Siah Proteins Induce the Epidermal Growth Factor-dependent Degradation of Phospholipase Cϵ*

Phospholipase Cϵ (PLCϵ) is activated by various growth factors or G-protein-coupled receptor ligands via different activation mechanisms. The Ras association (RA) domain of PLCϵ is known to be important for its ability to bind with Ras-family GTPase upon growth factor stimulation. In the present study, we identified Siah1 and Siah2 as novel binding partners of the PLCϵ RA domain. Both Siah1 and Siah2 interacted with the RA2 domain of PLCϵ, and the mutation of Lys-2186 of the PLCϵ RA2 domain abolished this association. Moreover, Siah induced the ubiquitination and degradation of PLCϵ upon epidermal growth factor (EGF) stimulation, and Siah proteins were phosphorylated on multiple tyrosine residues via an Src-dependent pathway upon EGF treatment. The Src inhibitor abolished the EGF-dependent ubiquitination of PLCϵ, and the Siah1 phosphorylation-deficient mutant could not increase the EGF-dependent ubiquitination and degradation of PLCϵ. The EGF-dependent degradation of PLCϵ was blocked in mouse embryonic fibroblast (MEF) cells derived from Siah1a/Siah2 double knockout mice, and the extrinsic expression of wild-type Siah1 restored the degradation of PLCϵ, whereas the phosphorylation-deficient mutant did not. Siah1 expression abolished PLCϵ-dependent potentiation of EGF-dependent cell growth. In addition, the expression of wild-type Siah1 in Siah1a/Siah2-double knockout MEF cells inhibited EGF-dependent cell growth, and this inhibition was abolished by PLCϵ knockdown. Our results suggest that the Siah-dependent degradation of PLCϵ plays a role in the regulation of growth factor-dependent cell growth.

PLC⑀ plays a role in the interplay between PLC and small GTPases. Various G-proteins directly activate PLC⑀. For example, RhoA was found to stimulate PLC⑀ activity by interacting with a 65-amino acid insert within the catalytic core of PLC⑀ (4). Lysophosphatidic acid and thrombin stimulates PLC⑀ by activating G␣ 12 and/or G␣ 13 and downstream RhoA (5). Moreover, Ras-family GTPases activate PLC⑀ by binding with the RA domain of PLC⑀. Adrenaline and prostaglandin E 1 have been reported to activate PLC⑀ by triggering adenylyl cyclase-coupled receptors, and Rap2B, which is regulated by Epac (a guanine-nucleotide-exchange factor regulated by cAMP), has been found to associate with the RA domain of PLC⑀ during PLC⑀ activation (6,7). In addition, EGF treatment was found to induce an association between the PLC⑀ RA domain and activated Ras, and this resulted in the recruitment of PLC⑀ into the plasma membrane for activation (8).
Several physiological studies have indicated that PLC⑀ is involved in development and cell growth. PLC⑀ knockout mice had cardiac dysfunction resulting from defective heart development or were susceptible to hypertrophy in response to chronic cardiac stress (9,10). Recently, mutations of PLC⑀ in individuals with severe nephrotic syndrome were identified, and PLC⑀ knockdown in zebra fish led to a loss of the filtration barrier maintained by glomerular podocytes, which in combination demonstrate the importance of PLC⑀ in kidney development and function (11). Moreover, ablation of PLC⑀ activity in mice led to reduced carcinogen-induced skin tumor formation, providing evidence that PLC⑀ plays a positive role in tumor cell growth (12). Furthermore, the overexpression of PLC⑀ in BaF3 cells induced platelet-derived growth factor-dependent cell growth (13).
Siah proteins are homologues of Drosophila SINA, which is a ring-finger protein involved in R7 cell development in the eye (14). Three murine (Siah1a, Siah1b, and Siah2) and two human (SIAH1 and SIAH2) homologues have been identified. The mammalian Siah proteins are highly homologous; Siah1a and Siah1b are 98% identical, whereas Siah1 proteins and Siah2 protein diverge significantly only at their N termini (15,16). Siah proteins are RING finger proteins with E3 ligase activity and have been implicated in the ubiquitination and proteasomedependent degradation of various substrate molecules. Substrates of Siah proteins are quite diverse and include transcriptional regulators (17)(18)(19), membrane receptors (20,21), a microtubule-associated motor protein (22), and other proteins. In particular, the involvement of Siah proteins in cell growth regulation has been suggested in many reports. Siah1 expression is induced by tumor suppressor p53 in mammals and the overexpression of Siah1 inhibits cell proliferation and promotes apoptosis (23)(24)(25). Moreover, Siah-induced ␤-catenin degradation is important for the negative regulation of cell proliferation (26,27), and the Siah-induced degradation of Kid is important for mitosis and contributes to cell growth arrest (28). Furthermore, mutations of Siah proteins in several cancers have been reported (29). These reports imply that Siah has tumor suppressor functions in some experimental settings.
