Loss of the tumor suppressor MMAC1 has been shown to be involved in breast, prostate and brain cancer. Consistent with its identification as a tumor suppressor, expression of MMAC1 has been demonstrated to reduce cell proliferation, tumorigenicity, and motility as well as affect cell–cell and cell–matrix interactions of malignant human glioma cells. Subsequently, MMAC1 was shown to have lipid phosphatase activity towards PIP3 and protein phosphatase activity against focal adhesion kinase (FAK). The lipid phosphatase activity of MMAC1 results in decreased activation of the PIP3-dependent, anti-apoptotic kinase, AKT. It is thought that this inhibition of AKT culminates with reduced glioma cell proliferation. In contrast, MMAC1's effects on cell motility, cell–cell and cell–matrix interactions are thought to be due to its protein phosphatase activity towards FAK. However, recent studies suggest that the lipid phosphatase activity of MMAC1 correlates with its ability to be a tumor suppressor. The high rate of mutation of MMAC1 in late stage metastatic tumors suggests that effects of MMAC1 on motility, cell–cell and cell–matrix interactions are due to its tumor suppressor activity. Therefore the lipid phosphatase activity of MMAC1 may affect PIP3 dependent signaling pathways and result in reduced motility and altered cell–cell and cell–matrix interactions. We demonstrate here that expression of MMAC1 in human glioma cells reduced intracellular levels of inositol trisphosphate and inhibited extracellular Ca2+ influx, suggesting that MMAC1 affects the phospholipase C signaling pathway. In addition, we show that MMAC1 expression inhibits integrin-linked kinase activity. Furthermore, we show that these effects require the catalytic activity of MMAC1. Our data thus provide a link of MMAC1 to PIP3 dependent signaling pathways that regulate cell–matrix and cell–cell interactions as well as motility. Lastly, we demonstrate that AKT3, an isoform of AKT highly expressed in the brain, is also a target for MMAC1 repression. These data suggest an important role for AKT3 in glioblastoma multiforme. We therefore propose that repression of multiple PIP3 dependent signaling pathways may be required for MMAC1 to act as a tumor suppressor.
MMAC1 (also known as PTEN or TEP-1) is mutated at a high frequency in brain, breast, and prostate tumors as well as in melanomas and endometrial carcinomas, (Guldberg et al., 1997; Kong et al., 1997; Li and Sun, 1997; Li et al., 1997; Steck et al., 1997). These observations suggest that MMAC1 acts as a tumor suppressor in multiple tissues. Indeed, subsequent studies showed that reintroduction of this gene into human glioma cells reduced cell growth, tumorigenicity in nude mice, and affected motility and cell–cell interactions, demonstrating that MMAC1 represents a bone fide tumor suppressor (Furnari et al., 1997; Cheney et al., 1998; Tamura et al., 1998; Morimoto et al., 1999). Interestingly, germ line mutations in MMAC1 have also been linked to the multiple hamartomatous predisposition syndromes, Cowden's disease and Bannayan–Zonana. These syndromes are also associated with increased susceptibility to breast and thyroid cancer (Liaw et al., 1997; Marsh et al., 1997).
Sequence analysis of MMAC1 indicated that this gene encodes motifs conserved in dual specificity phosphatases (Li and Sun, 1997; Li et al., 1997; Steck et al., 1997). MMAC1 has been shown to possess protein phosphatase activity towards focal adhesion kinase (FAK) (Tamura et al., 1998) and lipid phosphatase activity toward phosphatidylinositol 3,4,5-trisphosphate (PIP3) (Maehama et al., 1998). However, it appears that the lipid phosphatase activity of MMAC1 correlates with tumor suppression (Myers et al., 1998).
Many signaling molecules directly bind PIP3 through pleckstrin homology (PH) domains (Corvera and Czech, 1998) and could thus be affected by reduced levels of PIP3 due to expression of MMAC1. The binding of PIP3 to PH domain-containing proteins such as AKT, phospholipase C (PLC) and integrin linked kinase (ILK) is thought to facilitate membrane targeting and induce conformational changes that result directly, or indirectly, in activation (Aoki et al., 1998; Delcommenne et al., 1998; Falasca et al., 1998).
Recent studies have shown that PIP3 levels are indeed higher in cells lacking MMAC1 (Haas-Kogan et al., 1998; Stambolic et al., 1998). In addition, lack of endogenous MMAC1 expression correlated with elevated levels of activated AKT1 (Haas-Kogan et al., 1998; Myers et al., 1998; Stambolic et al., 1998; Suzuki et al., 1998), and ectopic MMAC1 expression resulted in decreased levels of activated AKT and phosphorylated BAD protein (Myers et al., 1997; Wu et al., 1998a). Therefore, these results are consistent with a model in which MMAC1 reduces AKT activity and thus increases apoptosis, resulting in tumor suppression. However, there are a number of observations that suggest that MMAC1 does not inhibit tumor suppression solely through repression of the AKT signaling pathway.
