fusion proteins were analyzed by Ponceau-staining of membrane. (d) HEK293T cells were transfected with expression vectors for SIAH1 and full-length wild-type (WT) or C-terminal truncated (wACK1) mutant of ACK1-Myc. Protein expression was analyzed by immunoblotting (asterisk indicates adjusted exposition signals of WT ACK1 and wACK1). (e) The core sequence of the putative SIAH-binding motif in ACK1 was mutated by a single amino acid substitution of valine to glycine at amino acid position 909 (ACK1V909G). Pulldown experiments were performed with lysates from wild-type ACK1 (WT) or ACK1V909G transfected HEK293T cells and GST or GST-SIAH1 as described in (c). The lower panel shows the densitometric analysis of precipitated ACK1 (WT) or ACK1V909G protein levels (means±s.d., n=3). (f) Myc-tagged wild-type ACK1 (WT) or mutant ACK1V909G were coexpressed with SIAH1 or SIAH2 in HEK293T cells and a following immunoblot analysis was done as stated.

It has been reported that SIAH1 targets T-STAR. The peptide sequence of murine and human T-STAR differs by a single amino acid in the degron VxP core consensus motif (glycine instead of a valine present in human T-STAR). This difference suffices to render murine T-STAR insensitive to SIAH1-mediated degradation.10 To further verify the SIAH1 binding site in ACK1, we mutated the core sequence of the degron in ACK1 by a single amino acid substitution of valine to glycine, at the position 909 (ACK1V909G). Of note, this V909G point mutation in the ACK1 degron abolished the interaction of ACK1V909G with GST-SIAH1 (Figure 4e). Consistently, the SIAH1 binding-deficient mutant ACK1V909G displayed an enhanced protein stability compared with wild-type ACK1 when coexpressed with SIAH1 or SIAH2 in HEK293T cells (Figure 4f). Interestingly, we likewise observed that ACK1V909G significantly increased SIAH1 and SIAH2 levels (Figure 4f). This was similarly observed for wACK1 (Figure 4d), which like ACK1V909G cannot bind to SIAHs (Figures 4c and e). Taken together, these data suggest that both SIAH proteins promote the proteasomal degradation of ACK1 dependent on an intact SIAH degron motif in ACK1. Furthermore, the inability of SIAHs to accelerate proteasomal degradation of ACK1V909G increases the stability of SIAH proteins.

Estrogen-induced SIAH2 regulates the levels of ACK1 in MCF-7 breast cancer cells

Since estrogen upregulates SIAH2 in various breast cancer cell lines,9, 38, 39 we examined the impact of induced SIAH2 on ACK1 expression in this system. We incubated MCF-7 breast cancer cells with different concentrations of estrogen and monitored SIAH2 and ACK1 protein expression by immunoblotting. The incubation of MCF-7 cells with estrogen upregulated SIAH2 expression and this correlated with a reduction of endogenous ACK1 protein levels (Figure 5a). We subsequently analyzed whether estrogen might have a differential effect on SIAH1 and SIAH2. Real-time PCR analyses and immunoblotting for the SIAHs revealed that estrogen increases the protein and mRNA expression of SIAH2 but not of SIAH1 (Figures 5b and c). Experiments with lactacystin verified that estrogen induces the proteasomal degradation of ACK1 (Figure 5d). This compound even reduced ACK1 mRNA levels strongly (Figure 5e), but stabilized the ACK1 protein in the presence of estrogen (Figure 5d). These data illustrate that ACK1 levels are mainly controlled at the protein level. The loss of ACK1 mRNA in the presence of a proteasomal inhibitor is likely due to the frequently observed stabilization of transcriptional corepressors by such agents.7, 40

