SH2D4A Regulates Cell Proliferation Via the Erα/PLC-Γ/PKC Pathway

BMB reports

SH2D4A regulates cell proliferation via the ERα/PLC-γ/PKC pathway

Tingting Li, Wei Li, Jingyu Lu, Hong Liu, Yinghui Li & Yanyan Zhao* Department of Medical Genetics, China Medical University, Shenyang 110001, China

SH2D4A, comprising a single SH2 domain, is a novel protein ing, Rual et al. identified four proteins that interacted with of the SH2 signaling protein family. We have previously dem- SH2D4A, including DBNL, HCLS1, MAGEA11, and SH3GL1 onstrated SH2D4A is expressed ubiquitously in various tissues (4). Through mass spectrometry, Ewing et al. found an inter- and is located in the cytoplasm. In this study we investigated action between PPP1CB and SH2D4A (5). Neither of them de- the function of SH2D4A in human embryonic kidney (HEK) tected the function of these interactions. Additionally, Lapinski 293 cells using interaction analysis, cell proliferation assays, et al. observed that SH2D4A was dispensable for T cell re- and kinase activity detection. SH2D4A was found to directly ceptor signal transduction (6). bind to estrogen receptor α (ERα), and prevent the recruitment Herein, we demonstrated an inhibitory effect of SH2D4A on of phospholipase C-γ (PLC-γ) to ERα. Moreover, we observed cell proliferation. We showed that this effect was due to sup- its inhibitory effects on estrogen-induced cell proliferation, in- pression of a non-genomic action of ERα via an ERα/PLC-γ/ volving the protein kinase C (PKC) signaling pathway. Toge- PKC signaling pathway. In addition, we confirmed a new rela- ther, these findings suggested that SH2D4A inhibited cell pro- tionship among SH2D4A, ERα, and PLC-γ. Our findings proliferation by suppression of the ERα/PLC-γ/PKC signaling path- vide insight into the biological function of SH2D4A. way. SH2D4A may be useful for the development of a new anti-cancer drug acting as an ER signaling modulator. [BMB re- RESULTS AND DISCUSSION ports 2009; 42(8): 516-522] SH2D4A interacts with ERα in vivo and in vitro To investigate the biological function of SH2D4A, we per- INTRODUCTION formed a yeast two-hybrid screen using SH2D4A as the bait. Among the proteins obtained, only ERα contains phosphotyr- SH2D4A is a novel protein of the SH2 signaling protein family osine residues (7). We verified the interaction between ERα that only contains a single SH2 domain. We cloned the SH2D4A and SH2D4A via a further yeast two-hybrid assay. As shown in gene from the human chromosome 8p22 region (GenBank ac- Fig. 1A, all yeasts grew similarly in medium with histidine and cession no. AY190323), and indicated SH2D4A protein is lo- adenine, and only yeast cells expressing both SH2D4A and cated in the cytoplasm and is expressed ubiquitously (1, 2). The ERα grew in medium without histidine and adenine. The find- SH2 domain is the prototype for protein-protein interaction mod- ings indicated that histidine-adenine auxotrophy resulted from ules, binds to a phosphotyrosine, and mediates the formation of the interaction between SH2D4A and ERα. multiprotein complexes in protein tyrosine kinase pathways (3). We further investigated the interaction between SH2D4A SH2 domains are typically found in multidomain signaling en- and ERα in vivo. We co-transfected HEK293 cells with pSH2D4A zymes or in adaptor proteins, where they mediate protein-pro- and pERα in the condition of estrogen, and then performed a tein interactions and regulate associated catalytic subunits. The co-immunoprecipitation assay. As shown in Fig. 1B, when cell lack of additional domains in SH2D4A suggests that it is unlikely lysates were immunoprecipitated with anti-ERα or anti-HA an- to function in either of these roles. tibodies, and blotted with anti-HA or anti-ERα, SH2D4A and However, only a few reports of SH2D4A have been pub- ERα could precipitate each other in bidirectional reactions. As lished so far, and its detailed biological function is still poorly a control of co-immunoprecipitation assay, both SH2D4A and understood. Using high-throughput yeast two-hybrid screen- ERα were detected in the non-immunoprecipitated samples. The data confirmed the interaction of SH2D4A with ERα in eu- karyotic cells. *Corresponding author. Tel: 86-24-23257322; Fax: 86-24-23257322; GST pull-down assays were used to determine the inter- E-mail: [email protected] action between SH2D4A and ERα in vitro. GST-ERα and GST- SH2D4A fusion proteins as well as GST were expressed, puri- Received 29 October 2008, Accepted 19 February 2009 fied, and tested for interaction with in vitro translated 35S-la- Keywords: Cell proliferation, Estrogen receptor, PKC, PLC-γ, SH2D4A beled SH2D4A or ERα. As shown in Fig. 1C, SH2D4A was