Expression of MMAC1 in malignant human glioma cells not only inhibits cell proliferation and tumorigenicity, but also affects motility and cell–cell and cell–matrix interactions (Furnari et al., 1997; Cheney et al., 1998; Tamura et al., 1998; Morimoto et al., 1999). In addition, loss of MMAC1 correlates with the progression of tumors to a metastatic state, suggesting that the tumor suppressor activity of MMAC1 affects cell motility and/or cell–matrix or cell–cell interactions in vivo. As the AKT signaling pathway has not been shown to affect cell–matrix and cell–cell interactions or cell motility, these observations suggest that repression of PIP3 regulated signaling pathways distinct from AKT may contribute to the ability of MMAC1 to suppress tumor formation.
In contrast to the AKT signaling pathway, both the phospholipase C (PLC) and the integrin linked kinase (ILK) signaling pathways regulate motility as well as cell–cell and cell–matrix interactions. The PLC signaling pathway has been linked to the motility of human glioma cells (Kyoshmomn et al., 1999). Similarly, integrin linked kinase (ILK) affects cell motility and cell–cell and cell–matrix interactions. Overexpression of this PIP3 regulated kinase is also sufficient to induce tumorigenicity in vivo (Hannigan et al., 1996; Radeva et al., 1997). As both ILK and PLC are PIP3 dependent enzymes (Hannigan et al., 1996; Falasca et al., 1998), they represent potential downstream targets for MMAC1 regulation. As such, MMAC1 could affect tumor cell motility, cell–cell and cell–matrix interactions through the inhibition of these enzymes and their signaling pathways.
We show here that reintroduction of MMAC1 into human glioblastoma cells reduces extracellular Ca2+ influx, intracellular inositol trisphosphate (IP3) levels, and ILK activity. These results thus provide a link between MMAC1 and PIP3 dependent signaling pathways that affect cell motility, cell–cell and cell–matrix interactions. As the PLC and the ILK signaling pathways are also involved in mitogenesis (Rhee et al., 1997; Hannigan et al., 1996), the repression of these pathways may also contribute to the ability of MMAC1 to inhibit cell proliferation. We also demonstrate that the activity of AKT3, an isoform of AKT highly expressed in the brain, is inhibited by MMAC1 expression. Therefore, inhibition of both AKT1 and AKT3 may be required for tumor suppression in the brain. Together our results suggest that the tumor suppressor activity of MMAC1 may be the result of pleotropic effects on multiple PIP3 regulated signaling pathways.
MMAC1 suppresses the PLC/Ca2+ signaling pathway
The presence of MMAC1 in vivo and in vitro correlates with decreased proliferation, tumorigenicity and motility of human glioma cells (Li et al., 1997; Steck et al., 1997; Furnari et al., 1997; Cheney et al., 1998; Tamura et al., 1998; Morimoto et al., 1999). The lipid phosphatase activity of MMAC1 appears to result in inhibition of AKT1 activity and a growth suppression phenotype (Haas-Kogan et al., 1998; Stambolic et al., 1998; Suzuki et al., 1998). The protein phosphatase activity results in FAK dephosphorylation which is thought to then cause decreased cell motility and cell–matrix interaction (Tamura et al., 1998). However, it appears that the lipid phosphatase activity of MMAC1 correlates with tumor suppressor activity (Myers et al., 1998). Thus far, the AKT signaling pathway has not been linked to either cell–cell, cell–matrix interactions or cell motility. This suggested that other PIP3 dependent signaling pathways may be inhibited by the tumor suppressor activity of MMAC1 resulting in decreased cell motility and/or altered cell–matrix and cell–cell interactions.
We therefore examined whether MMAC1 expression affects signaling pathways that are involved in such processes and are also regulated by PIP3. One such candidate is the phospholipase C (PLC) signaling pathway. PLC activation generates inositol trisphos-phate (IP3) and diacylglycerol (DAG) which result in a flux of intracellular calcium and stimulation of protein kinase C (PKC) in a PIP3 dependent manner, respectively (Falasca et al., 1998; Rameh et al., 1998; Rhee et al., 1997). In addition, the PLC signaling pathway has been linked to the motility of human glioma cells (Khoshyomn et al., 1999). We therefore examined whether expression of MMAC1 affected the intracellular levels of IP3 in human glioma cells.