To identify novel PLC⑀ regulatory proteins, we performed yeast two-hybrid analysis using the RA domain of PLC⑀, and Siah1 and Siah2 were identified as PLC⑀-binding proteins. Here, we demonstrate that Siah proteins induce the proteasomal degradation of PLC⑀ after EGF stimulation. Src-dependent phosphorylation was found to be required for the EGF-dependent degradation of PLC⑀. Moreover, the Siah-dependent degradation of PLC⑀ was found to act as an important negative regulator of PLC⑀-dependent cell growth.

MATERIALS AND METHODS
Antibodies-Rabbit polyclonal antibody of PLC⑀ was obtained from Dr. Tohru Kataoka (Kobe University, Japan). Mouse monoclonal antibody of Siah1 was described previously (30). Other antibodies used were: goat polyclonal anti-Siah2 antibody and goat polyclonal anti-AH FIGURE 1. Interaction between PLC⑀ and the Siah proteins. A, COS-7 cells were transfected with HA-Siah1/ FLAG-PLC⑀ or HA-Siah2/FLAG-PLC⑀. The proteasomal inhibitor MG132 was added to the media 12 h before cell lysis to inhibit the self-degradation of the Siah proteins. Siah proteins were immunoprecipitated with ␣-HA antibody, and immunocomplexes were subjected to immunoblotting with the indicated antibodies (TCL, total cell lysates). B, COS-7 cells were transfected with Myc-Siah2 and the indicated PLC isozymes. PLC isozymes were immunoprecipitated with ␣-FLAG antibody and the immunocomplexes were subjected to immunoblotting with ␣-Myc antibody to detect co-immunoprecipitated Siah2. C, MEF cells were incubated with MG132 (10 M) for 12 h. Cell lysates were prepared, and Siah2 was immunoprecipitated with ␣-Siah2 antibody. The immunocomplexes were subjected to immunoblotting with the indicated antibodies. Goat ␣-AH receptor antibody was used for the control antibody. receptor antibody (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-FLAG antibody (Sigma), mouse monoclonal anti-Myc antibody (Invitrogen), mouse monoclonal anti-HA antibody (Sigma, St. Louis, MO), Rhodamine-conjugated goat anti-rabbit IgG and fluorescein isothiocyanate-conjugated goat antimouse IgG, were purchased from Sigma. Horseradish peroxidaseconjugated goat anti-rabbit IgG and goat anti-mouse IgA, IgM, and IgG were from Kirkegaard & Perry Laboratories (Gaithersburg, MD).
Cell Culture-COS-7 cells were grown in DMEM containing 10% bovine calf serum, antibiotics, and glutamine. Wild-type and Siah-deficient MEF cells were grown in DMEM containing 10% fetal bovine serum, 0.02 mM ␤-mercaptoethanol, antibiotics, and glutamine as previously described (30). Cells were grown to ϳ90% confluence for immunoprecipitation and Western blot experiments.
Yeast Two-hybrid Screening-PLC⑀ RA domains (amino acids 1990 -2218) were cloned into the pLexA (BD Clontech) in-frame with the LexA DNA-binding domain (referred to as pLexA-PLC⑀). The yeast strain, EGY48, carrying a reporter gene was cotransformed with the bait plasmid, pLexA-PLC⑀, and a human HeLa cDNA library fused to the VP16 activation domain. Transformation was carried out using the lithium acetate method (31). Leucine-positive colonies were identified by a filter-lifting assay for ␤-galactosidase activity. Library-derived DNA was prepared from candidate clones and analyzed by DNA sequencing.