Figure 5
figure5

SIAH2 regulates ACK1 stability in the breast cancer cell line MCF-7. (a) MCF-7 cells were left untreated or incubated with estrogen (E2; 10 nM, 20 nM) for 48 h and ACK1 protein levels were examined by immunoblotting. For detection of SIAH2 protein, SIAH2 was immunoprecipitated (IP) followed by immunoblotting (IB). (b) Cells were treated with estrogen (20 nM) for 48 h and SIAH1 and SIAH2 protein levels were detected as described in (a). (c) mRNA levels of SIAH1 and SIAH2 were analyzed by real-time qPCR (n=3; ***P<0,001). Treatment conditions were as in (b). (d) Cells were incubated with estrogen (20 nM) for 48 h in presence or absence of lactacystin (10 μM), added 24 h prior cell lysis and ACK1 protein levels were examined by immunoblotting. (e) After treatment of cells as in (d), mRNA levels of ACK1 were analyzed by real-time qPCR. (f) MCF-7 cells were transfected witch siRNAs directed against SIAH1/SIAH2 mRNA (siSIAH) or nontargeting control siRNA (siControl). Cells were treated with estrogen (20 nM, 48 h) and cell lysates were examined for endogenously expressed ACK1 level by immunoblotting. (g) MCF-7 cells were transfected with ACK1-Myc cDNA and SIAH2 specific shRNA plasmids (or non-targeting control shRNA) and further treated and analyzed as described in (f). Ectopical ACK1-Myc levels were detected by immunoblotting. (h) Wild-type ACK1 (WT) or SIAH-binding deficient ACK1V909G were expressed in MCF-7 cells and cells were left untreated or treated for 48 h with estrogen (20 nM). Immunoblotting was performed (asterisk indicates adjusted exposition signals of WT ACK1 and ACK1V909G). (i) ER-negative MDA-MB-231 and ER-positive T47D cells were incubated with estrogen (20 nM) for 48 h and protein levels of ACK1, SIAH2, and tubulin were analyzed as stated in (a).

Next, we investigated whether the estrogen-induced downregulation of ACK1 depends on increased SIAH2 expression in MCF-7 cells. We observed that endogenous ACK1 levels were significantly reduced upon estrogen stimulation of cells transfected with control siRNA, whereas the reduction in ACK1 was prevented in cells transfected with siRNA targeting SIAH2 (Figure 5f and Supplementary Figure S3). Similar to endogenous ACK1, cotransfection of MCF-7 cells with ACK1-Myc and shRNA specifically targeting SIAH2 blocked the estrogen-mediated downregulation of ACK1 (Figure 5g), indicating that induced SIAH2 negatively regulates ACK1. The knockdown efficiency of SIAH2 mRNA by shRNA or siRNA is demonstrated in Supplementary Figures S4 and S5. To further verify the role of SIAH2 expression in reducing the levels of ACK1, we transfected MCF-7 cells with wild-type ACK1 or SIAH-binding deficient ACK1V909G. The cells were then treated with estrogen and ACK1 protein stability was assessed. Remarkably, wild-type ACK1 was strongly degraded upon estrogen treatment and the SIAH-binding deficient mutant ACK1V909G remained stable (Figure 5h).

Finally, we tested whether the control of ACK1 by SIAH2 is limited to estrogen-responsive breast cancer cells. We used MDA-MB-231 basal-like breast cancer cells lacking a functional estrogen receptor (ER) and T47D breast cancer cells. Like MCF-7 cells, T47D cells are luminal breast cancer cells harboring a functional ER.41, 42, 43 We found that estrogen induces SIAH2 and reduces ACK1 in T47D cells, but not in MDA-MB-231 cells (Figure 5i). Moreover, MDA-MB-231 cells reconstituted with ERα have reduced ACK1 levels (Supplementary Figure S6). These data suggest that intact ER signaling is necessary to induce SIAH2 and to reduce ACK1.

In summary, our results suggest that estrogen and SIAH2 act as novel regulatory factors for ACK1 in breast cancer cells. The model shown in Figure 6 summarizes the findings of the work we present here.

Figure 6
figure6

Model summarizing our findings for a proteasomal mechanism dictating ACK1 protein stability. In the absence of estrogen breast cancer cells express ACK1 and low levels of SIAH2 (left panel). Induction of ER signaling with estrogen induces SIAH2 to mediate proteasomal degradation of ACK1. SIAH2 interacts with ACK1 and can regulate the ubiquitinylation of ACK1 and its subsequent degradation by proteasomes (right panel).

Discussion

ACK1 is a 140 kDa Cdc42-associated nonreceptor tyrosine kinase that is activated by multiple extracellular stimuli.32, 33, 44, 45 It is overexpressed in many cancer types and is involved in tumorigenesis.31, 33, 46 For example, activated ACK1 promotes prostate cancer progression by activation of the androgen receptor and degradation of the tumor suppressor WWOX.31, 32, 47 Moreover, ACK1 enhances oncogenic epidermal growth factor (EGFR) signaling and was shown to increase proliferation and invasiveness of renal and breast cancer cells.48, 49 In a murine breast cancer metastasis model, ACK1 overexpression tied in with increased mortality of the mice.46 Due to these oncogenic properties, ACK1 has emerged as a valid target for cancer therapy.34, 50