As a control, we first examined whether IP3 levels are sensitive to the PI3 kinase inhibitor, LY294002, in U373 cells. Asynchronously growing U373 control cells were treated with vehicle or the PI3 kinase inhibitor, LY294002, and the amount of IP3 present in the lysates was determined. As shown in Figure 1a, the levels of IP3 were reduced by over 50% when cells were treated with LY294002. To determine whether MMAC1 similarly affects IP3 levels, we also examined IP3 levels in cells expressing WT MMAC1 or a catalytically inactive form of MMAC1, C124S MMAC1 (Figure 1b). Expression of wild type MMAC1 consistently reduced the levels of IP3 in U373 cells by 50% or more in two independently isolated clones of cells stably expressing WT MMAC1. In contrast, cells expressing the catalytically inactive form of MMAC1 had IP3 levels comparable to control cells that lack endogenous MMAC1. The pretreatment of U373 cells with LY294002, expression of wild type or C124S MMAC1 did not appear to alter the expression level of PLCγ1 (Figure 1c) the major isoform of PLC expressed in U373 cells (data not shown), suggesting that MMAC1 affects the enzymatic activity of PLC. These data indicated that, similar to a PI3 kinase inhibitor, MMAC1 reduces the intracellular levels of IP3 and that the catalytic activity of MMAC1 is required for this effect.
To further examine the potential effect of MMAC1 on the PLC signaling pathway, we addressed whether MMAC1 affects Ca2+ flux in human glioblastoma cells. Intracellular Ca2+ flux can occur in response to the generation of IP3 by PLC. Therefore, we first examined the ability of U373 cells to release Ca2+ from the endoreticulum (ER) stores, to flux extracellular calcium into the cells and whether these events require PIP3. U373 control cells were suspended in calcium free media, incubated with the Ca2+-binding dye indo-1AM, washed and incubated with EGTA. After 380 s, cells were exposed to a source of extracellular calcium to measure Ca2+ influx (Figure 2a; control). We found that U373 cells exhibit a constitutive extracellular calcium influx. We next examined the ability of U373 cells to release Ca2+ from the ER stores in response to serum (Figure 2a; control+serum). We found that U373 control cells release Ca2+ from the ER stores in response to serum, but that this response is minimal as compared to the extracellular influx of Ca2+ (compare Figure 2a, control versus control+serum). The use of other stimuli such as EGF or thapsigargin produced similar profiles for Ca2+ efflux from the ER stores (data not shown) suggesting that the predominant Ca2+ flux in U373 cells is extracellular influx. We next examined whether the efflux of Ca2+ from the ER stores and the influx of extracellular Ca2+ are affected by the PI3 kinase inhibitor LY294002 (Figure 2a, control; LY294002+serum). Whereas there is minimal inhibition of the ER store release of Ca2+, the influx of extracellular calcium was significantly inhibited by treatment with LY294002. These data indicated that the predominant flux of Ca2+ in U373 cells is the influx of extracellular Ca2+, and that this event is PI3 kinase-dependent.
We next examined the effect of expression of wild type MMAC1 or the catalytically inactive C124S mutant of MMAC1 on the ability of U373 cells to flux calcium. Similar to LY294002 pretreatment of cells (Figure 2a, control; LY294002+serum), expression of wild type MMAC1 significantly inhibited the extracellular influx of Ca2+ but did not appear to significantly affect the efflux of calcium out of the ER stores (Figure 2b, compare control to WT). In contrast, cells expressing a catalytically inactive form of MMAC1 exhibit an extracellular Ca2+ influx similar to that of control cells (Figure 2b, C124S). Interestingly, the C124S MMAC1 mutant-expressing cells consistently exhibited a calcium influx profile slightly different than that of control cells (compare Figure 2b control to C124S). We have observed this phenotype with multiple independently isolated clones of C124S MMAC1 expressing cells. Similar enhanced responses have been observed with cells expressing this mutant form of MMAC1 (Li and Sun, 1998; Myers et al., 1998; Morimoto et al., 1999) and may be due to stabilization of PIP3 or other components of this signaling pathway by MMAC1. Our results demonstrate that expression of catalytically active MMAC1 inhibits a PI3 kinase dependent influx of extracellular Ca2+. Together, our findings are consistent with MMAC1 inhibiting the PLC signaling pathway resulting in decreased amounts of intracellular IP3 and Ca2+ influx.
MMAC1 regulates integrin linked kinase activity
Integrin linked kinase (ILK) has been implicated in the regulation of cell–cell and cell–matrix interactions (Hannigan et al., 1996; Radeva et al., 1997; Wu et al., 1998b). This serine/threonine kinase is also a PIP3-dependent signaling protein (Delcommenne et al., 1998), making it a candidate target for regulation by MMAC1. To examine a potential link between MMAC1 and ILK, we first asked whether the expression patterns of ILK and MMAC1 are similar. Normal adult mouse organs were homogenized and proteins were immune precipitated with cross-linked anti-MMAC1 antibody. The immune precipitates were then Western blotted with anti-MMAC1 antibody. MMAC1 protein was detected in all tissues tested, with the highest levels in brain and barely detectable levels in the heart. Interestingly, a doublet of MMAC1 protein was consistently detected in mouse liver tissue (Figure 3a). Protein lysates were immune precipitated and then Western blotted with anti-ILK antibody (Figure 3b). High levels of ILK protein were detected in every tissue examined. Importantly, ILK protein was easily detected in the brain (Figure 3b) where MMAC1 is expressed (Figure 3a) and is thought to act as a tumor suppressor (Li et al., 1997; Steck et al., 1997).
