PP2C family members play key roles in regulation of cell survival and apoptosis

Abstract

Although unlimited proliferation of cancer cells is supported by multiple signaling pathways involved in the regulation of proliferation, survival, and apoptosis, the molecular mechanisms coordinating these different pathways to promote the proliferation and survival of cancer cells have remained unclear. SAPK and integrin‐ILK signaling pathways play key roles in the promotion of apoptosis and cell proliferation/survival, respectively. Studies of TNFα‐ and H₂O₂‐induced apoptosis revealed that ASK1, a component of the SAPK system, mediates the TNFα and H₂O₂ signaling of apoptosis. ASK1 is activated by autophosphorylation of a specific threonine residue (T845) following TNFα stimulation. Our recent studies indicate that PP2Cɛ, a member of the PP2C family, associates with and inactivates ASK1 by dephosphorylating T845. In contrast, PP2Cδ/ILKAP, a second PP2C family member, activates ASK1 by enhancing cellular phosphorylation of T845. PP2Cδ/ILKAP also forms a complex with ILK1 to inhibit the GSK3β‐mediated integrin‐ILK1 signaling in vivo, inhibiting cell cycle progression. These observations raise the possibility that PP2Cδ/ILKAP acts to control the cross‐talk between integrin‐induced and TNFα‐induced signaling pathways, inhibiting the former and stimulating the latter, thereby inhibiting proliferation and survival and promoting the apoptosis of cancer cells. (Cancer Sci 2006; 97: 563–567)

Abbreviations:

ASK1

apoptosis signal‐regulating kinase 1

GSK‐3β

glycogen synthase kinase‐3β

ILK

integrin‐linked kinase

JNK

c‐Jun N‐terminal kinase

LNCaP

lymphnode carcinoma of prostate

MAPK

mitogen‐activated protein kinase

MEF

mouse embryonic fibroblasts

MEK

mitogen extracellular signal‐regulated kinase

MKK

mitogen‐activated protein kinase kinase

MKKK

mitogen‐activated protein kinase kinase kinase

PP2C

protein serine/threonine phosphatase 2C

ROS

reactive oxygen species

SAPK

stress‐activated protein kinase

siRNA

short interfering RNA

TAK1

transforming growth factor‐activated kinase 1

TNF‐α

tumor necrosis factor‐α.

Unlimited proliferation is typical of cancer cells, a phenotype supported by several distinct biological events, including enhanced cell proliferation, increased cell survival, and decreased cell death. Studies examining the molecular bases of these events have identified a variety of signaling pathways that are involved in their regulation. The mechanisms by which these different signaling pathways are coordinated to regulate proliferation and survival of cancer cells has remained poorly understood.

ROS are normally generated by mitochondria during respiration, but can also be generated by inducible enzyme systems in mammalian cells.^{( 1 )} Cellular ROS levels increase following exposure to a variety of stress agents, such as anticancer drugs.^{( 2 )} Upregulation of these highly reactive molecules promotes apoptosis by stimulating the activation of pro‐apoptotic signaling molecules, such as JNK and p38 (Fig. 1).^{( 3} , ^{4 )} ROS also function in p53‐induced apoptosis.^{( 5 )}

Figure 1.

Diverse effects of reactive oxygen species (ROS) on behavior of cancer cells.

In contrast, low levels of ROS stimulate normal cell proliferation under non‐stress conditions; ROS production was reported to be increased in cancer cells^{( 6} , ^{7 )} (Fig. 1). Elevated ROS appear to maintain the constitutive activation of transcription factors (NF‐κB and AP‐1) during tumor progression.^{( 8 )} Overexpression of Mox1, the catalytic subunit of NADPH oxidase, induces superoxide generation, leading to the transformation of NIH 3T3 cells.^{( 9 )} Thus, ROS might play a critical role in the pathogenesis of cancer. The diverse, and even opposing, effects of ROS on cell behavior reflect the complex nature of the biological functions of these highly reactive molecules.