PLC⑀ Phosphorylation Analysis-COS-7 cell-transfected Siah proteins were serum-starved for 20 h and then incubated for another 12 h. Cells were treated with EGF (100 ng/ml) for the indicated times. Cell lysates were prepared, and PLC⑀ was immunoprecipitated with ␣-FLAG antibody. The immunocomplexes were subjected to immunoblotting with ␣-Myc antibody to detect co-immunoprecipitated Siah2. B, COS-7 cells were transfected with Myc-Siah2 and FLAG-PLC⑀ in the presence or absence of HA-Ras mutants (RasV12, constitutively active mutant; RasN17, dominant negative mutant). After serum starvation and incubation with MG132 (10 M), the cells were stimulated with EGF (100 ng/ml) for 30 min. Cell lysates were prepared, and PLC⑀ was immunoprecipitated with ␣-FLAG antibody.

FIGURE 4. EGF-induced ubiquitination and degradation of PLC⑀.
A, COS-7 cells were transfected with HAubiquitin, FLAG-PLC⑀, and Myc-Siah2. After serum starvation for 12 h, the cells were incubated with MG132 (10 M) for an additional 12 h. The cells were stimulated with EGF (100 ng/ml) for the indicated times. PLC⑀ was immunoprecipitated with ␣-FLAG antibody, and PLC⑀ ubiquitination was detected with ␣-HA antibody. B, COS-7 cells were transfected with Myc-Siah2 and FLAG-PLC⑀. After serum starvation for 24 h, the cells were pretreated with cycloheximide (10 g/ml) for 1 h in the presence or absence of MG132 (10 M), and then EGF (100 ng/ml) was added to media for the indicated times (CHX, cycloheximide). Cell lysates were prepared, and the level of PLC⑀ was monitored by immunoblotting with ␣-PLC⑀ antibody. C, COS-7 cells were transfected with wild-type FLAG-PLC⑀ or the K2186A mutant in the presence of HA-ubiquitin and Myc-Siah2. PLC⑀ ubiquitination was detected after stimulation with EGF (100 ng/ml) for 60 min. D, COS-7 cells were transfected with FLAG-PLC⑀ wild type or the K2186A mutant in the presence of Myc-Siah2. After serum starvation for 24 h, cells were pretreated with cycloheximide (10 g/ml) for 1 h and then treated with EGF (100 ng/ml) for the indicated times. Cell lysates were prepared and subjected to SDS-PAGE and immunoblotting with ␣-PLC⑀ antibody. The remaining PLC⑀ level after EGF stimulation for the indicated time was quantified and expressed as a percentage of the PLC⑀ level of unstimulated control cells. The results are shown as the means Ϯ S.D. (n ϭ 3). with 1 mCi of [ 32 P]orthophosphate in 2 ml of phosphate-free DMEM for 4 h at 37°C. Cells were treated with 100 ng/ml EGF for the indicated times. Cell lysates were prepared, and Siah proteins were immunoprecipitated. The immunocomplexes were subjected to SDS-PAGE and autoradiography. The amounts of immunoprecipitated Siah proteins were measured by immunoblotting and used for the quantitation of relative Siah phosphorylation.
Cell Growth Assay-MEF cells were seeded in triplicate into 6-well plates at a density of 2 ϫ 10 5 cells per well and were transfected with PLC⑀ siRNA or control scrambled siRNA. After 24 h, cells were incubated with serum-free DMEM for 24 h to reach quiescence. The cells were incubated in serum-free medium supplemented with 100 ng/ml EGF for 18 h prior to addition of 3 H-labeled thymidine for additional 6 h. Thymidine incorporation was measured as previously reported (32).
Plasmid Construction and Mutagenesis-FLAG-tagged mouse PLC⑀ DNA is a generous gift from Dr. Tohru Kataoka (Kobe University, Japan). For the construction of Siah binding-deficient mutant, evolutionally conserved VLK (2173-2175) sequence was changed into AAA (called PLC⑀ 3A) or Lys-2186 residue was changed into alanine (called PLC⑀ K2186A) by site-directed mutagenesis. PLC⑀ K2151E/K2153E is a Ras bindingdeficient mutant. Siah1 wild-type and Y100F/Y126F mutant DNA was kindly provided by Dr. Zhiheng Xu (Columbia University) and introduced into pcDNA-HA vector by PCR amplification. To construct phosphorylation-deficient mutant of Siah1, Tyr-47, Tyr-199, and Tyr-223 were changed into phenylalanines (called Siah1 TM). For expression of Siah1 in knockout MEF cells, wild-type Siah1 or Siah1 TM were PCR-amplified and cloned into a lentivirusderived C-FUW vector (33).