To examine whether MMAC1 affects ILK activity, lysates from U373 cells expressing wild type MMAC1, C124S MMAC1 or control cells were immune precipitated for ILK protein and kinase assays were performed using myelin basic protein as a substrate (Figure 3c). Kinase assays were quantitated using a phosphoimager. Expression of wild type MMAC1 suppressed ILK activity by 40%, and the PI3 kinase inhibitor LY294002 inhibited ILK activity by 28%. Inhibition of ILK kinase activity (by approximately 40%) was consistently observed in two independently isolated clones of cells expressing wild type MMAC1. In contrast, cells expressing C124S MMAC1 consistently exhibited ILK kinase activity slightly above (18%) that of control cells (Figure 3c). These results are consistent with the enhanced Ca2+ flux observed in C124S MMAC1-expressing cells (Figure 2b, C124S). In addition, treatment of cells expressing C124S MMAC1 with LY294002 reduced ILK activity, demonstrating that ILK activity in C124S MMAC1-expressing cells is PI3 kinase-dependent (Figure 3c). As a control, U373 cells were also treated with the MEK inhibitor, PD 98059. No significant effect on precipitable ILK activity was detected indicating that MAPK activity was not present at detectable levels in anti-ILK immune precipitates (Figure 3c). Together, these results indicate that expression of MMAC1 in human glioblastoma cells down-modulates ILK activity.
All AKT isoforms are expressed in normal brain tissue
MMAC1 has been shown to repress the activity of AKT1, however it is not known whether MMAC1 affects all three AKT isoforms. To address whether MMAC1 affects all AKT isoforms, we first examined whether the isoforms exhibit expression patterns similar to MMAC1. Protein lysates were immune precipitated and then Western blotted with AKT isoform-specific antibodies (Figure 4). AKT2 was expressed at the highest levels in the liver, testes and spleen whereas AKT1 expression was high in the brain, heart, lung, spleen and testes and AKT3 expression was highest in brain, testes, spleen and lung. All isoforms were also detected at low levels in the remaining tissues tested. Importantly, AKT isoforms were detected in all tissues in which MMAC1 protein was detected (compare Figure 4 to Figure 3a). As AKT1 and AKT3 expression were high in brain tissue and the loss of MMAC1 appears to be involved in approximately 40% of glioblastoma multiforme tumors (Li et al., 1997; Steck et al., 1997), these results suggested that both AKT1 and AKT3 represent potential targets for MMAC1 in the brain.
All AKT isoforms are expressed and active in GBM cells
To determine whether all AKT isoforms are expressed and active in glioblastoma multiforme (GBM) cells that lack endogenous MMAC1 expression (Li et al., 1997; Steck et al., 1997), U373 and A172 cells were grown in serum free media overnight and then stimulated with serum. Lysates were immune precipitated with AKT isoform-specific antibodies and kinase assays were performed using histone H2B as a substrate (Figure 5a,b). AKT1 exhibited very low basal activity which is stimulated by serum twofold in U373 cells and fivefold in A172 cells (Figure 5b), in agreement with recently published studies (Haas-Kogan et al., 1998; Myers et al., 1998; Stambolic et al., 1998). In both cell lines AKT2 activity was very low and not altered significantly by serum stimulation. In U373 cells, basal AKT3 activity was high and not significantly serum inducible. Similarly, AKT3 basal activity was high and only increased by 18% upon serum stimulation of A172 cells. Consistent with previously published observations, serum stimulated AKT1 activity and basal AKT3 activity are inhibited by the PI3 kinase inhibitor wortmannin in U373 cells (Downward 1998 and references within; Nakatani et al., 1999 and data not shown). Therefore, all three AKT isoforms are active in GBM cells that lack endogenous expression of MMAC1. However, the isoforms exhibit distinct activation profiles with AKT3 possessing high basal activity and little response to serum stimulation, whereas AKT1 activity is serum inducible.
