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.
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.
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.
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).
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.
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
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
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.
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).
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.
House CM, Möller A, Bowtell DD . Siah proteins: novel drug targets in the Ras and hypoxia pathways. Cancer Res 2009; 69: 8835–8838.
Krämer OH, Stauber RH, Bug G, Hartkamp J, Knauer SK . SIAH proteins: Critical roles in leukemogenesis. Leukemia 2012, (in press).
Liu M, Hsu J, Chan C, Li Z, Zhou Q . The ubiquitin ligase SIAH1 controls Ell2 stability and formation of super elongation complexes to modulate gene transcription. Mol Cell 2012; 46: 325–334.
Metzger MB, Hristova VA, Weissman AM . HECT and RING finger families of E3 ubiquitin ligases at a glance. J Cell Sci 2012; 125: 531–537.
Mogk A, Schmidt R, Bukau B . The N-end rule pathway for regulated proteolysis: prokaryotic and eukaryotic strategies. Trends Cell Biol 2007; 17: 165–172.
Zhao HL, Ueki N, Hayman MJ . The Ski protein negatively regulates Siah2-mediated HDAC3 degradation. Biochem Biophys Res Commun 2010; 399: 623–628.
Perissi V, Scafoglio C, Zhang J, Ohgi KA, Rose DW, Glass CK et al. TBL1 and TBLR1 phosphorylation on regulated gene promoters overcomes dual CtBP and NCoR/SMRT transcriptional repression checkpoints. Mol Cell 2008; 29: 755–766.
Wu H, Lin Y, Shi Y, Qian W, Tian Z, Yu Y et al. SIAH-1 interacts with mammalian polyhomeotic homologues HPH2 and affects its stability via the ubiquitin-proteasome pathway. Biochem Biophys Res Commun 2010; 397: 391–396.
Frasor J, Danes JM, Funk CC, Katzenellenbogen BS . Estrogen down-regulation of the corepressor N-CoR: mechanism and implications for estrogen derepression of N-CoR-regulated genes. Proc Natl Acad Sci USA 2005; 102: 13153–13157.
Venables JP, Dalgliesh C, Paronetto MP, Skitt L, Thornton JK, Saunders PT et al. SIAH1 targets the alternative splicing factor T-STAR for degradation by the proteasome. Hum Mol Genet 2004; 13: 1525–1534.
Nagano Y, Fukushima T, Okemoto K, Tanaka K, Bowtell DD, Ronai Z et al. Siah1/SIP regulates p27(kip1) stability and cell migration under metabolic stress. Cell Cycle 2011; 10: 2592–2602.
Fukushima T, Zapata JM, Singha NC, Thomas M, Kress CL, Krajewska M et al. Critical function for SIP, a ubiquitin E3 ligase component of the beta-catenin degradation pathway, for thymocyte development and G1 checkpoint. Immunity 2006; 24: 29–39.
Bursen A, Moritz S, Gaussmann A, Dingermann T, Marschalek R . Interaction of AF4 wild-type and AF4.MLL fusion protein with SIAH proteins: indication for t(4;11) pathobiology? Oncogene 2004; 23: 6237–6249.
Habelhah H, Frew IJ, Laine A, Janes PW, Relaix F, Sassoon D et al. Stress-induced decrease in TRAF2 stability is mediated by Siah2. EMBO J 2002; 21: 5756–5765.
Buchwald M, Pietschmann K, Müller JP, Böhmer FD, Heinzel T, Krämer OH . Ubiquitin conjugase UBCH8 targets active FMS-like tyrosine kinase 3 for proteasomal degradation. Leukemia 2010; 24: 1412–1421.
Krämer OH, Müller S, Buchwald M, Reichardt S, Heinzel T . Mechanism for ubiquitylation of the leukemia fusion proteins AML1-ETO and PML-RARalpha. Faseb J 2008; 22: 1369–1379.
Pietschmann K, Buchwald M, Müller S, Knauer SK, Kögl M, Heinzel T et al. Differential regulation of PML-RARalpha stability by the ubiquitin ligases SIAH1/SIAH2 and TRIAD1. Int J Biochem Cell Biol 2012; 44: 132–138.
Pietschmann K, Bolck HA, Buchwald M, Spielberg S, Polzer H, Spiekermann K et al. Breakdown of the FLT3-ITD/STAT5 axis and synergistic apoptosis induction by the histone deacetylase inhibitor Panobinostat and FLT3-specific inhibitors. Mol Cancer Ther 2012, (in press).