Integrins, a large family of adhesion receptors including more than 20 members, mediate highly dynamic cell–cell and cell–extracellular matrix interactions. Integrin‐mediated cell adhesion regulates a wide variety of biological processes, including cell migration, survival, and proliferation. The association and dissociation of integrin and their ligands are achieved by changes in the conformations of integrin molecules. Adoption of the active conformation triggered by intracellular signaling and cytoskeletal assembly, resulting in ligand binding, integrin clustering, and recruitment of cytoplasmic plaque proteins to integrin attachment sites, called focal adhesions.^{( 10} , ^{11 )} ILK plays a central role in integrin activation and signaling.^{( 12 )} ILK is composed of three structurally distinct domains, three ankyrin repeats near the N‐terminus, a short linker sequence, and a C‐terminal kinase domain.

Functional studies of ILK1 revealed that GSK3β is a target of ILK1.^{( 13 )} GSK3β, an important mediator of developmental signaling through the Wnt/wingless pathway, alters the transcriptional activity of Tcf/Lef factors through phosphorylation of the transcriptional cofactor β‐catenin.^{( 14} , ^{15 )} GSK3β‐mediated phosphorylation targets β‐catenin for degradation. Overexpression of ILK1 in epithelial cells induces the phosphorylation of GSK3β at Ser9, an inhibitory modification that results in the stabilization and nuclear translocation of β‐catenin, with concomitant activation of Tcf/Lef transcription factors. Thus, ILK1 is an important intracellular regulator of Wnt/wingless signaling, acting specifically through the modulation of GSK3β activity. Activation of ILK1 leads to cell survival and proliferation.

In this article, we propose a novel mechanism governing the cooperation of ROS induction of apoptosis with the integrin‐induced cellular proliferation.

SAPKs play key roles in ROS‐induced activation of both proliferation and apoptosis

Studies examining ROS‐induced activation of cell growth and apoptosis revealed that SAPKs function in these opposing effects. SAPKs, a subfamily of the MAPK superfamily, are highly conserved from yeast to mammals (Fig. 2).^{( 16 )} Two distinct classes of SAPKs are present in mammalian cells, the JNK and p38 kinases. Both JNK and p38 are activated by a variety of environmental stresses, regulating diverse cellular functions in response to these challenges. SAPK activation requires phosphorylation by dual‐specificity protein kinases, members of the MKK family, at conserved tyrosine and threonine residues within the catalytic domain. MKK3 and MKK6 specifically phosphorylate p38, whereas MKK7 selectively modifies JNK. MKK4 recognizes both classes of stress‐activated kinases. The MKKs are activated by the phosphorylation of conserved serine and threonine residues. MKKKs, including ASK1, TAK1, the MEK kinase group, and the mixed lineage kinase group of kinases, have been identified, some of which are also activated by phosphorylation (Fig. 2).

Figure 2.

Regulation of stress‐activated protein kinase (SAPK) signal pathways by PP2C family members. The protein kinase cascade of SAPK signaling pathways and the points where the PP2C family members can interfere with the signals are shown. ASK1, apoptosis signal‐regulating kinase 1; ATF2, activating transcription factor; JNK, c‐Jun N‐terminal kinase; MAPK, mitogen‐activated protein kinase; MKK, mitogen‐activated protein kinase kinase; MKKK, mitogen‐activated protein kinase kinase kinase; PP2C, protein serine/threonine phosphatase 2C; TAK1, transforming growth factor‐activated kinase 1.