MMAC1 suppresses AKT3 activity in human glioma cells
To examine whether MMAC1 has an effect on AKT1 and AKT3, we examined the activity of each AKT isoform in U373 cells ectopically expressing wild type MMAC1 (Figure 6a). U373 cells were immune precipitated with AKT isoform-specific antibodies and kinase assays were performed using histone H2B as a substrate. Kinase assays were quantitated using a phosphoimager. Expression of wild type MMAC1 consistently reduced AKT3 kinase activity by 30%, whereas expression of catalytically inactive MMAC1 did not significantly alter AKT3 activity (Figure 6b). The basal activity of AKT2 was extremely low and expression of MMAC1 did not considerably alter its activity (Figure 6b). Expression of MMAC1 also suppressed AKT1 activity by 41% (Figure 6b), in agreement with previous studies (Haas-Kogan et al., 1998; Myers et al., 1998; Stambolic et al., 1998; Suzuki et al., 1998). These results demonstrate that the activity of the two AKT isoforms that are highly expressed in the brain, AKT3 and AKT1, is suppressed by MMAC1.
Here we have shown that expression of the tumor suppressor MMAC1 in human glioblastoma cells reduces the intracellular levels of IP3, inhibits extracellular Ca2+ influx, and inhibits ILK activity. In addition, we have identified AKT3 as an additional target for MMAC1 regulation. We have also demonstrated that the catalytic activity of MMAC1 is required for these effects. We and others have previously shown that expression of catalytically active MMAC1 is required to inhibit not only the proliferation of glioma cells, but also to reduce saturation density, motility, and anchorage-independent growth (Tamura et al., 1998; Li and Sun, 1998; Morimoto et al., 1999). Although the inhibition of AKT1 by MMAC1 (Haas-Kogan et al., 1998; Stambolic et al., 1998; Suzuki et al., 1998) may contribute to MMAC1's anti-proliferative phenotype, the AKT signaling pathway has not been linked to cell–matrix and cell–cell interactions or motility. Dephosphorylation of FAK by MMAC1 is thought to affect cell motility and cell–matrix interactions (Tamura et al., 1998). However, it appears that the lipid phosphatase activity of MMAC1 correlates with tumor suppressor activity (Myers et al., 1998). The loss of MMAC1 correlates with the progression of tumors to a metastatic state (Li et al., 1997; Steck et al., 1997). This suggests that the tumor suppressor activity of MMAC1 affects cell motility and/or cell–matrix or cell–cell interactions in vivo. Therefore, inhibition of PIP3 dependent signaling pathways involved in cell motility and cell–matrix interactions may be required for MMAC1's function as a tumor suppressor.
The studies presented here identify a connection between MMAC1 and PIP3 dependent signaling molecules that affect cell–cell and cell–matrix interactions and cellular motility. MMAC1 affects the levels of intracellular IP3 and the influx of extracellular Ca2+. These results suggest that MMAC1 inhibits PLC activity. The PLCγ signaling pathway has previously been linked to the migration of endothelial cells in response to PDGF (Ronnstrand et al., 1999). In addition, inhibition of the PLCγ signaling pathway has been shown to decrease tumor cell motility and invasiveness (Turner et al., 1997; Khoshyomn et al., 1999). Therefore the inhibition of the PLC signaling pathway by MMAC1 may affect the motility of malignant human glioma cells.
Repression of the PLC signaling pathway may also contribute to the anti-proliferative phenotype induced by MMAC1. Decreased intracellular levels of the PLC product, DAG, could reduce PKC activity. PKC activation occurs in response to multiple mitogenic signals (Rhee et al., 1997), and amplification of multiple isoforms of PKC have been found in malignant gliomas (Baltuch et al., 1996). We are currently examining whether expression of MMAC1 represses activation of various PKC isoforms in GBM cells. In addition, PLC is commonly activated in response to stimulation of growth factor receptor tyrosine kinases (Rhee et al., 1997). Therefore, repression of the PLC pathway by MMAC1 may also down-modulate signaling from tyrosine kinase pathways constitutively activated in gliomas (Vassbotn et al., 1994). Interestingly, calcium has also been shown to regulate Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) which represses AKT in a PIP3-independent manner (Yano et al., 1998), indicating that PLC signaling may overlap with the AKT pathway (Figure 7). Therefore, by repressing the PLC signaling pathway, MMAC1 could effectively alter cell motility as well as proliferation signals in AKT-dependent as well as AKT-independent pathways.
ILK may similarly affect cell motility as well as cell–cell and cell–matrix interactions of glioma cells. Overexpression of ILK induces phosphorylation and inactivation of glycogen synthase kinase-3 (GSK3) and may thus affect levels of the cell–cell interaction protein, β-catenin (Delcommenne et al., 1998). Overexpression of ILK also reduces expression of E-cadherin (Wu et al., 1998Wu et al., 1998) and decreases adhesion of cells to the extracellular matrix (Hannigan et al., 1996; Wu et al., 1998Wu et al., 1998). In this manner, MMAC1 inhibition of ILK activity may affect cell–cell contacts as well as cellular motility. ILK has also been linked to cellular proliferation through its effect on the G1-S transition of the cell cycle (Radeva et al., 1997). This effect has been attributed to phosphorylation of AKT1 on serine 473 by ILK (Delcommenne et al., 1998). Human AKT3 has recently been cloned and appears to have the PDK2 serine phosphorylation site, similar to AKT1 (Brodbeck et al., 1999). If ILK indeed has PDK2 activity, then the inhibition of AKT1 and AKT3 by MMAC1 expression could be due to combined effects on membrane recruitment of AKT and/or downregulation of ILK. We are currently generating a dominant-negative mutant of ILK to examine whether ILK repression affects both cellular proliferation and cell–cell interactions.