516 BMB reports http://bmbreports.org An antiproliferative effect of SH2D4A on HEK293 cells Tingting Li, et al.

Fig. 1. SH2D4A interacts with ERα. (A) SH2D4A interacts with ERα in yeast. Yeast AH109 was co-transformed with pBD/SH2D4A and pAD/ ERα or with the corresponding vehicle (as a negative control). Only yeast cells expressing SH2D4A and ERα grew in medium without histidine and adenine. (B) SH2D4A interacts with ERα in HEK293 cells. pSH2D4A was co-transfected with pERα into HEK293 cells (ES), un-transfected cells (N) were used as a control, and both cells were treated with 10-10M estrogen. The immunoprecipitated proteins (IPs) and cell lysate (input) were probed by Western blots with anti-ERα and anti- HA antibodies. (C) SH2D4A interacts with ERα in vitro. GST-tagged fusion protein GST- ERα or GST-SH2D4A was incubated with 35S-labeled SH2D4A or ERα, respectively. To control the specificity the full-length GST was also incubated with the corresponding recombinant protein. All experiments were done in triplicate. specifically retained by GST-ERα, and ERα was also retained plasm (2), may interact with the membrane or cytoplasmic by GST-SH2D4A. The negative control experiment with GST ERα, affect the non-genomic action of estrogen, and partic- confirmed that the GST tag was not responsible for the inter- ipate in estrogen-mediated cell proliferation. action between SH2D4A and ERα. Therefore, the interaction SH2D4A with a single SH2 domain is homologous to Src. It between SH2D4A and ERα was direct and independent on ad- has been shown that Src is an initial step in ER-mediated cell ditional proteins in the cells. signaling, and can interact with tyrosine 537 of ER (12). ERs are known to localize to several sites within cells. In the Moreover, Robert et al. observed that SH2 domains of Shc in- nucleus, ERs induce the genomic action of estrogen, activating teracted with ER, but tyrosine 537 of ER was not involved in the transcription of certain estrogen-sensitive genes (8). This this interaction (13). The binding property of SH2 domains is action occurs after a time lag of at least two hours after estro- determined by its ability to discriminate between different gen stimulation. In addition, a small pool of ERs localizes to phosphorylated motifs (14), and can be flexible in some cases. the membrane or cytoplasm, and induces a non-genomic ac- For example, SH2D1A (also termed SAP), a similar protein to tion of estrogen (9-11). This rapid membrane-initiated response SH2D4A, is unusual in binding stably to unique motifs, even happens within seconds or minutes. SH2D4A, located in cyto- in their un-phosphorylated state (15); it acts as an inhibitor in http://bmbreports.org BMB reports 517 An antiproliferative effect of SH2D4A on HEK293 cells Tingting Li, et al.

SLAM-mediated signal transductions and controls communica- assays. As shown in Fig. 2A, estrogen-induced cell proliferation tion between T and B cells (16, 17). In the present study, we reached peak activities on the second day. However, SH2D4A demonstrated the interaction between SH2D4A and ERα in the arrested the estrogen effect by 60%. To detect whether SH2D4A condition of estrogen as evidence for phosphorylation of ERα affected the non-genomic action of estrogen in cell proliferation, by estrogen-dependent tyrosine kinase has emerged in many we performed a flow cytometry assay. As illustrated in Fig. 2B, cells (18-20). Further experiments must be undertaken to de- HEK293 cells were transfected with different constructs, and termine if the SH2 domain of SH2D4A binds to phosphotyr- then treated with or without estrogen. In agreement with pre- osine 537 of ERα. We nowaday are more interested in the role vious reports (21), we found that treating HEK293 cells with of SH2D4A in ERs pathway because of its SH2 domain. It may estrogen promoted the entry of cells into S phase. In contrast, function as a signaling inhibitor, like SH2D1A, by blocking or after SH2D4A vector treatment, a marked decrease of cell pop- regulating the binding of other signaling proteins to ERα. ulation in S phase was observed (from 17.2% in ER+E2 to 5.71% in ES+E2). The results indicated that SH2D4A inhibited SH2D4A inhibits estrogen-induced cell proliferation the non-genomic action of estrogen on cell proliferation through To identify the effects of SH2D4A in estrogen-induced actions, arresting S phase entry. However, the mechanism by which we tested cell proliferation using MTT and flow cytometry SH2D4A arrests the proliferative effect of estrogen would be identified.