SAPKs have been studied primarily in stress responses and apoptosis.^{( 16 )} Following cellular stress, SAPK activation is important in promoting apoptosis in wide range of cell types, including primary tumor cells and transformed cell lines. Signaling through the JNK pathway is critical in the activation of the mitochondria‐dependent apoptotic pathway, but appears to be dispensable for the apoptotic mechanisms induced by activation of death receptors.^{( 17 )}

Accumulating genetic and biochemical data, however, suggest that SAPKs, especially JNK, contribute to proliferative responses in the absence of stress. JNK1^−/– and JNK1^−/–JNK2^−/–MEFs proliferate more slowly than wild‐type MEFs, achieving lower saturation densities. These results establish that JNK is required for normal MEF proliferation.^{( 17 )}

ASK1 mediates ROS‐induced cell apoptosis

Recent studies have suggested several mechanisms linking ROS induction and SAPK activity. ASK1, an upstream regulator of SAPKs, is inhibited in non‐stressed cells by association with thioredoxin.^{( 18 )} Increased ROS levels dissociate this complex, enabling the activation of ASK1 and downstream SAPKs.^{( 19 )} ROS induction is also implicated in TNFα‐induced activation of ASK1.

Studies of the mechanisms controlling TNFα‐ or H₂O₂‐induced signaling have suggested that early/transient activation of JNK by TNFα or H₂O₂ correlates with cell survival/proliferation, whereas late/sustained activation leads to apoptosis, raising the possibility that different pathways of JNK activation might regulate cell survival/proliferation and apoptosis.^{( 20} , ^{21 )} Using wild‐type and ASK1^−/–MEFs, Tobiume et al. demonstrated that ASK1 does not participate in the initial TNFα‐ or H₂O₂‐induced activation of JNK, but does function in sustained activation of JNK.^{( 22 )} These results support the idea that ASK1 is specifically responsible for promoting TNFα‐ or H₂O₂‐induced apoptosis. The induction of apoptosis by TNFα was restored in ASK1^−/–MEFs following infection with adenoviruses encoding ASK1.

PP2Cɛ participates in negative regulation of ASK1

Phosphorylation events participate in both the activation and inactivation of SAPK signaling. ASK1 is activated by autophosphorylation at Thr845 following stimulation of upstream signals, but is inactivated by phosphorylation at Ser83 by Akt, suggesting a direct link between Akt and the stress‐activated kinase family.^{( 23 )} Phosphorylation of both Ser967 and Ser1034 has also been implicated in ASK1 inactivation, although the protein kinases responsible for this phosphorylation event have not been identified.^{( 24} , ^{25 )} These observations suggest that protein phosphatases function in both the negative and positive regulation of SAPK signaling.

PP2C is one of the four major protein serine/threonine phosphatase families, PP1, PP2A, PP2B, and PP2C, in eukaryotes. At least 12 distinct PP2C gene products, 2 Cα, 2Cβ, 2 Cγ/FIN13, 2Cδ/ILKAP, 2Cɛ, 2Cζ, 2Cη, Wip1, CaMKP/hFEM2/POPX2, CaMKP‐N/POPX1, NERPP‐2C, and SCOP/PHLPP, have been identified in mammalian cells (Table 1).^{( 26} , ²⁷ , ²⁸ , ²⁹ , ³⁰ , ³¹ , ³² , ³³ , ³⁴ , ³⁵ , ³⁶ , ³⁷ , ³⁸ , ³⁹ , ⁴⁰ , ⁴¹ , ⁴² , ⁴³ , ⁴⁴ , ⁴⁵ , ⁴⁶ , ⁴⁷ , ⁴⁸ , ⁴⁹ , ⁵⁰ , ^{51 )} These 12 PP2C gene products all exhibit Mg² ± and/or Mn²⁺‐dependent protein phosphatase activities, sharing six conserved structural motifs. Functional studies of PP2C family members indicated that four of these enzymes, PP2Cα, PP2Cβ, PP2Cɛ, and Wip1, participate in the negative regulation of SAPK signaling (Fig. 2).^{( 29} , ⁴⁷ , ⁵² , ⁵³ , ^{54 )} PP2Cβ‐1 and PP2Cɛ suppress SAPK signaling by associating with and dephosphorylating TAK1 at different phosphorylation sites^{( 47} , ^{54 )} (Li et al., unpublished observations, 2004).

Table 1.