Interaction between PLC⑀ and
Siah Proteins-We sought to identify binding partners of the PLC⑀ RA domain and performed a yeast two-hybrid analysis using bait containing the serial RA1 and RA2 domain of PLC⑀. Our yeast two-hybrid analysis revealed that the RA domain of PLC⑀ interacts with various proteins other than small GTPases. Siah1 and Siah2 were identified as novel PLC⑀ RA domain-binding proteins. The positive clones obtained from a HeLa cell cDNA library contained the substrate binding domain of the Siah proteins (Siah1, 177-282; Siah2, 217-324). We examined whether PLC⑀ interacts with Siah proteins in cells by using co-immunoprecipitation analysis. Both Siah1 and Siah2 were found to associate with PLC⑀ in COS-7 cells (Fig. 1A), but Siah2 did not interact with PLC-␥1 or PLC-␤1 (Fig. 1B). These results indicate that Siah proteins interact with PLC⑀ by specifically recognizing the RA domain, which is present only in the PLC⑀ isozyme.
We then investigated whether endogenous PLC⑀ and Siah2 can form a complex in MEF cells. As shown in Fig. 1C, Siah2 was immunoprecipitated with ␣-Siah2 antibody and the immunocomplex contained PLC⑀, which indicates that PLC⑀-Siah2 complex exists under physiological conditions.  JANUARY (36). In the present study, we explored the binding region of the RA domains in detail. Glutathione S-transferasepulldown analysis revealed that the RA2 domain is responsible for the interaction with Siah2, and further deletion analysis enabled us to narrow this down to several amino acids ( Fig. 2A). We mutated several evolutionally conserved amino acids and examined their ability to interact with Siah2. In particular, the mutation of Lys-2186 into Ala led to the 85% inhibition of the interaction between PLC⑀ and Siah2, whereas mutation of VLK (2173-2175) into AAA had little effect on the interaction with Siah2. In addition, the Ras binding-deficient mutant of PLC⑀ (K2151E/K2153E) had almost the same affinity for Siah2 as did the wild type (Fig. 2B). According to previous structural analysis of the PLC⑀ RA domain (36), the Siah binding region of PLC⑀ corresponds to the loop between ␤3 and ␤4 of the RA2 domain, which is removed from the Ras binding region (␤1, ␤2, and the ␣1-␤3 loop of RA2). In fact, the K2186A mutant showed no difference in binding affinity for the constitutively active H-RasV12 mutant (Fig. 2C). Taken together, these results support the possibility that the RA2 domain has a specific Siah binding region that is quite distinct from the Ras binding region.

Regulation of PLC⑀ by Siah-mediated Ubiquitination
EGF Stimulation Induced the Interaction between Siah2 and PLC⑀-EGF stimulation evokes PLC⑀ activity through Ras-dependent translocation to the plasma membrane (8). In the present study, we investigated whether EGF treatment affects the interaction between Siah and PLC⑀. Interestingly, EGF treatment induced an association between PLC⑀ and Siah2 in COS-7 cells (Fig. 3A). The interaction was initially detected at 5 min after EGF stimulation and was most prominent at 30 min after stimulation. We then investigated whether Rasdependent activation of PLC⑀ is required for the EGF-dependent association between Siah2 and PLC⑀. To this end, PLC⑀ activation was blocked by the expression of a dominant negative mutant, RasN17. The expression of the RasN17 mutant abolished EGF-dependent Siah2 binding with PLC⑀ (Fig. 3B), which indicates that PLC⑀ activation is required for the association with Siah2. Our findings demonstrate that Siah2 associates with activated PLC⑀ after EGF stimulation.
EGF Stimulation Induced the Siah-dependent Degradation of PLC⑀-Siah proteins function as E3 ubiquitin ligases and mediate the ubiquitination of various substrate molecules by direct interaction (17)(18)(19)(20)(21)(22). Thus, we investigated whether the EGF-dependent association between Siah proteins and PLC⑀ can lead to the ubiquitination of PLC⑀. We found that PLC⑀ was efficiently ubiquitinated in COS-7 cells upon EGF stimulation (Fig. 4A). Many Siah substrates are known to be subjected to degradation by the ubiquitin-proteasome pathway (17)(18)(19)(20)(21)(22), and in the present study, the expression of Siah2 also led to the EGF-dependent degradation of PLC⑀, which was blocked by co-incubation with MG132 (a proteasomal inhibitor), indicating that Siah-mediated ubiquitination leads to the proteasomal degradation of PLC⑀ (Fig. 4B). The PLC⑀ K2186A mutant was not ubiquitinated by EGF stimulation (Fig. 4C). Concomitantly, the PLC⑀ K2186A mutant was not efficiently degraded by EGF stimulation (Fig. 4D), which demonstrates that the interaction between PLC⑀ and Siah2 is required for the EGF-induced PLC⑀ degradation. These results suggest that PLC⑀ is subjected to Siah-dependent ubiquitination and degradation upon EGF stimulation.