We show here for the first time that ACK1 can be subjected to SIAH-dependent proteasomal degradation. Earlier, lysosomal degradation of ACK1 was reported. The HECT E3 ubiquitin ligase Nedd4-1 was shown to mediate an EGFR activation-dependent degradation of ACK1 via this pathway.28 Interestingly, the downregulation of ACK1 by Nedd4-2 requires ACK1 kinase activity.51 SIAH proteins interact with and degrade ACK1 regardless of its kinase activity. The results of our study hence suggest that two distinct biochemical pathways (that is, via lysosomes or proteasomes) exist for ACK1 in vivo. We additionally show that the SIAH binding motif located in the very C-terminus of ACK1 dictates its SIAH-dependent turnover. This so-called SIAH degron motif is an established interaction surface for SIAHs and their targets.1, 2, 35 Nevertheless, we cannot exclude that additional factors may be required for the interaction of SIAHs and ACK1. An example is SIAH1 interacting protein/CacyBP that has been shown to mediate interactions of SIAH1 with other proteins.11, 12 Experiments addressing this possibility are underway and will decipher the details of the SIAH-ACK1 interaction in more detail. Interestingly, we detected lower SIAH1 and SIAH2 levels when coexpressed with degradable wild-type ACK1, and higher levels of the SIAHs in the presence of nondegradable, binding-deficient wACK1 or ACK1V909G. Apparently, ACK1 has a negative effect on SIAHs.

Activation of the ER has been reported to induce the expression of SIAH2 in breast cancer cells.9, 38, 39 Here, we demonstrate that the upregulation of SIAH2 by estrogen negatively affects the levels of ACK1 in the luminal breast cancer cells MCF-7 and T47D. These findings suggest that induced SIAH2 regulates endogenous ACK1 by accelerating its proteasomal turnover. Since both SIAH1 and SIAH2 can reduce ACK1, modulating their expression with small molecular compounds may attenuate oncogenic ACK1 expression. Furthermore, SIAH1 and SIAH2 are induced by the tumor suppressor p531, 19, 20 and this may regulate ACK1 and thereby cancer cell growth. To test a putative role for p53 in the estrogen-dependent control of SIAH2, we used T47D cells. Like MCF-7 cells, T47D cells harbor a functional ER41 but contrary to MCF-7 cells, T47D cells lack functional p53.42 We found that T47D cells induce SIAH2 and reduce ACK1 when exposed to estrogen. These data suggest that estrogen does not require intact p53 to induce SIAH2 in luminal breast cancer cells. On the other hand, it needs to be stressed that these data do not exclude that p53 is necessary to activate SIAHs in response to other stimuli.

Data collected with ER-negative MDA-MB-231 cells43 propose that estrogen requires functional ER signaling to activate the expression of SIAH2. Consistently, it was found that the ER antagonist ICI182,780 prevents the accumulation of SIAH2 in estrogen-stimulated MCF-7 cells and that SIAH2 is induced by estrogen in several human ER-positive breast cancer cell lines (MCF-7, ZR75-1, BT474).9, 38, 39 Curiously, the stimulation of estrogen-dependent ZR75-1/HERc cells with EGFR opposed the induction of SIAH2 by estrogen.38 It is possible that modulators of estrogen signaling disrupt such mechanisms and this might in turn promote ACK1 induction linked to EGFR signaling.38, 52 Truly, other effects such as the suppressive effect of SIAH2 on the tumor suppressor C/EBPδ53 may also play an important role in the vast complexity of breast tumorigenesis. Further experiments are necessary to fully elucidate the many roles that SIAH proteins play during cancer development.

In summary, we reveal a novel molecular mechanism regulating the expression of ACK1. We demonstrate that the ubiquitin ligases SIAH1 and SIAH2 control the protein stability of this kinase. These results might have implications for tumor therapy, especially in designing small molecules targeting ACK1 activity or stability.

Materials and methods

Drugs and chemicals

The proteasome inhibitor Z-Leu-Leu-Leu-al (MG132), N-ethyl-maleimide, estrogen (17β-estradiol), doxycycline hyclate (doxocycline), cycloheximide and polyethylenimine were purchased from Sigma-Aldrich (Steinheim, Germany); clasto-lactacystin β-lactone (lactacystin) was purchased from Axxora Alexis (Lörrach, Germany).

Cell culture

HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 1% penicillin/streptomycin and 2% L-glutamine. MCF-7 cells were maintained in Rosewell Park Memorial Institute (RPMI) medium with the same additives. For experiments with estrogen, MCF-7, T47D and MDA-MB-231 cells were cultured for a minimum of 6 days as described,9 in phenol-red free RPMI medium containing 1% penicillin/streptomycin, 2% L-glutamine and 5% charcoal stripped fetal calf serum (PAA Laboratories, Cölbe, Germany). All cell lines were cultured at 37 °C in a 5% CO2 atmosphere. MCF-7 and T47D cells are of the luminal A type (ER+, prolactin receptor+/– and EGFR HER2–) and MDA-MB-231 cells are of the claudin-low type (ER–, prolactin receptor– and EGFR HER2–).41, 43 MCF-7 cells express wild-type p53 and T47D cells lack functional p53.42

Construction of DNA Plasmids and mutagenesis; Yeast two-hybrid screening

Details on these materials and methods are provided in the Supplementary Methods section accompanying the article.