Winter M, Sombroek D, Dauth I, Moehlenbrink J, Scheuermann K, Crone J et al. Control of HIPK2 stability by ubiquitin ligase Siah-1 and checkpoint kinases ATM and ATR. Nat Cell Biol 2008; 10: 812–824.
Calzado MA, de la Vega L, Möller A, Bowtell DD, Schmitz ML . An inducible autoregulatory loop between HIPK2 and Siah2 at the apex of the hypoxic response. Nat Cell Biol 2009; 11: 85–91.
Yun S, Möller A, Chae SK, Hong WP, Bae YJ, Bowtell DD et al. Siah proteins induce the epidermal growth factor-dependent degradation of phospholipase Cepsilon. J Biol Chem 2008; 283: 1034–1042.
Wen YY, Yang ZQ, Song M, Li BL, Yao XH, Chen XL et al. The expression of SIAH1 is downregulated and associated with Bim and apoptosis in human breast cancer tissues and cells. Mol Carcinog 2010; 49: 440–449.
Wen YY, Yang ZQ, Song M, Li BL, Zhu JJ, Wang EH . SIAH1 induced apoptosis by activation of the JNK pathway and inhibited invasion by inactivation of the ERK pathway in breast cancer cells. Cancer Sci 2010; 101: 73–79.
Nadeau RJ, Toher JL, Yang X, Kovalenko D, Friesel R . Regulation of Sprouty2 stability by mammalian Seven-in-Absentia homolog 2. J Cell Biochem 2007; 100: 151–160.
Depaux A, Regnier-Ricard F, Germani A, Varin-Blank N . Dimerization of hSiah proteins regulates their stability. Biochem Biophys Res Commun 2006; 348: 857–863.
Xu Z, Sproul A, Wang W, Kukekov N, Greene LA . Siah1 interacts with the scaffold protein POSH to promote JNK activation and apoptosis. J Biol Chem 2006; 281: 303–312.
Ahmed AU, Schmidt RL, Park CH, Reed NR, Hesse SE, Thomas CF et al. Effect of disrupting seven-in-absentia homolog 2 function on lung cancer cell growth. J Natl Cancer Inst 2008; 100: 1606–1629.
Lin Q, Wang J, Childress C, Sudol M, Carey DJ, Yang W . HECTE3 ubiquitin ligase Nedd4-1 ubiquitinates ACK and regulates epidermal growth factor (EGF)-induced degradation of EGF receptor and ACK. Mol Cell Biol 2010; 30: 1541–1554.
Korzeniewski N, Cuevas R, Duensing A, Duensing S . Daughter centriole elongation is controlled by proteolysis. Mol Biol Cell 2010; 21: 3942–3951.
Khurana A, Nakayama K, Williams S, Davis RJ, Mustelin T, Ronai Z . Regulation of the ring finger E3 ligase Siah2 by p38 MAPK. J Biol Chem 2006; 281: 35316–35326.
Mahajan NP, Whang YE, Mohler JL, Earp HS . Activated tyrosine kinase Ack1 promotes prostate tumorigenesis: role of Ack1 in polyubiquitination of tumor suppressor Wwox. Cancer Res 2005; 65: 10514–10523.
Mahajan NP, Liu Y, Majumder S, Warren MR, Parker CE, Mohler JL et al. Activated Cdc42-associated kinase Ack1 promotes prostate cancer progression via androgen receptor tyrosine phosphorylation. Proc Natl Acad Sci USA 2007; 104: 8438–8443.
Mahajan K, Coppola D, Challa S, Fang B, Chen YA, Zhu W et al. Ack1 mediated AKT/PKB tyrosine 176 phosphorylation regulates its activation. PLoS One 2010; 5: e9646.
Mahajan K, Mahajan NP . Shepherding AKT and androgen receptor by Ack1 tyrosine kinase. J Cell Physiol 2010; 224: 327–333.
House CM, Hancock NC, Möller A, Cromer BA, Fedorov V, Bowtell DD et al. Elucidation of the substrate binding site of Siah ubiquitin ligase. Structure 2006; 14: 695–701.
Twomey E, Li Y, Lei J, Sodja C, Ribecco-Lutkiewicz M, Smith B et al. Regulation of MYPT1 stability by the E3 ubiquitin ligase SIAH2. Exp Cell Res 2010; 316: 68–77.