We also show that MMAC1 expression represses AKT3 activity but does not appear to significantly affect AKT2 activity. Basal AKT2 activity was extremely low in both cell lines examined, so it is difficult to address whether we would be able to detect subtle effects on AKT2 by MMAC1. Thus far, few differences between the AKT isoforms have been found (Konishi et al., 1995; Brodbeck et al., 1999). Importantly, our results show that the AKT3 isoform exhibits high basal activity in two human glioma cell lines that lack endogenous MMAC1 expression. As MMAC1 has been implicated in the etiology of both glioblastoma multiforme and prostate cancer, it is interesting to note that AKT3 is also constitutively active in prostate cancer cells lines (Nakatani et al., 1999). In contrast to AKT3, the majority of AKT1 activity is serum inducible, suggesting that these isoforms are differentially regulated. Lastly, our finding that the AKT isoforms have distinct tissue specific patterns of expression suggests that the isoforms have unique functions that result in cell type specific effects downstream of MMAC1.
Results from in vivo experiments indicate a role for MMAC1 in cell–cell and cell–matrix interactions and motility. Mouse embryos lacking MMAC1 exhibit severely disorganized blastocysts (Stambolic et al., 1998), suggesting that expression of MMAC1 is required for proper cell–cell signaling. Furthermore, mutations in MMAC1 predominate in advanced invasive tumors (Li et al., 1997; Steck et al., 1997). These findings suggest a role for MMAC1 in metastatic progression, consistent with its putative role in adhesion and motility. Interestingly, ILK overexpression resulted not only in cell transformation, but also in increased metastasis in in vitro models (Hannigan et al., 1996; Radeva et al., 1997; Delcommenne et al., 1998; Wu et al., 1998Wu et al., 1998), consistent with the possibility that ILK may mediate many of the tumor suppressor functions of MMAC1.
Materials and methods
A172 and U373 cells were from ATCC. U373 cells stably expressing wild type MMAC1 or C124S MMAC1 were isolated as described previously (Morimoto et al., 1999). Cells were maintained in Dulbecco's modified eagle's media (DMEM) with 10% fetal calf serum, penicillin (250 U/ml), streptomycin (25 μg/ml) and L-glutamine (5 mg/ml). For serum stimulation, cells were maintained in serum free DMEM (minimal media) for 16 h, washed three times with PBS and maintained in minimal media for an additional 3 h. Cells were then treated with DMEM containing 20% serum for 30 min at 37°C. For inhibition studies, cells were pretreated with 100 nM wortmannin (Calbiochem) or 50 μM LY294002 (Calbiochem) or 50 μM PD 98059 (Calbiochem) for 30 min at 37°C.
Cross linked anti-MMAC1 antibodies were generated as described previously (Morimoto et al., 1999). AKT isoform specific antibodies were purchased from UBI (AKT1, AKT2, AKT3) and a rabbit polyclonal antibody generated against the N-terminus of AKT3 (Nakatani et al., 1999). Anti-ILK antibody and anti-ERK1 and ERK2 MAP kinase antibody was purchased from UBI.
Immune precipitations and Westerns
Cells were lysed in 1% Nonidet P-40, 50 mM MOPS pH 7.0, 150 mM NaCl, 5% Glycerol, 0.4 mM EDTA pH 8 (lysis buffer). Lysates were immune precipitated with and washed three times in lysis buffer. Protein was eluted from the beads with SDS sample buffer (Novex) containing 10% β-mercaptoethanol at 37°C. Proteins were separated by SDS–PAGE and Western blotted in 5% blocking solution (BioRad) in 50 mM Tris pH 8, 150 mM NaCl, 0.05% Tween-20. HRP-linked Protein A was used with ECL (Amersham) as a detection agent.
Organs were harvested from normal mice, Dounce homogenized in lysis buffer, and centrifuged for 10 min at 14 000 r.p.m. at 4°C three times. Lysates were then incubated with formalin fixed S. aureus (Calbiochem) for 1 h at 4°C and centrifuged for 10 min at 14 000 r.p.m. at 4°C three times. Equivalent amounts of total protein (2 mg) from each tissue was used for the anti-ILK, anti-AKT and anti-MMAC1 immune precipitates.
