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The MMAC1 Tumor Suppressor Phosphatase Inhibits Phospholipase C and Integrin-Linked Kinase Activity

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The MMAC1 Tumor Suppressor Phosphatase Inhibits Phospholipase C and Integrin-Linked Kinase Activity Oncogene (2000) 19, 200 ± 209 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc The MMAC1 tumor suppressor phosphatase inhibits phospholipase C and integrin-linked kinase activity Alyssa M Morimoto1, Michael G Tomlinson1, Kaname Nakatani2, Joseph B Bolen3, Richard A Roth2 and Ronald Herbst*,1 1Department of Cell Signaling, DNAX Research Institute, 901 California Ave, Palo Alto, California, CA 94304, USA; 2Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California, CA 94305, USA; 3Department of Oncology, Hoechst Marion Roussel, Bridgewater, New Jersey, NJ 08807, USA Loss of the tumor suppressor MMAC1 has been shown Introduction to be involved in breast, prostate and brain cancer. Consistent with its identi®cation as a tumor suppressor, MMAC1 (also known as PTEN or TEP-1) is mutated at expression of MMAC1 has been demonstrated to reduce a high frequency in brain, breast, and prostate tumors cell proliferation, tumorigenicity, and motility as well as as well as in melanomas and endometrial carcinomas, aect cell±cell and cell±matrix interactions of malignant (Guldberg et al., 1997; Kong et al., 1997; Li and Sun, human glioma cells. Subsequently, MMAC1 was shown 1997; Li et al., 1997; Steck et al., 1997). These to have lipid phosphatase activity towards PIP3 and observations suggest that MMAC1 acts as a tumor protein phosphatase activity against focal adhesion suppressor in multiple tissues. Indeed, subsequent kinase (FAK). The lipid phosphatase activity of studies showed that reintroduction of this gene into MMAC1 results in decreased activation of the PIP3- human glioma cells reduced cell growth, tumorigenicity dependent, anti-apoptotic kinase, AKT. It is thought that in nude mice, and aected motility and cell ± cell this inhibition of AKT culminates with reduced glioma interactions, demonstrating that MMAC1 represents a cell proliferation. In contrast, MMAC1's eects on cell bone ®de tumor suppressor (Furnari et al., 1997; Cheney motility, cell ± cell and cell ± matrix interactions are et al., 1998; Tamura et al., 1998; Morimoto et al., 1999). thought to be due to its protein phosphatase activity Interestingly, germ line mutations in MMAC1 have also towards FAK. However, recent studies suggest that the been linked to the multiple hamartomatous predisposi- lipid phosphatase activity of MMAC1 correlates with its tion syndromes, Cowden's disease and Bannayan ± ability to be a tumor suppressor. The high rate of Zonana. These syndromes are also associated with mutation of MMAC1 in late stage metastatic tumors increased susceptibility to breast and thyroid cancer suggests that eects of MMAC1 on motility, cell ± cell (Liaw et al., 1997; Marsh et al., 1997). and cell ± matrix interactions are due to its tumor Sequence analysis of MMAC1 indicated that this suppressor activity. Therefore the lipid phosphatase gene encodes motifs conserved in dual speci®city activity of MMAC1 may aect PIP3 dependent signaling phosphatases (Li and Sun, 1997; Li et al., 1997; Steck pathways and result in reduced motility and altered cell ± et al., 1997). MMAC1 has been shown to possess cell and cell ± matrix interactions. We demonstrate here protein phosphatase activity towards focal adhesion that expression of MMAC1 in human glioma cells kinase (FAK) (Tamura et al., 1998) and lipid reduced intracellular levels of inositol trisphosphate and phosphatase activity toward phosphatidylinositol 2+ inhibited extracellular Ca in¯ux, suggesting that 3,4,5-trisphosphate (PIP3) (Maehama et al., 1998). MMAC1 aects the phospholipase C signaling pathway. However, it appears that the lipid phosphatase activity In addition, we show that MMAC1 expression inhibits of MMAC1 correlates with tumor suppression (Myers integrin-linked kinase activity. Furthermore, we show et al., 1998). that these eects require the catalytic activity of Many signaling molecules directly bind PIP3 through MMAC1. Our data thus provide a link of MMAC1 to pleckstrin homology (PH) domains (Corvera and PIP3 dependent signaling pathways that regulate cell ± Czech, 1998) and could thus be aected by reduced matrix and cell ± cell interactions as well as motility. levels of PIP3 due to expression of MMAC1. The Lastly, we demonstrate that AKT3, an isoform of AKT binding of PIP3 to PH domain-containing proteins such highly expressed in the brain, is also a target for as AKT, phospholipase C (PLC) and integrin linked MMAC1 repression. These data suggest an important kinase (ILK) is thought to facilitate membrane role for AKT3 in glioblastoma multiforme. We therefore targeting and induce conformational changes that propose that repression of multiple PIP3 dependent result directly, or indirectly, in activation (Aoki et al., signaling pathways may be required for MMAC1 to 1998; Delcommenne et al., 1998; Falasca et al., 1998). act as a tumor suppressor. Oncogene (2000) 19, 200 ± Recent studies have shown that PIP3 levels are 209. indeed higher in cells lacking MMAC1 (Haas-Kogan et al., 1998; Stambolic et al., 1998). In addition, lack of Keywords: MMAC1; PTEN; TEP1; ILK; PLC 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 *Correspondence: R Herbst phosphorylated BAD protein (Myers et al., 1997; Wu Received 6 May 1999; revised 30 September 1999; accepted 13 October 1999 et al., 1998a). Therefore, these results are consistent MMAC1 inhibits PLC and ILK AM Morimoto et al 201 with a model in which MMAC1 reduces AKT activity and motility of human glioma cells (Li et al., 1997; and thus increases apoptosis, resulting in tumor Steck et al., 1997; Furnari et al., 1997; Cheney et al., suppression. However, there are a number of observa- 1998; Tamura et al., 1998; Morimoto et al., 1999). tions that suggest that MMAC1 does not inhibit tumor The lipid phosphatase activity of MMAC1 appears to suppression solely through repression of the AKT result in inhibition of AKT1 activity and a growth signaling pathway. suppression phenotype (Haas-Kogan et al., 1998; Expression of MMAC1 in malignant human glioma Stambolic et al., 1998; Suzuki et al., 1998). The cells not only inhibits cell proliferation and tumor- protein phosphatase activity results in FAK depho- igenicity, but also aects motility and cell ± cell and sphorylation which is thought to then cause decreased cell ± matrix interactions (Furnari et al., 1997; Cheney cell motility and cell ± matrix interaction (Tamura et et al., 1998; Tamura et al., 1998; Morimoto et al., al., 1998). However, it appears that the lipid 1999). In addition, loss of MMAC1 correlates with the phosphatase activity of MMAC1 correlates with progression of tumors to a metastatic state, suggesting tumor suppressor activity (Myers et al., 1998). Thus that the tumor suppressor activity of MMAC1 aects far, the AKT signaling pathway has not been linked cell motility and/or cell ± matrix or cell ± cell interac- to either cell ± cell, cell ± matrix interactions or cell tions in vivo. As the AKT signaling pathway has not motility. This suggested that other PIP3 dependent been shown to aect cell ± matrix and cell ± cell signaling pathways may be inhibited by the tumor interactions or cell motility, these observations suggest suppressor activity of MMAC1 resulting in decreased that repression of PIP3 regulated signaling pathways cell motility and/or altered cell ± matrix and cell ± cell distinct from AKT may contribute to the ability of interactions. MMAC1 to suppress tumor formation. We therefore examined whether MMAC1 expres- In contrast to the AKT signaling pathway, both sion aects signaling pathways that are involved in the phospholipase C (PLC) and the integrin linked such processes and are also regulated by PIP3. One kinase (ILK) signaling pathways regulate motility as such candidate is the phospholipase C (PLC) well as cell ± cell and cell ± matrix interactions. The signaling pathway. PLC activation generates inositol PLC signaling pathway has been linked to the trisphos-phate (IP3) and diacylglycerol (DAG) which motility of human glioma cells (Kyoshmomn et al., result in a ¯ux of intracellular calcium and 1999). Similarly, integrin linked kinase (ILK) aects stimulation of protein kinase C (PKC) in a PIP3 cell motility and cell ± cell and cell ± matrix interac- dependent manner, respectively (Falasca et al., 1998; tions. Overexpression of this PIP3 regulated kinase is Rameh et al., 1998; Rhee et al., 1997). In addition, also sucient to induce tumorigenicity in vivo the PLC signaling pathway has been linked to the (Hannigan et al., 1996; Radeva et al., 1997). As motility of human glioma cells (Khoshyomn et al., both ILK and PLC are PIP3 dependent enzymes 1999). We therefore examined whether expression of (Hannigan et al., 1996; Falasca et al., 1998), they MMAC1 aected the intracellular levels of IP3 in represent potential downstream targets for MMAC1 human glioma cells. regulation. As such, MMAC1 could aect tumor cell As a control, we ®rst examined whether IP3 levels motility, cell ± cell and cell ± matrix interactions are sensitive to the PI3 kinase inhibitor, LY294002, in through the inhibition of these enzymes and their U373 cells. Asynchronously growing U373 control cells signaling pathways. were treated with vehicle or the PI3 kinase inhibitor, We show here that reintroduction of MMAC1 into LY294002, and the amount of IP3 present in the lysates human glioblastoma cells reduces extracellular Ca2+ was determined. As shown in Figure 1a, the levels of in¯ux, intracellular inositol trisphosphate (IP3) levels, IP3 were reduced by over 50% when cells were treated and ILK activity. These results thus provide a link with LY294002. To determine whether MMAC1 between MMAC1 and PIP3 dependent signaling similarly aects IP3 levels, we also examined IP3 levels pathways that aect cell motility, cell ± cell and cell ± in cells expressing WT MMAC1 or a catalytically matrix interactions. As the PLC and the ILK signaling inactive form of MMAC1, C124S MMAC1 (Figure pathways are also involved in mitogenesis (Rhee et al., 1b).
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