Phosphorylation of glycogen synthase kinase-3 β at serine-9 by phospholipase C γ 1 through protein kinase C in rat 3Y1 fibroblasts

Phospholipase C γ 1 (PLC γ 1) plays an important role in controlling cellular proliferation and differentiation. PLC γ 1 is overexpressed in some tumors, and its overexpression induces solid tumors in nude mice. However, the regulatory mecha-nisms underlying PLC γ 1-induced cell proliferation are not fully understood. Here we show that overexpression of PLC γ 1 highly phosphorylated glycogen synthase kinase-3 β (GSK-3 β ) at serine-9 in 3Y1 fibroblasts. Inhibition of protein kinase C (PKC)s with GF109203X abrogated GSK-3 β phosphorylation by PLC γ 1. We also found that steady-state level of cyclin D1 protein, but not cyclin D1 mRNA, was highly elevated in response to serum stimulation in PLC γ 1-transfected cells as compared with vector-transfected cells. Since GSK-3 β is involved in cyclin D1 proteolysis in response to mitogenic stimulation, PLC γ 1-mediated GSK-3 β phosphorylation may function as a regulation of cyclin D1 accumulation in PLC γ 1-overexpressing cells. of GSK-3b in 3Y1 fibroblasts.


Introduction
The hydrolysis of membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdInsP2), by phospholipase C (PLC) is one of the earliest key events in the regulation of various cell functions (Rhee and Bae, 1997). To date four types (β, γ, δ, and ε) and 11 PLC isozymes (β1-4, γ1-2, δ1-4, ε) are identified (Rhee and Bae, 1997). Among them, PLCγ1 isozyme is a direct substrate of the certain receptor tyrosine kinases such as epidermal growth factor (EGF) receptor and PDGF receptor (Margolis et al., 1989;Meisenhelder et al., 1989;Nishibe et al., 1989;Wahl et al., 1989) and may play critical roles in cell proliferation and differentiation (Smith et al., 1989;Smith et al., 1990;Valius and Kazlauskas, 1993;Roche et al., 1996;Bae et al., 1998;Kim et al., 2000). Several papers have reported that PLCγ1 is overexpressed in some human hyperproliferative tissues, including breast carcinoma (Arteaga et al., 1991), human skin under hyperproliferative conditions (Nanney et al., 1992), colorectal carcinoma Lee et al., 1995), familial adenomatous polyposis (Park et al., 1994), and highly metastatic colorectal tumor cell lines (Yeatman et al., 1994). Overexpression of PLCγ1 shows anchorage-independent growth in soft agar and induces tumors after injection into nude mice (Chang et al., 1997;Smith et al, 1998). Recently, we have found that overexpression of PLCγ1 downregulates the transcription factor Egr-1 gene (Shin et al., 2002), which plays a key regulatory role in cell growth, differentiation, and development (Cao et al., 1992;Gashler and Sukhatme, 1995), and functions as a tumor suppressor (Huang et al., 1994). These results suggest that PLCγ1 is a necessary component for mitogenic signaling pathway and that abnormal expression of PLCγ1 may be associated with tumor development in some tumors. However, it is not fully understood how PLCγ1 regulates cell proliferation.
Glycogen synthase kinase-3β (GSK-3β) is a protein kinase that regulates multiple biological processes, Phosphorylation of glycogen synthase kinase-3β at serine-9 by phospholipase Cγ1 through protein kinase C in rat 3Y1 fibroblasts including body patterning during embryonic development, protein synthesis, cell adhesion, cell proliferation, cell differentiation, and cell death (reviewed in Frame and Cohen, 2001). In resting state, GSK-3β is active and negatively regulates some transcription factors including c-Jun (Nikolakaki et al., 1993), and NFAT (Beals et al., 1997). GSK-3β also phosphorylates cyclin D1 at threonine-286 which is required for nuclear export and ubiquitin-dependent degradation in the cytoplasm (Diehl et al., 1998). Upon growth factor stimulation such as insulin or EGF, GSK-3β is phosphorylated at N-terminal serine-9 residue by protein kinase B (PKB, also called Akt) (Cross et al., 1995), leading to inhibition of its kinase activity. GSK-3β is also a negative regulatory component of Wnt-dependent signaling. In Wnt signaling, control of β-catenin accumulation is critical for regulation of Wnt-dependent transcriptional activation. In the resting state, GSK-3β phosphorylates β-catenin, leading to subsequent degradation via the proteasome-dependent pathway (Aberle et al., 1997;Ikeda et al., 1998). When cells are activated by Wnt ligands, GSK-3β activity is inhibited through Dishevelled protein (Kishida et al., 1999;Miller et al., 1999;Smalley et al., 1999). This leads to stabilization and accumulation of cytosolic β-catenin. The accumulated βcatenin interacts with Tcf/Lef family of transcription factors and translocates to nucleus to activate Wnt target genes, including c-myc and cyclin D1 (He et al., 1998;Tetsu and McCormick, 1999).
In this study, we investigated whether PLCγ1 regulates the phosphorylation of GSK-3b in 3Y1 fibroblasts.