AKT kinase assays
Protein lysates were incubated with the appropriate AKT antibody for at least 2 h at 4°C. Protein A-agarose beads (UBI) were added for an additional 2 h incubation. Immune precipitates were washed three times with 25 mM HEPES pH 7, 1 M Nacl, 0.1% BSA, 10% Glycerol, 1% Triton X-100 and once with 20 mM HEPES pH 7, 10 mM MgCl2, 10 mM MnCl2, 0.2 mM EGTA (kinase buffer). Beads were resuspended in kinase buffer containing 1 mM DTT, 5 μM ATP, 10 μCi[γ-32P]ATP and 500 ng histone H2B (Boehringer Mannheim). Reactions were incubated at 30°C for 30 min and terminated by the addition of 2Ã—SDS sample buffer containing 10% β-MeOH. Kinase assay reactions were quantitated using a phosphoimager and ImageQuant software, Molecular Dynamics.
ILK kinase assays
Cells were resuspended in lysis buffer and incubated with 4 μg of ILK antibody and protein A agarose for 2 h at 4°C. Beads were washed three times with lysis buffer and once with 50 mM HEPES pH 7, 10 mM MnCl2, 10 mM MgCl2, 2 mM NaF, 1 mM Na3VO4 (ILK kinase buffer). Beads were resuspended in ILK kinase buffer containing 5 μM cold ATP, 10 μCi[γ-32P]ATP and 30 μg myelin basic protein (UBI). The reactions were incubated at 30°C for 20 min and terminated by the addition of 2Ã—SDS sample buffer. The kinase assay reactions were quantitated as described above.
IP3 binding assays
Cells were incubated in lysis buffer for 10 min on ice and 0.2 volumes of ice cold 20% perchloric acid were added. Lysates were incubated an additional 20 min on ice and centrifuged at 14 000 r.p.m. for 15 min at 4°C. Supernatants were neutralized with ice cold KOH to a pH of 7.5 and centrifuged for 15 min at 14 000 r.p.m. at 4°C. Supernatants were equalized for protein concentration and IP3 levels were quantitated using an IP3 binding assay (Amersham TRK1000) as per manufacturer's instructions.
Calcium flux assays
U373 cells (2.5Ã—106) were incubated with 1 μM indo-1AM (Molecular Probes, Eugene, OR, USA) for 30 min at room temperature in calcium free HBSS. After labeling, cells were washed and resuspended in calcium free HBSS supplemented with 20 mM HEPES buffer. Cells were preincubated with 5 mM EDTA for 5 min and/or 50 μM LY294002 (Calbiochem) for 30 min and then stimulated with media containing 20% serum or PBS and subsequently incubated with a molar excess of calcium chloride. Measurement of calcium flux was performed using a FACSVantage (Becton Dickinson, Mountain View, CA, USA).
Aoki M, Batista O, Bellacosa A, Tsichlis P and Vogt PK . 1998 Proc Natl Acad Sci USA 95: 14950–14955.
Baltuch GH and Yong VW . 1996 Brain Res 71: 143–149.
Brodbeck D, Cron P and Hemmings BA . 1999 J Biol Chem 274: 9133–9136.
Cheney IW, Johnson DE, Vaillancourt M-T, Avanzini J, Morimoto A, Demers GW, Wills KN, Shabram PW, Bolen JB, Tavtigian SV and Bookstein R . 1998 Cancer Res 58: 2331–2334.
Corvera S and Czech MP . 1998 Trends in Cell Biol 8: 442–446.
Delcommenne M, Tan C, Gary V, Rue L, Woodgett J and Dedhar S . 1998 Proc Natl Acad Sci USA 95: 11211–11216.
Downward J . 1998 Science 279: 673–674.
Falasca M, Logan SK, Lehto VP, Baccante G, Lemmon MA and Schlessinger J . 1998 EMBO J 17: 414–422.
Furnari FB, Lin H, Su Huang H-J and Cavenee WK . 1997 Proc Natl Acad Sci 94: 12479–12484.
Guldberg P, Straten P-t, Birck A, Ahrenkiel V, Kirkin AF and Zeuthen J . 1997 Cancer Res 57: 3660–3663.
Haas-Kogan D, Shalev N, Wong M, Mills G, Yount G and Stokoe D . 1998 Curr Biol 8: 1195–1198.
Hannigan GE, Leung-Hagesteijn C, Fitz-Gibbon L, Coppolino MG, Radeva G, Filmus J, Bell JC and Dedhar S . 1996 Nature 379: 91–96.
Kong D, Suzuki A, Zou T-T, Sakurada A, Kemp LW, Wakatsuki S, Yokoyama T, Yamakawa H, Furukawa T, Sato M, Ohuchi N, Sato S, Yin J, Wang S, Abraham JM, Souza RF, Smolinski KN, Meltzer SJ and Horii A . 1997 Nat Genet 17: 143–144.