Yokoyama N, Miller WT . Biochemical properties of the Cdc42-associated tyrosine kinase ACK1. Substrate specificity, authphosphorylation, and interaction with Hck. J Biol Chem 2003; 278: 47713–47723.
Jansen MP, Ruigrok-Ritstier K, Dorssers LC, van Staveren IL, Look MP, Meijer-van Gelder ME et al. Downregulation of SIAH2, an ubiquitin E3 ligase, is associated with resistance to endocrine therapy in breast cancer. Breast Cancer Res Treat 2009; 116: 263–271.
Stebbing J, Filipovic A, Lit LC, Blighe K, Grothey A, Xu Y et al. LMTK3 is implicated in endocrine resistance via multiple signaling pathways. Oncogene 2012, (in press).
Krämer OH, Zhu P, Ostendorff HP, Golebiewski M, Tiefenbach J, Peters MA et al. The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2. Embo J 2003; 22: 3411–3420.
Lu M, Mira-y-Lopez R, Nakajo S, Nakaya K, Jing Y . Expression of estrogen receptor alpha, retinoic acid receptor alpha and cellular retinoic acid binding protein II genes is coordinately regulated in human breast cancer cells. Oncogene 2005; 24: 4362–4369.
Li C, Lin M, Liu J . Identification of PRC1 as the p53 target gene uncovers a novel function of p53 in the regulation of cytokinesis. Oncogene 2004; 23: 9336–9347.
Holliday DL, Speirs V . Choosing the right cell line for breast cancer research. Breast Cancer Res 2011; 13: 215.
Prieto-Echague V, Miller WT . Regulation of ack-family nonreceptor tyrosine kinases. J Signal Transduct 2011; 2011: 742372.
Pao-Chun L, Chan PM, Chan W, Manser E . Cytoplasmic ACK1 interaction with multiple receptor tyrosine kinases is mediated by Grb2: an analysis of ACK1 effects on Axl signaling. J Biol Chem 2009; 284: 34954–34963.
van der Horst EH, Degenhardt YY, Strelow A, Slavin A, Chinn L, Orf J et al. Metastatic properties and genomic amplification of the tyrosine kinase gene ACK1. Proc Natl Acad Sci USA 2005; 102: 15901–15906.
Mahajan K, Challa S, Coppola D, Lawrence H, Luo Y, Gevariya H et al. Effect of Ack1 tyrosine kinase inhibitor on ligand-independent androgen receptor activity. Prostate 2010; 70: 1274–1285.
Chua BT, Lim SJ, Tham SC, Poh WJ, Ullrich A . Somatic mutation in the ACK1 ubiquitin association domain enhances oncogenic signaling through EGFR regulation in renal cancer derived cells. Mol Oncol 2010; 4: 323–334.
Howlin J, Rosenkvist J, Andersson T . TNK2 preserves epidermal growth factor receptor expression on the cell surface and enhances migration and invasion of human breast cancer cells. Breast Cancer Res 2008; 10: R36.
Mahajan K, Coppola D, Chen YA, Zhu W, Lawrence HR, Lawrence NJ et al. Ack1 tyrosine kinase activation correlates with pancreatic cancer progression. Am J Pathol 2012; 180: 1386–1393.
Chan W, Tian R, Lee YF, Sit ST, Lim L, Manser E . Down-regulation of active ACK1 is mediated by association with the E3 ubiquitin ligase Nedd4-2. J Biol Chem 2009; 284: 8185–8194.
Osborne CK, Neven P, Dirix LY, Mackey JR, Robert J, Underhill C et al. Gefitinib or placebo in combination with tamoxifen in patients with hormone receptor-positive metastatic breast cancer: a randomized phase II study. Clin cancer res 2011; 17: 1147–1159.
Sarkar TR, Sharan S, Wang J, Pawar SA, Cantwell CA, Johnson PF et al. Identification of a Src tyrosine kinase/SIAH2 E3 ubiquitin ligase pathway that regulates C/EBPdelta expression and contributes to transformation of breast tumor cells. Mol Cell Biol 2012; 32: 320–332.
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).
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
About this article
Cite this article
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
BioMed Research International (2019)
Cell Reports (2019)
Design and biological evaluation of tetrahydro-β-carboline derivatives as highly potent histone deacetylase 6 (HDAC6) inhibitors
European Journal of Medicinal Chemistry (2018)
Biochimica et Biophysica Acta (BBA) - General Subjects (2017)