Transfection assays and RNA interference

HEK293T cells were transfected with polyethylenimine as described.16 Unless otherwise stated, transient protein expression in HEK293T cells was carried out for 48 h using the following amounts of cDNA: Myc-tagged ACK1, caACK1, kdACK1, wACK1 and ACK1V909G (0.5 μg); SIAH1, SIAH2, MDM2, RLIM-HA and TRIAD-Myc (0.2 μg). A plasmid encoding green fluorescent protein (green fluorescent protein, 0.05 μg) was cotransfected to monitor transfection efficiency. Empty vector pcDNA3.1 was used to obtain equal amounts of transfected DNA (total of 1 μg per 5 × 105 cells). The knockdown of SIAH1 and SIAH2 levels in HEK293T cells was performed by transfecting 40 pmol siRNA targeting SIAH1 and SIAH2 or nontargeting control siRNA (siSIAH1/2, sc-44102; siRNA-A, sc-37007; Santa Cruz Biotechnology, Heidelberg, Germany) with lipofectamine2000 for 48 h (manufacturer’s protocol; Invitrogen, Darmstadt, Germany). Myc-tagged ACK1, ACK1V909G and the SIAH2 specific shRNA were expressed in MCF-7 cells by transfection of 2–4 μg plasmid-DNA with Attractene (Qiagen, manufacturer’s protocol). siRNA-mediated knockdown in MCF-7 cells was carried out by nucleofection. 40 pmol siRNA or 2 μg plasmid were transfected with the Amaxa Nucleofector Kit as recommended by the manufacturer (Lonza, Köln, Germany) using solution V and program E-14.

Immunoblotting and immunoprecipitation

Cell lysis, SDS–PAGE and immunoblotting were described by us previously.15, 17 All the lysates were assessed by Bradford assay for protein concentrations. Tubulin served as loading control for all immunoblots.

For immunoprecipitation of SIAH1 or SIAH2, the harvested cells were lysed in NaCl-EDTA-Tris-Nonidet (NETN) buffer.15, 17 300 μg of total lysate protein was added to 1 μg of antibody and 10 μl protein-G sepharose beads (GE Healthcare, München, Germany). For the detection of phosphorylated ACK1, the buffers were supplemented with 5 mM sodium-fluoride (NaF) and 1 mM sodium-orthovanadate (Na3OV4). Immunoprecipitation volumes were adjusted to 600 μl with NETN buffer. Precipitation was performed under continuous rotation for 16 h at 4 °C. The beads were washed and bound proteins were eluted with Laemmli buffer and analyzed by immunoblotting. For the detection of ubiquitinylated ACK1, all the buffers were supplemented with 10 mM N-ethyl-maleimide to inhibit deubiquitinylating isopeptidases and 10 μM MG132 to block proteasome-mediated degradation.

Antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany; ACK1, sc-28336; SIAH1, sc-5506; SIAH2, sc-5507; green fluorescent protein, sc-9996), New England Biolabs (Frankfurt/Main, Germany; Myc-Tag, #2276; Phospho-Tyrosine p-Tyr-100, #9411), Sigma-Aldrich (tubulin, T5168; Ubiquitin, U5379), Covance (Freiburg im Breisgau, Germany; HA-Tag, MMs-101P); Calbiochem (Darmstadt, Germany; MDM2, OP115).

GST pulldown assays and quantitative real-time PCR

Details on these methods are provided in the Supplementary Methods section accompanying the article.

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Acknowledgements

We thank C. Kosan, M. Korfei and S. Scheiding for their discussions and their excellent help. We are grateful to M. Kögl, N. Varin-Blank, Z. Ronai, R. Marschalek, H. Bursen, A. Baniahmad, O. Werz and O. Huber for providing material. Grant support: German Cancer Aid (FKZ102362); Wilhelm-Sander Foundation (No. 2010.078.1).

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Correspondence to O H Krämer.

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Buchwald, M., Pietschmann, K., Brand, P. et al. SIAH ubiquitin ligases target the nonreceptor tyrosine kinase ACK1 for ubiquitinylation and proteasomal degradation. Oncogene 32, 4913–4920 (2013). https://doi.org/10.1038/onc.2012.515

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Keywords

  • ACK1
  • estrogen
  • proteasome
  • SIAH1
  • SIAH2
  • TNK2

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