PP2C family members

	Size (kDa)	Isoforms	Tissue distribution	References
PP2Cα	42, 36	α‐1, α‐2	ubiquitous	27, 29, 33
PP2Cβ	42–55	β‐1(βs)‐β‐5,	β‐1, βx: ubiquitous	31, 32, 34–36
		βx (βl)	β‐2: brain, heart β‐3–5: testis, liver, intestine
PP2Cγ (FIN13)	59		ubiquitous	37, 38
PP2Cδ (ILKAP)	43		ubiquitous	39, 40
PP2Cɛ	34		rich in brain, heart, testis	47
PP2Cζ	56		testis	48
PP2Cη	45		ubiquitous	49
Wip1	66			41
CaMKP (hFEM2, POPX2)	49		ubiquitous	42, 43, 45
NERPP‐2C	55	NERPP‐55	brain	46
	80	NERPP‐80
CaMKP‐N (POPX1)	83		rich in brain, testis	44, 45
SCOP (PHLPP)	140			50, 51

Recent evidence suggested that PP2Cɛ participates in the regulation of both TAK1 and ASK1 signaling (Fig. 3) (Saito et al., unpublished observations, 2006). In coexpression and coimmunoprecipitation studies, ASK1 was able to associate with PP2Cɛ, as seen with TAK1. The in vivo binding of endogenous PP2Cɛ to endogenous ASK1 was also observed in mouse brain. PP2Cɛ inhibited ASK1‐induced AP‐1 activation in a dose‐dependent manner. PP2Cɛ also suppressed the H₂O₂‐enhanced phosphorylation of ASK1 Thr845, suggesting that PP2Cɛ is involved in the negative regulation of ASK1 (Fig. 3).

Figure 3.

Regulation of TNF‐α‐ASK1 and integrin‐ILK1 signal pathways by PP2Cɛ, PP5 and PP2Cδ/ILKAP. ASK1, apoptosis signal‐regulating kinase 1; GSK‐3β, glycogen synthase kinase‐3β; ILK, integrin‐linked kinase; JNK, c‐Jun N‐terminal kinase; MKK, mitogen‐activated protein kinase kinase; PP2C, protein serine/threonine phosphatase 2C; ROS, reactive oxygen species; TNF‐α, tumor necrosis factor‐α.

Morita et al. previously reported that ASK1 was negatively regulated by PP5, a protein Ser/Thr phosphatase that is distinct from the PP2C family (Fig. 3).^{( 55 )} Interestingly, PP5 was also able to dephosphorylate Thr845 of ASK1. Furthermore, H₂O₂ treatment of cells induced the association of PP5 with ASK1, suggesting that PP5 participates in the negative feedback regulation of the ASK1 signaling pathway. In contrast, PP2Cɛ bound to ASK1 under non‐stress conditions and dissociated from ASK1 on H₂O₂ treatment of cells, suggesting that PP2Cɛ contributes to keeping the ASK1 signaling pathway in an inactive state by associating with and dephosphorylating ASK1 (Saito et al., unpublished observations, 2006). Thus, PP2Cɛ and PP5 appear to play different roles in regulating the ASK1 pathway, although they both dephosphorylate the same site of ASK1.

PP2Cδ/ILKAP participates in positive regulation of ASK1

In contrast to PP2Cɛ, PP2Cδ/ILKAP acts as an activator of TNFα‐induced SAPK signaling (Fig. 3). The expression of PP2Cδ/ILKAP, originally identified by Tong et al., is induced by cellular stresses, including ethanol treatment or ultraviolet irradiation.^{( 39 )} Recently, we determined that expression of PP2Cδ/ILKAP in 293 cells enhanced both the TNFα‐induced activation of AP1 and the phosphorylation of JNK and p38. PP2Cδ expression in HEK293 cells, however, had no effect on TNFα‐induced, early/transient activation of JNK or p38, but further enhanced the sustained activation of these MAPKs (Toriumi et al., unpublished observations, 2006). In these cells, PP2Cδ/ILKAP associated with ASK1, activating this kinase by enhancing the phosphorylation of T845. These observations suggest that PP2Cδ might act as a positive regulator of TNFα‐induced apoptosis (Fig. 3). It remains unknown, however, whether PP2Cδ mediates this activation by dephosphorylating any of the inhibitory phosphorylation sites (Ser83, Ser967, and Ser1034) of ASK1.