The EGF-dependent Activation of Src Leads to the Phosphorylation of Siah Proteins-The growth factor-dependent ubiquitination of substrate molecules by Siah proteins has not been reported to date. We attempted to identify the upstream mediator responsible for the activation of Siah proteins upon EGF stimulation. It was recently reported that Siah1 is phosphorylated on tyrosine residues by a JNK-dependent pathway after camptothecin treatment, and that this tyrosine phosphorylation plays an important role in the stability and function of Siah1 (37). Thus, we examined whether EGF treatment could induce the tyrosine phosphorylation of Siah proteins. Interestingly, both Siah1 and Siah2 were found to be phosphorylated on tyrosine residues after EGF treatment in COS-7 cells (Fig. 5A). We tested several pharmacological inhibitors to identify poten- , the cells were pretreated with the indicated inhibitors and then treated with EGF (100 ng/ml) for 60 min. PLC⑀ was immunoprecipitated and the immunocomplexes were probed with ␣-HA antibody to detect ubiquitinated PLC⑀. B, COS-7 cells were transfected with FLAG-PLC⑀ and HA-Siah1 wild-type or Siah1 TM. After serum starvation, the cells were pretreated with MG132 (10 M) and then treated with EGF (100 ng/ml) for 60 min. PLC⑀ was immunoprecipitated with ␣-FLAG antibody, and the immunocomplexes were subjected to immunoblotting with ␣-HA antibody. C, COS-7 cells were transfected with FLAG-PLC⑀ and HA-Siah1 wild-type or Siah1 TM. After serum starvation for 24 h, cells were pretreated with cycloheximide (10 g/ml) for 1 h and then treated with EGF (100 ng/ml) for the indicated times. Cell lysates were prepared and subjected to immunoblotting with ␣-PLC⑀ antibody. The remaining PLC⑀ level after EGF stimulation for the indicated time was quantified and expressed as a percentage of the PLC⑀ level of unstimulated control cells. Multiple bands below PLC⑀ position are PLC⑀ degradation products. tial upstream kinases responsible for the EGF-induced phosphorylation of Siah proteins. Src tyrosine kinase inhibitor PP2 blocked the EGF-induced phosphorylation of Siah2 (Fig. 5B), whereas the EGF-dependent phosphorylation of Siah2 was unaffected by SP600125 (a JNK inhibitor), which suggested a novel phosphorylation-dependent means of Siah protein regulation. There are five conserved tyrosine residues in Siah1 and Siah2, and Tyr-100 and Tyr-126 of Siah1 have been reported to be phosphorylated by camptothecin treatment. Thus, we investigated whether Tyr-100 and Tyr-126 of Siah1 are also phosphorylated by EGF stimulation. Mutation of both residues to phenylalanines had no effect on the EGF-dependent phosphorylation of Siah1 (Fig. 5C). Thus, we substituted Tyr-47, Tyr-199, and Tyr-223 for phenylalanine and re-examined EGF-induced phosphorylation. Individual mutations of these tyrosine residues resulted in a partial reduction of the tyrosine phosphorylation of Siah1, but the mutation of all three tyrosine residues completely blocked this phosphorylation (Fig. 5C). These results imply that Siah is phosphorylated at multiple tyrosine residues via a Src-dependent pathway after EGF stimulation.