SH2D4A competes with PLC-γ for binding to ERα There is abundant evidence suggesting that the non-genomic action of estrogen plays a significant role in cell proliferation (22, 23), and the MAPK signal transduction is involved in this action (10, 22). Therefore, the PLC/PKC pathway, acting up- stream of MAPK signal transduction, is participated in the non- genomic action of estrogen (24, 25). To assess the relationship between SH2D4A and PLC/PKC in the estrogen-induced cell proliferation, we carried out co-immunoprecipitation assay in vivo. After transfected with pERα, or both pERα and pSH2D4A in the condition of estrogen, HEK293 cells were lysised. Cell lysates were then immunoprecipitated with anti-ERα and blotted with anti-PLC-γ, and vice versa. As shown in Fig. 3, PLC-γ interacted with ERα in bidirectional reactions, this interaction was suppressed by SH2D4A and PLC-γ, ERα and SH2D4A were detected in the non-immunoprecipitated samples. These findings suggested a new relationship among SH2D4A, PLC-γ and ERα󰠏i.e., ERα could directly bind to PLC-γ through its

Fig. 2. SH2D4A inhibits estrogen-induced cell proliferation. Cells were transfected with pcDNA3.1 (K), pERα (ER), or both pERα and pSH2D4A (ES), and treated with or without 10-10 M estrogen (E2) 24 hours after transfection. (A) Cell growth was assessed by MTT absorbance among HEK293 cells, which was performed once every two days for six days. Fig. 3. SH2D4A competes with PLC-γ for binding to ERα. HEK293 SH2D4A inhibited the estrogen-induced cell proliferation. (B) SH2D4A cells were transfected with pERα (ER), or both pERα and pSH2D4A inhibited the non-genomic action of estrogen in cell proliferation. One (ES). The PLC-γ-ERα association was measured by immunoprecipitation hour after estrogen stimulation, the proportion of cells in S-phase of the of PLC-γ or ERα, and detection of ERα or PLC-γ by immunoblotting. cell cycle was determined by flow cytometry. Data are the means ± SD of The cell lysates (input) were blotted with anti-PLC-γ antibody, anti-ERα three independent experiments. *P ＜ 0.05, compared with respective antibody or anti-HA antibody as control. The experiment was done control values. in triplicate.

518 BMB reports http://bmbreports.org An antiproliferative effect of SH2D4A on HEK293 cells Tingting Li, et al.

phosphotyrosine and SH2D4A competed this binding. It has been showed that estrogen stimulates the rapid tyrosine phosphorylation of PLC-γ and activates MAPK through receptor tyrosine kinase (RTK) pathway where RTK-Shc-ERα is formed as signalsome (26, 27). Herein, our data suggested a new signalsome RTK-PLC-ERα could be involved in the pathway. PLC-γ contains two SH2 domains and one SH3 domain. One SH2 domain of PLC-γ binds to the phosphotyrosine of RTK, and the other SH2 domain probably binds to the phosphotyrosine of ERα, leading to the tyrosine phosphorylation of PLC-γ and an increase in its enzymatic activity (28, 29). Acti- vated PLC-γ increases the amounts of DAG and Ca2+ whereby PKC becomes activated and eventually leads to the activation of MAPK. Herein, SH2D4A may compete with both SH2 domains of PLC-γ, blocking RTK pathway.