Northern blot analysis
Total RNA was isolated as described previously (Lee and Seo, 2002). Isolated total RNA (5-10 µg) was separated on 1.2% agarose gel containing 6% formaldehyde in 0.02 M Mops, pH 7.0, 8 mM sodium acetate, and 1 mM EDTA, then transferred to Hybond N + nylon membrane (Amersham Pharmacia Biotech., Piscataway, NJ) by the capillary method. Cross-linking was then performed by UV irradiation. cyclin D1 and GAPDH cDNAs were labeled with [α-32 P]dCTP (6,000 Ci/mmol, DuPont NEN) by standard random primer method. After prehybridization, blots were hybridized overnight at 42 o C in Northern-Max hybridization solution (Ambion, Austin, TX). Blots were then washed with 2 SSC/0.1% SDS for 20 min at room temperature, 2 SSC/0.1% SDS at 42 o C for 30 min, and 0.5 SSC/0.1% SDS for 30 min at 52 o C. For rehybridization, the probes were stripped from the membrane by boiling in 0.1 SSC/0.5% SDS.

Tcf/Lef-1-dependent transcription assay
The pTOPflash reporter plasmid (Korinek et al., 1998), which contains three copies of consensus Tcf/Lef-1-binding sites was purchased from Upstate Biotechnology (Lake Placid, NY). pTOPflash reporter (50 ng-1 µg) was transfected into HEK293T cells without or with expression plasmids (pCMV/flag-PLCγ1, pQNCII/ flag-Dvl-1) using LipofectAMINE 2000 reagents according to manufacturer's instruction. pCMV/β-gal plasmid (100 ng) was included all the samples to monitor the transfection efficiency. At 24 h after transfection, cells were harvested and protein extracts were prepared by three cycles of freezing and thawing. One to two µg of protein was assayed for luciferase activity. Luminescence was measured using a luminometer model TD 2020 (Berthold, Tubingen, Germany). Trans-fection efficiencies were normalized by a ratio of luciferase activity to β-galactosidase activity obtained from the same sample.

Results and Discussion
Overexpression of PLCγ1 enhances GSK-3β phosphorylation at serine-9 in response to mitogenic stimulation Previous studies have demonstrated that GSK-3β is involved in the control of cell growth in response to growth factors stimulation (Diehl et al., 1998;Alt et al., 2000;Sears et al., 2000). As PLCγ1 is overexpressed in some human cancer tissues, including colon and breast cancers (Yeatman et al., 1994;Lee et al., 1995), and has been implicated mitogenic signaling, we wondered if PLCγ1 is involved in mitogen-induced phosphorylation of GSK-3β. To determine the effect of PLCγ1 on GSK-3β phosphorylation, we used PLCγ1-transfected 3Y1 fibroblast cell lines (Chang et al., 1997). Quiescent, serum-starved, vec-tor-transfected 3Y1 cells (Vector-3Y1) and PLCγ1transfected 3Y1 cells (PLCγ1-3Y1) were treated with 20% serum, and GSK-3β phosphorylation was assessed by Western blotting using anti-phospho-GSK-3β (Ser 9) antibody. Treatment with serum-induced time-dependent phosphorylation of GSK-3β at serine-9 in both transfectants. Increased phosphorylation of GSK-3β was evident at 30 min, after which the level of phosphorylation gradually reduced in Vector-3Y1 cells ( Figure 1A). Basal level of GSK-3β phosphorylation was approximately 7-fold higher in PLCγ1-3Y1 cells compared with Vector-3Y1 cells. In PLCγ1-3Y1 cells, serum-induced increase of GSK-3β phosphorylation was occurred as early as 15 min and was sustained for more than 120 min after serum stimulation ( Figure 1A). During this time course, notable increases in GSK-3β phosphorylation were observed in PLCγ1-3Y1 cells compared with Vector-3Y1 cells. When the serum-starved cells were treated with EGF for 30 min, phosphorylation of GSK-3β at serine-9 also strongly induced in PLCγ1-overexpressed cells as compared to Vector-3Y1 cells ( Figure 1B). These data suggest that PLCγ1 may contribute to the elevation of mitogen-induced GSK-3β phosphorylation at serine-9.