Konishi H, Kuroda S, Tanaka M, Matsuzaki H, Ono Y, Kameyama K, Haga T and Kikkawa U . 1995 Biochem Biophys Res Comm 216: 526–534.
Khoshyomn S, Penar PL, Rossi J, Wells A, Abramson DL and Bhushan A . 1999 Neurosurg 44: 568–577.
Li D-M and Sun H . 1997 Cancer Res 57: 2124–2129.
Li D-M and Sun H . 1998 Proc Natl Acad Sci USA 95: 15406–15411.
Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanela BC, Ittman M, Tycko B, Hibshoosh H, Wigler MH and Parsons R . 1997 Science 275: 1943–1947.
Liaw D, Marsh DJ, Li J, Dahia PLM, Wang SI, Zheng Z, Bose S, Cal KM, Tsou HC, Peacocke M, Eng C and Parsons R . 1997 Nature Genet 16: 64–67.
Marsh DJ, Dahia PLM, Zheng Z, Liaw D, Parsons R, Gorlin RJ and Eng C . 1997 Nat Genet 16: 333–334.
Maehama T and Dixon J . 1998 J Biol Chem 273: 13375–13378.
Myers MP, Stolarov JP, Eng C, Li J, Wang SI, Wigler MH, Parsons R and Tonks NK . 1997 Proc Natl Acad Sci USA 94: 9052–9057.
Myers M, Pass I, Batty IH, Van der Kaay J, Stolarov JP, Hemmings BA, Wigler MH, Downes CP and Tonks NK . 1998 Proc Natl Acad Sci USA 95: 13513–13518.
Morimoto AMM, Berson AE, Fujii GH, Steck PA, Tavtigian SV, Bookstein R and Bolen JB . 1999 Oncogene 18: 1261–1266.
Nakatani K, Thompson DA, Barthel A, Sakaue H, Liu W, Weigel RJ and Roth RA . 1999 J Biol Chem 274: 21528–21532.
Radeva G, Petrocells T, Behrend E, Leung-Hagesteijn C, Films J, Slingerland J and Dedhar S . 1997 J Biol Chem 272: 13937–13944.
Rameh LE, Rhee SG, Spokes K, Kazlauskas A, Cantley LC and Cantley LG . 1998 J Biol Chem 273: 23750–23757.
Rhee SG and Bae YS . 1997 J Biol Chem 272: 15045–15048.
Ronnstrand L, Siegbahn A, Rorsman C, Johnell M, Hansen K and Heldin CH . 1999 J Biol Chem 274: 22089–22094.
Steck PA, Pershouse MA, Jasser SA, Yung WKA, Lin H, Ligon AH, Langford LA, Baumgard ML, Hattier T, Davis T, Frye C, Hu R, Swelund B, Teng DH-F and Tavtigain SV . 1997 Nature Genet 15: 356–362.
Suzuki A, de la Pompa JL, Stambolic V, Elia AJ, Sasaki T, del Barco Barrantes I, Ho A, Wakeman A, Itie A, Khoo W, Fukumoto M and Mak TW . 1998 Curr Biol 8: 1169–1178.
Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C, Sasaki T, Ruland J, Penninger JM, Siderovski DP and Mak TM . 1998 Cell 95: 29–39.
Tamura M, Gu J, Matsumoto K, Aota S, Parsons R and Yamada KM . 1998 Science 280: 1614–1617.
Turner T, Epps-Fung MV, Kassis J and Wells A . 1997 Clin Cancer Res 3: 2275–2282.
Vassbotn FS, Ostman A, Langeland N, Holmsen H, Westermark B, Heldin C-H and Nister M . 1994 J Cell Physiol 158: 381–389.
Wu X, Senechal K, Neshat M, Whang YE and Sawyers CL . 1998a Proc Natl Acad Sci USA 95: 15587–15591.
Wu C, Keightley SY, Leung-Hagesteijn C, Radeva G, Coppolino M, Goicoechea S, McDonald JA and Dedhar S . 1998b J Biol Chem 273: 528–536.
Yano S, Tokumitsu H and Soderling TR . 1998 Nature 396: 584–587.
We are grateful to Madeline Fort for providing mouse tissues and thank the FACS facility for help with the calcium flux assays. We also thank Emma Lees and Jing Wang for helpful comments on the manuscript and Maribel Andonian and Gary Burget for graphics support. DNAX Research Institute is fully supported by Schering-Plough Corporation.
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Morimoto, A., Tomlinson, M., Nakatani, K. et al. The MMAC1 tumor suppressor phosphatase inhibits phospholipase C and integrin-linked kinase activity. Oncogene 19, 200–209 (2000). https://doi.org/10.1038/sj.onc.1203288
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