SH2D4A suppresses estrogen-induced release of Ca2+ and activation of PKC To address SH2D4A resulted in blockage of the ERα/PLC-γ signal transduction, Ca2+ efflux and PKC activity were assessed. We tested the concentration of intracellular Ca2+ using a fluorescent probe. HEK293 cells were transfected with vehicle control, ERα, SH2D4A, or both SH2D4A and ERα, respectively, and estrogen was added just before the measurement. As shown in Fig. 4A, the treatment of estrogen (10-10 M) 2+ triggered a 3-fold [Ca ]i of the control. However, SH2D4A 2+ significantly reduced the [Ca ]i response to estrogen. Thereafter, the activity of PKC was measured by PepTag Protein Kinase Assay. Cells were transfected with different ve- hicles as described in Fig. 4. Consistent with earlier ob- servations that estrogen effected PKC signal pathway (22, 25), estrogen did stimulate PKC activity within 30 min (Fig. 4B). Furthermore, SH2D4A arrested the estrogen-induced activation of PKC. Two hours later, the activity of PKC returned to the basal level (Fig. 4C). Ca2+, one of the major activators of PKC, is released from the calcium ions pool by activated PLC. Many groups have 2+ shown that estrogen up-regulates [Ca ]i and activates PKC by 2+ non-genomic action (22, 25, 30). Our results indicated that Fig. 4. SH2D4A blocks the estrogen-induced release of Ca and 2+ activation of PKC. HEK293 cells were transfected with pcDNA3.1 SH2D4A inhibited the estrogen-induced release of Ca and (K), pERα (ER), or both pERα and pSH2D4A (ES). Cells transfected activation of PKC, by blocking access of positive regulator with pcDNA3.1 were used as a control. (A) Changes in intracellular 2+ such as PLC-γ to ERα. Ca concentration were measured by fluorescent probe Fluo-3/AM. Estrogen (E2) was added just before detection. (B) Twenty-four hours after transfection, cells were stimulated with estrogen for 30 minutes Conclusion and perspectives or 2 hours. The agarose gel was visualized under UV light. (C) In this report, we have confirmed an antiproliferative effect of Fluorescent intensity for each lysate was determined. The data are SH2D4A on HEK293 cells, which involves an ERα/PLC-γ/PKC presented as mean ± SD of three experiments. *P ＜ 0.05, com- signaling cascade. We demonstrated the interaction between pared with control. PLC-γ and ERα in vivo. This complex may be concerned with the estrogen-induced activation of RTK. SH2D4A, a novel pro- may also have an inhibitory effect on other cells. Besides ERα, tein of the SH2 signaling protein family, interacted with ERα, SH2D4A perhaps interacts with various receptors, such as and competed with PLC-γ for binding to ERα, thereby blocking RTK, and affects other signal pathways. Further studies will be the effect of estrogen on PKC, and repressing estrogen-induced required to confirm these speculations. SH2D4A has an in- cell proliferation. hibitory effect on cell proliferation, suggesting that it may be As SH2D4A inhibited estrogen-induced cell proliferation, it developed as a new anti-cancer drug. http://bmbreports.org BMB reports 519 An antiproliferative effect of SH2D4A on HEK293 cells Tingting Li, et al.