Figure 2.
Inhibition of PKC abrogates serum-induced GSK-3β phosphorylation in 3Y1 cells. Vector-3Y1 cells (Vector) and PLCγ1-3Y1 cells (PLCγ1) were serum-starved with 0.5% for 24 h, and then treated with the indicated concentrations of GF109203X before 30 min of exposure to 20% serum. After an additional incubation for 30 min, cells were harvested and whole cell lysates were prepared for Western blotting with anti-PLCγ1 (top), anti-phospho-GSK-3β (Ser 9) (middle), or anti-GSK-3β (bottom) antibodies. stimulates the release of Ca 2+ from internal stores (Nishizuka, 1984). To determine whether PKC activity is required for PLCγ1-mediated increase of GSK-3β phosphorylation, we examined the effect of GF109203X, a selective inhibitor of PKCs (Toulle et al., 1991), on the serum-induced GSK-3β phosphorylation. As shown in Figure 2, pre-incubation of GF109203X abrogated the serum-induced GSK-3β phosphorylation at serine-9 in a dose-dependent manner in both cell lines. This result suggests that PKC activity is necessary for PLCγ1-mediated GSK-3β phosphorylation. This conclusion was further supported by experiments demonstrating that down-regulation of PKC by long-term treatment with 1 µM phorbol 12-myristate 13-acetate (PMA) for 24 h failed to induce the GSK-3β phosphorylation (data not shown).

PLCγ1-mediated GSK-3β phosphorylation is not linked to Wnt signaling
Wnt signaling plays an important role in embryonic development (Cardigan, 1997) and deregulation of Wnt signaling considered as a significant factor causing cancer development (Bienz and Clevers, 2000). Control of β-catenin stability is central to Wnt signal pathway. Upon stimulation by Wnt signaling, the activity of GSK-3β is inhibited, leading to stabilization and accumulation of β-catenin in the nucleus. Accumulated β-catenin interacts with Tcf/lef-1 transcription factors to activate transcription of Wnt target genes such as c-myc and cyclin D1 (He et al., 1998;Tetsu and McCormick, 1999). To investigate whether inhibition of GSK-3β by PLCγ1-mediated phosphorylation at serine-9 is linked to Wnt signaling, we examined cytosolic and nuclear β-catenin levels in Vector-and PLCγ1-3Y1 cells. After exposure to X-ray film, the relative intensities of β-catenin bands in each lane were determined by quantitative scanning densitometer. The amount of cytosolic free β-catenin in PLCγ1-3Y1 cells was much less than in Vector-3Y1 cells ( Figure 3A middle panel and Figure 3B). Basal level of nuclear β-catenin in PLCγ1-3Y1 cells was reduced to 42% of that in Vector-3Y1 cells ( Figure  3A bottom panel and Figure 3B). Translocation of β-catenin to the nucleus in response to serum stimulation is increased in a time-dependent manner after serum stimulation in Vector-3Y1 cells, but very marginal in PLCγ1-3Y1 cells ( Figure 3A bottom panel). The amount of nuclear β-catenin in Vector-3Y1 cells was 5-fold higher than that in PLCγ1-3Y1 cells by 6 h of stimulation ( Figure 3B). This finding suggested that PLCγ1-mediated phosphorylation of GSK-3β by serum is not associated with the regulation of β-catenin. The mechanism by which PLCγ1 decreases free β-catenin pools is not understood at present.
To further determine whether PLCγ1-mediated GSK-3β phosphorylation is not involved in Wnt signaling, we examined the effect of transient expression of PLCγ1 on Tcf/Lef-1-mediated transcription. pTOPflash reporter constructs, which contain three repeats of the Tcf/Lef-1 binding elements (Korinek et al., 1997), was transfected into HEK293T cells. Transfection of pTOPflash reporter produced Tcf/Lef-1-dependent transcription in a DNA concentration-dependent manner (Figure 4A). Expression of flag-tagged Dvl-1, as a positive control, induced pTOPflash reporter activity by 3-fold, while PLCγ1 had no effect on the reporter activity ( Figure 4B), suggesting that Tcf/Lef-1-mediated transcription is not dependent on PLCγ1. Taken together, these results suggest that PLCγ1-mediated GSK-3β phosphorylation is not linked to Wnt signaling.