PP2Cδ/ILKAP suppresses integrin signaling by inhibiting ILK1

Leung‐Hagesteijin et al. isolated PP2Cδ/ILKAP in a yeast two‐hybrid screen designed to identify proteins interacting with ILK1.^{( 40 )} PP2Cδ/ILKAP and ILK1 were coprecipitated from HEK293 cell lysates, independent of ILK1 and PP2Cδ/ILKAP catalytic activities. Exogenous expression of recombinant, catalytically active PP2Cδ/ILKAP in HEK293 cells inhibited endogenous ILK1 protein kinase activity (Fig. 3). PP2Cδ/ILKAP did not affect Raf‐1 kinase activity. Inactive PP2Cδ/ILKAP did not affect ILK1 activity. PP2Cδ/ILKAP expression inhibited integrin‐stimulated phosphorylation of GSK3β at Ser9, leading to inactivation of Tcf/Lef. Conversely, silencing of endogenous PP2Cδ/ILKAP with siRNA stimulated GSK3β phosphorylation at Ser9. These results suggested that PP2Cδ/ILKAP selectively inhibits GSK3β‐mediated ILK1 signaling in vivo by forming a complex with ILK1 (Fig. 3).^{( 40 )}

PP2Cδ/ILKAP suppresses oncogenic transformation by inhibition of ILK1

The function of PP2Cδ/ILKAP in the regulation of oncogenic transformation was investigated by Kumar et al.^{( 56 )} In LNCaP prostate carcinoma cells, both transient and stable expression of PP2Cδ/ILKAP suppressed ILK kinase activity; PP2Cδ/ILKAP‐mediated inhibition of ILK downregulated GSK3β Ser9 phosphorylation, which could be reversed by overexpression of wild‐type, but not dominant‐negative, ILK. The expression levels of cyclin D1, a target of ILK‐GSK3β signaling, were enhanced by siRNA‐mediated downregulation of PP2Cδ/ILKAP, suggesting that ILK1 antagonism modulates cell cycle progression. Exogenous expression of PP2Cδ/ILKAP increased the proportion of LNCaP cells in G1 phase in comparison to that seen in cells transfected with vector alone; siRNA suppression of PP2Cδ/ILKAP expression increased the entry of cells into S phase, consistent with ILK antagonism. Anchorage‐independent growth of LNCaP cells was inhibited by PP2Cδ/ILKAP overexpression, suggesting a critical role for this protein in the suppression of cellular transformation.^{( 56 )} Taken together, these results indicate that endogenous PP2Cδ/ILKAP activity inhibits ILK‐GSK3β signaling, suggesting that PP2Cδ/ILKAP activity inhibits oncogenic transformation (Fig. 3).

Conclusions

In this article, we discussed the regulation by PP2C family members of the TNFα‐ASK1‐JNK and integrin‐ILK‐GSK3β‐β‐catenin signaling pathways, which participate in the stimulation of apoptosis and cell proliferation/survival, respectively. The TNFα‐ASK1‐JNK pathway is inactivated by PP2Cɛ and activated by PP2Cδ/ILKAP. Both of these PP2C family members regulate ASK1 activity, although they exert opposite effects (Fig. 3). These observations suggest that PP2Cδ/ILKAP functions as an apoptosis‐promoting factor. PP2Cδ/ILKAP, however, is also able to suppress the integrin‐ILK‐GSK3β‐β‐catenin pathway by inhibition of ILK1. Accumulating evidence indicates that PP2Cδ/ILKAP inhibits cell proliferation and oncogenic transformation through its effects on this pathway. These observations raise the possibility that PP2Cδ might control the cross‐talk between the integrin‐induced and TNFα‐induced signaling pathways, inhibiting the former and stimulating the latter to promote the apoptosis of cancer cells (Fig. 3).