added to the mixtures. The bound proteins were eluted from MATERIALS AND METHODS the beads by boiling in SDS loading buffer, separated by SDS- PAGE, and visualized by autoradiography. Plasmids construction The human SH2D4A cDNA was cloned into expression vector Microculture tetrazolium (MTT) assay pcDNA3.1 to form pSH2D4A (expressing SH2D4A with an The MTT assay was performed as previously reported (31). HA tag) (2). pBD/SH2D4A and pAD/ ERα contained Gal4BD HEK293 cells were cultured in 96-well plates. Twenty-four and SH2D4A or Gal4AD and ERα, respectively, were con- hours after transfection, cells were incubated in absence structed using the Matchmaker yeast two-hybrid system3 (control) or presence of estrogen for 0, 2, 4 and 6 days. At the (Clontech, Mountain View, CA, USA), according to the manu- end of incubation, cells were loaded with 0.5 mg/ml MTT. facturer’s instructions. pGST/SH2D4A and pGST/ERα were Before the detection, MTT was replaced with DMSO. The opti- generated by inserting the SH2D4A or ERα into pGST cal density at 490 nm was measured on a Bio-Rad microplate (Amersham Pharmacia Biotech, UK). All constructs were veri- spectrophotometer. fied by sequencing. Flow-cytometric analyses Cells culture and transfection Cells cultured in 6-well plates were collected by trypsiniza- HEK293 cells were grown in DMEM supplemented with 10% tion, fixed in 70% ethanol and stored at 4oC until staining for FBS (HyClone, Logan, UT, USA), 100 U/ml penicillin, and 100 flow cytometry. Before being analyzed on a flow cytometer U/ml streptomycin. Cells were transfected with the various (BD FACSCalibur, San Jose, CA, USA), cells were stained with constructs using the Lipofectamine 2000 Reagent (Invitrogen, 5 μg/ml propidium iodide (PI; Sigma). The proliferative frac- Carllsbad, CA, USA), according to the manufacturer’s instruc- tion in the culture was determined as cells in the S phase of tions. Estrogen (Sigma, Louis, MO, USA) was dissolved in etha- the cell cycle. nol with a final concentration of ethanol in the medium of less 2+ than 0.01%. Measurement of [Ca ]i Changes in Ca2+ concentration were measured by the Ca2+- Yeast two-hybrid screen and assay sensitive fluorescent probe Fluo-3/AM (Sigma). Transfected cell In the yeast two-hybrid screen, yeast AH109 was co-trans- suspensions were loaded with 5μM Fluo-3/AM in the dark, formed with a human kidney cDNA library (Clontech) and and then washed in PBS. The fluorescence intensities of Fluo-3 pBD/SH2D4A. In the assay, yeast was co-transformed with (F) were measured with a spectrofluorometer (HITACHI F- pBD/SH2D4A and pAD/ERα. The colonies were isolated on 4500, Japan) at an excitation wavelength of 488 nm and an SD medium deficient in leucine and tryptophan, and then emission wavelength of 520 nm. Estrogen was added just be- streaked on the same medium lacking histidine and adenine. fore fluorescence detection. Afterward, 2 mM CaCl2 was add- Colonies were selected for their ability to grow in SD/-his/-ade ed and the Fmax obtained. 2 mM EGTA was then added and medium. the Fmin calculated. The concentration of Ca2+ was calculated 2+ by the formula: [Ca ]i=Kd *(F - Fmin)/(Fmax - F); Kd=400. Co-immunoprecipitation HEK293 cells were transfected with ERα, or co-transfected PKC activity assay with SH2D4A and ERα, and then were treated with 10−10M The estrogen-induced activation of PKC was measured by the estrogen. Cell lysates were prepared and incubated with an- PepTag Protein Kinase Assay (Promega). The assay was per- ti-HA (Santa Cruz Biotech, Santa Cruz, CA, USA; sc-805), or formed according to the manufacturer’s instructions. A mix- anti-ERα (Santa Cruz Biotech; sc-7207), or anti-PLC-γ (Abcam, ture, containing cells lysates, PepTag, PKC substrate, and PKC Cambridge, UK; ab41433). Then Protein A/G Plus agarose activating solution, was incubated at 30oC. The reaction was (Santa Cruz Biotech) was added. At the end of the incubation, stopped by boiling the assay medium in a water bath. The immunoprecipitated proteins (IPs) were eluted from the beads samples were electrophoresed on an agarose gel and vi- by boiling in SDS sample buffer. Cell lysate (input), and IPs sualized under UV light. The phosphorylated peptides’ fluo- were probed by Western blots with anti-ERα, anti-HA, or an- rescence intensities were quantified by spectrofluorometer at ti-PLC-γ antibodies. 570 nm (HITACHI F-4500).

GST pull-down assay Statistical analysis Expression of fusion proteins in E.coli strain BL21 was induced All values are expressed as means ± SD, unless otherwise with 0.1 mM IPTG. Fusion proteins were purified on gluta- indicated. One-way analysis of variance (ANOVA) was used to thione-Sepharose 4B beads (GE Healthcare). 35S-labeled assess statistical differences among different groups using the SH2D4A or ERα was generated by TNT coupled Transcription/ statistical software SPSS13.0. A P value ＜ 0.05 was consid- Translation Systems (Promega, Madison, WI, USA), and then ered significant.

520 BMB reports http://bmbreports.org An antiproliferative effect of SH2D4A on HEK293 cells Tingting Li, et al.

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