Effect of PLCγ1 overexpression on serum-induced cyclin D1 expression
GSK-3β can reduce abundance of cyclin D1 protein through phosphorylation of cyclin D1 at threonine-286, which target for ubiquitination-dependent proteolytic degradation (Diehl et al., 1998). GSK-3β is constitutively active in the resting state and its enzyme activity is inhibited by phosphorylation at serine-9 upon growth factor stimulation, resulting in accumulation of cyclin D1. Thus, we next examined whether increase of GSK-3β phosphorylation in PLCγ1-3Y1 cells regulates the cyclin D1 level. As shown in Figure  5A, cyclin D1 protein levels gradually increased up to 12 h after serum stimulation, and declined there after in Vector-3Y1 cells. After 36 h, amount of cyclin D1 protein had fallen back to near control levels. During this time course, notable increases in the cyclin D1 level were observed in PLCγ1-3Y1 cells compared with Vector-3Y1 cells. In contrast, cyclin E levels were not changed between two cell lines. To examine whether this increase in cyclin D1 protein levels results from a increase in mRNA levels, Northern blot analysis was performed. Induction of cyclin D1 mRNA by serum stimulation showed similar kinetics between two cell lines ( Figure 5B). This finding suggests that PLCγ1 controls cyclin D1 abundance at the post-transcriptional level, probably through phosphorylation of GSK-3β.
In this report, we demonstrate for the first time that GSK-3β is a down-stream target of PLCγ1-dependent signals. PLCγ1 overexpression resulted in increase of GSK-3β phosphorylation at serine-9 by serum stimulation, which is abrogated by inhibition of PKC activity by treatment of GF109203X. It has been reported that GSK-3β is active in unstimulated state and phosphorylates cyclin D1 at threonine-286, thereby resulting ubiquitination and subsequent proteolytic de-   (PLCγ1) were serum-starved with 0.5% for 24 h, and then treated with 20% serum for indicated time periods. (A) Proteins were extracted, separated on 10% SDS-polyacrylamide gels (20 µg/lane) and transferred to nitrocellulose membrane. Cyclin D1 (top), cyclin E (middle) and p42/p44 MAPK (bottom) protein levels were detected by Western blotting. (B) Total RNA was isolated from cells, electrophoresed on 1% agarose-gel (10 µg/lane), capillary transferred to a nylon membrane, and subjected to Northern blotting. The blot was hybridized with the 32 P-labeled cyclin D1 probe (top). The same blot was stripped and reprobed with 32 P-labeled GAPDH probe (bottom). gradation of cyclin D1 (Diehl et al., 1997;Diehl et al., 1998). Upon mitogenic stimulation, GSK-3β is phosphorylated and inactivated through several signaling pathways including Ras, phosphatidylinositol-3-OH kinase (PI-3K) and PKB/Akt. Inhibition of GSK-3β activity results in the elevation of cyclin D1 level (Diehl et al., 1998). This may enhance the activities of cyclin-dependent protein kinase (CDK)-4 and CDK-6, allowing them to phosphorylate pRb and stimulate cell cycle progression. Given the facts that (1) GSK-3β phosphorylation is increased by PLCγ1; (2) GSK-3β directly regulates cyclin D1 proteolysis in response to mitogenic stimulation; (3) Cylin D1 protein level, but not mRNA level, is up-regulated in PLCγ1-transfected cells, it is plausible to suggest that the functional role of PLCγ1-mediated GSK-3β phosphorylation may be associated with the accumulation of cyclin D1 in PLCγ1-overexpressing cells.
In summary, this study demonstrates that PLCγ1 mediate GSK-3β phosphorylation at serine-9 via PKC activation. Since phosphorylation of GSK-3β is responsible for the accumulation of cyclin D1 in response to mitogenic stimulation, PLCγ1 overexpression might contribute to mitogen-induced cell cycle progression through phosphorylation of GSK-3β at serine-9, at least in part.