Siah Phosphorylation Is Required for the EGF-induced
Degradation of PLC⑀-To elucidate the role of the Src-dependent tyrosine phosphorylation of Siah proteins, we pretreated cells with Src tyrosine kinase inhibitor PP2 before EGF stimulation and examined the ubiquitination of PLC⑀ in these cells. Src inhibition led to the suppression of the EGFdependent ubiquitination of PLC⑀, whereas pretreatment of SP600125 had no effect on PLC⑀ ubiquitination (Fig. 6A). To further confirm the role of Siah phosphorylation, we utilized a phosphorylation-deficient mutant of Siah1 (Siah1 TM) that had phenylalanine substitutions at Tyr-47, Tyr-199, and Tyr-223. Siah1 TM did not effectively induce the ubiquitination of PLC⑀ after EGF treatment, whereas wild-type and the Siah1 Y100F/Y126F mutant efficiently ubiquitinated PLC⑀ (Fig. 6B). Concomitantly, the EGF-dependent degradation of PLC⑀ was impaired in the cells transfected with Siah1 TM (Fig.  6C), as compared with the cells transfected with wild-type Siah1. Taken together, these results show that the Src-dependent phosphorylation of Siah is required for the EGF-induced ubiquitination and degradation of PLC⑀. The cells were serum-starved and stimulated with EGF (100 ng/ml) and cycloheximide (10 g/ml) for the indicated times. Cell lysates were prepared, and the level of PLC⑀ was measured by immunoblotting with ␣-PLC⑀ antibody. The remaining PLC⑀ level after EGF stimulation for the indicated time was quantified and expressed as a percentage of the PLC⑀ level of unstimulated control cells. B, MEF cells were treated with MG132 (10 M) for 12 h, and then cell lysates were prepared. Endogenous and exogenous expression of Siah1and Siah1 constructs in MEF cells was detected by immunoblotting with ␣-Siah1 antibody or ␣-FLAG antibody.
Siah Is Required for the EGF-dependent Degradation of PLC⑀ in MEFs-We attempted to determine whether the EGF-dependent degradation of PLC⑀ is impaired in the absence of the Siah proteins. To this end, we utilized Siah1a/Siah2 double knockout MEF cells. PLC⑀ was subjected to EGF-dependent degradation in MEF cells derived from wild-type mice (Fig. 7A,  panel a), but the degradation of PLC⑀ was blocked in the absence of Siah1a and Siah2 (Fig. 7A, panel b). To clarify the involvement of Siah, we expressed wild-type Siah1 or Siah1 TM in knockout cells by lentiviral infection (Fig. 7B). Add-back of wild-type Siah1 restored the EGF-dependent degradation of PLC⑀ (Fig. 7A, panel c), however, the add-back of Siah1 TM did not (Fig. 7A, panel d). These results confirm the role of endogenous Siah proteins in the EGF-dependent degradation of PLC⑀.
Siah Suppressed PLC⑀-induced Cell Growth-Various reports suggest that PLC⑀ can promote cell growth (12,13), and our previous results revealed that EGF-dependent cell growth was enhanced by PLC⑀. Thus, we speculated that the Siah-dependent degradation of PLC⑀ can contribute to the negative regulation of EGF-dependent cell growth. To test this hypothesis, we first examined whether Siah1 can inhibit the EGF-dependent cell growth potentiated by PLC⑀ expression. As shown in Fig. 8A, PLC⑀ expression enhanced EGF-dependent cell growth in HEK293 cells and co-expression of Siah1 abolished the PLC⑀-dependent enhancement of cell growth. We then measured the EGF-dependent cell growth in Siah1a/Siah2double knockout cells and in Siah1-add-back cells. The addback of Siah1 in double knockout cells suppressed EGFdependent cell growth (Fig. 8C). We then reduced PLC⑀ in Siah1a/Siah1-double knockout cells and in Siah1-add-back cells to investigate whether Siah-dependent growth inhibition is attributable to PLC⑀ degradation (Fig. 8B). PLC⑀ reduction in double knockout cells led to EGF-dependent growth inhibition, and notably, the suppression of Siah-dependent cell growth was abolished by PLC⑀ knockdown (Fig. 8C). Taken together, these results suggest that Siah inhibits EGF-dependent cell growth by removing PLC⑀.

DISCUSSION
Although the activation mechanisms and physiological functions of PLC⑀ have been largely revealed, little is known about the negative regulation of PLC⑀. The present study provides evidences that growth factor-activated PLC⑀ is subjected to proteasomal degradation. Siah proteins interact with the RA2 domain of PLC⑀ and promote PLC⑀ ubiquitination in the process. Our findings demonstrate that the RA2 domain of PLC⑀ plays a role in the inactivation of PLC⑀ as well as in the activation of PLC⑀ after growth factor stimulation. The RA2 domain of PLC⑀ binds with activated Ras or Rap, and this binding is critical for the growth factor-dependent translocation to the membrane and the activation of PLC⑀ (8,36). It is interesting to note that the blockade of the Ras-dependent activation of PLC⑀ led to the inhibition of the EGF-dependent interaction between Siah2 and PLC⑀, which implies that PLC⑀ activation may be a prerequisite for the EGF-induced ubiquitination and degradation of PLC⑀. In addition, the EGF-dependent interaction between Siah2 and PLC⑀ is most prominent at 30 min after EGF stimulation (Fig. 3A), whereas the EGF-induced translocation of PLC⑀ into the membrane for activation reportedly begins at 5 min after stimulation and ends at 40 min after stimulation (8), which indicates that the Siah-induced ubiquitination of PLC⑀ is induced after the activation of PLC⑀. Thus, we speculate that activated PLC⑀ is subjected to Siah-dependent ubiquitination and degradation to terminate PLC⑀ downstream signaling. This is the first study to explore the molecular mechanism underlying the negative regulation of PLC⑀.
Several studies have reported that PLC⑀ promotes cell growth. For example, carcinogen-induced tumor formation was inhibited in PLC⑀-deficient mice (12), and the overexpression of PLC⑀ in BaF3 cells potentiated platelet-derived growth factor-dependent cell growth (13). Consistent with previous reports, we found that PLC⑀ knockdown in MEFs led to the suppression of EGF-dependent cell growth. We conclude that it is necessary to control the level of PLC⑀ in cells to prevent aberrant cell growth. Many reports have implicated Siah proteins in cell growth control. Siah is a transcriptional target of p53 and contributes to p53-induced apoptosis and tumor suppression (23)(24)(25). Moreover, ␤-catenin degradation by genotoxic stress was mediated by Siah1, which leads to the suppres- sion of cancer cell growth (26,27). Siah1 was also found to induce growth arrest by inhibiting cytokinesis via the degradation of kinesin (Kid) (28). The involvement of Siah proteins in the regulation of growth factor-dependent cell growth was newly revealed in the present study. PLC⑀ was found to be a novel substrate of Siah proteins, and it was degraded by Siah proteins after EGF stimulation. We demonstrated that Siah proteins contribute to the negative regulation of growth factorinduced MEF cell growth by mediating PLC⑀ degradation. The fact that PLC⑀ reduction abolished Siah-dependent growth inhibition (Fig. 7) suggests that PLC⑀ is a major substrate of Siah proteins, which needs to be degraded to regulate cell growth in MEF cells.
The present study suggests a novel regulatory mechanism for Siah-mediated substrate degradation. We found that the Srcdependent phosphorylation of Siah is required for the EGFinduced degradation of PLC⑀. Whether Src directly phosphorylates Siah proteins or activates other protein kinases remains unclear. Previous reports have shown that Siah proteins are phosphorylated in different environments. For example, Siah2 was phosphorylated on Thr-24 and Ser-29 by p38 MAPK under hypoxia, and this was found to be important for the degradation of PHD-3 (38). Greene et al. (37) reported that Siah1 is phosphorylated on Tyr-100 and Tyr-126 upon camptothecin treatment via the activation of the JNK pathway. This phosphorylation increased the stability of Siah1 and its association with the adaptor protein POSH. Our analysis revealed that Siah1 is phosphorylated on Tyr-47, Tyr-199, and Tyr-223 via Src activation. We speculate that the EGF-induced phosphorylation of Siah proteins contributes to the binding of proteins with PLC⑀ and the subsequent degradation of PLC⑀, because pretreatment with the Src tyrosine kinase inhibitor PP2 reduced the EGFinduced interaction between PLC⑀ and Siah proteins as well as the ubiquitination of PLC⑀ (data not shown). Thus, Siah phosphorylation appears to be an important regulatory mode that mediates various interactions with substrate molecules or adaptor proteins in different cellular contexts.
In summary, we identified Siah1 and Siah2 as negative regulators of PLC⑀. PLC⑀ is subjected to EGF-dependent degradation via Siah-induced ubiquitination and proteasomal degradation processes. Siah is phosphorylated by EGF stimulation, and this phosphorylation is required for PLC⑀ degradation. Physiologically, the Siah-induced degradation of PLC⑀ contributes to the negative regulation of the EGF-dependent cell growth potentiated by PLC⑀.