Mammalian Sterile 20–Like Kinases in Tumor Suppression: an Emerging Pathway

Review

Mammalian Sterile 20–Like Kinases in Tumor Suppression: An Emerging Pathway

Eric E. O’Neill,1 David Matallanas,1 and Walter Kolch1,2

1Beatson Insitute for Cancer Research and 2Sir Henry Welcome Functional Genomics Facility, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom

Abstract dimerization domain that is thought to mediate a trans-autophos- Emerging evidence suggests that the proapoptotic kinase phorylation event that is essential for kinase activity (4). At later times, mammalian sterile 20–like kinase 2 (MST2) acts in a novel usually several hours after initial activation, ensuing caspase activity tumor suppression pathway. Recently, we showed that Raf-1 severs the carboxyl-terminal regulatory domain from the kinase kinase sequesters and inhibits MST2 and that this event is domain, further enhancing MST1/2 catalytic activity. critical for Raf-mediated cell survival. In this review, we Raf Regulates MST2 Signaling summarize Raf control of MST2 and we outline a novel Raf-MAPK signaling mediates diverse cellular fates, including pathway involving the downstream effector proteins Salvador proliferation, differentiation, and survival. Activation of Raf occurs and Warts/Lats that may act to limit the positive effects of as a result of GTP loading of Ras in response to mitogens, which Raf–mitogen-activated protein kinase signaling in cancer allows a cascade of activation through Raf and MEK kinases to the cells. (Cancer Res 2005; 65(13): 5485-7) MAP kinases, also known as the extracellular signal-regulated kinases (ERK; ref. 7). The MAPK pathway does provide mechanisms The Mammalian Sterile 20–Like Kinases to generate specific biological responses that can be imparted The kinase mammalian sterile 20 (Ste20)–like kinase 2 (MST2, through the overall strength and duration of the ERK signal, allowing Krs1) and its close homologue MST1 (Krs2) are members of the sustained versus transient activation of ERK substrates. The germinal center kinase group II (GCK II) family of mitogen-activated existence of multiple isoforms at each step in the pathway allows protein kinase (MAPK)–related kinases that includes the more further specificity through distinct binding partners and, hence, distantly related kinases MST3, MST4, LOK, SOK, and SLK (1). This integration of additional signals that could modulate the overall GCK II family also shares homology to the yeast Ste20 kinase in the response (7). The kinase cascade consists of three Raf isoforms, Raf- kinase domain. GCK II kinases seem to be mainly involved in 1, B-Raf, and A-Raf, which are able to phosphorylate both isoforms of apoptotic signaling in response to stress signals, including Ste20 MEK (MEK1/2), which in turn can activate either ERK1 or ERK2 (7). itself (2). Both MST1 and MST2 have been described as MAPK kinase Ras activates a group of pathways, but is the only known signal that kinase kinases that lead to c-Jun-NH2-kinase (JNK) activation in activates the Raf kinases (7) and the importance of this step in response to stress and Fas signaling, apparently through a MEKK1/ cellular events has been established through the identification of MKK7 route (3). Activation of JNK and subsequent activation of mutation in Ras and the B-Raf kinase, which lead to increased ERK caspases seems to explain the original description of MST1 and signaling in human cancers (8). Genetic ablation of the Raf isoforms MST2 as proapoptotic kinases. Once activated, caspase-3 removes in mice allowed us to separate some unique aspects of individual Raf the carboxyl-terminal portion of MST2; this also occurs on MST1 isoform signaling. Most notably, mice in which the raf1 gene was together with a distinct caspase-6/7–mediated cleavage event (4). deleted died in utero due to an overwhelming level of apoptosis, The carboxyl-terminal region contains an inhibitory region together which occurred despite normal expression of the alternate Raf with the Salvador and RASSF homology (SARAH) protein-protein isoforms and undisturbed ERK activation (9). interaction domain, dimerization domain, and a nuclear export The fact that ERK activation was unaffected in cells from Raf -1À/À signal (4). Caspase cleavage results in a constitutively active kinase mice pointed to a survival signal, other than Ras-induced ERK domain residing in the nucleus that phosphorylates histone 2B and signaling, that is defective and that led to the apoptotic phenotype of potentiates the DNA condensation characteristic of apoptosis (5). these mice (10, 11). A survival function for Raf-1 that is independent The yeast homologue, Ste20, has been shown recently to function in of ERK-promoting activity was confirmed by phenotypic rescue of a similar manner to promote an apoptotic-like response in yeast, the Raf-1 null mice with a Raf-1-YY340/341FF derivative that is not which do not have caspase-like activity or Ste20 cleavage (2). activated by mitogens and that has low basal kinase activity (10). Caspase regulation of MST1/2 may have arose later in evolution as Mouse embryo fibroblasts derived from the Raf-1À/À mice also the full-length kinases seem to be able to be activated in cells by exhibited a hyperapoptotic phenotype; furthermore, the sensitivity caspase-independent mechanisms. However, activation of the full- was specific to certain stimuli (i.e., serum starvation and engagement length kinase is not readily observed, in agreement with the idea that of the death receptor Fas). Using this system, we identified the only a small pool of MST1/2 is active at one time (4). Activation of full- pathway that leads to the proapoptotic phenotype, defining MST1 length MST1/2 occurs in response to stress conditions or treatment and MST2 in proteomic screens for potential pathways through with staurosporine or okadaic acid (6), requiring a functional which Raf-1 could provide a survival signal (12). The interaction of Raf-1 and MST2 in cells occurred in the absence of serum growth factors and was dissociated by signaling Requests for reprints: Walter Kolch, Beatson Insitute for Cancer Research, by mutant Ras, growth factors, or proapoptotic stimuli, only the Garscube Estate, Switchback Road, Glasgow, Scotland G61 1BD, United Kingdom. Phone: 44-141-330-3983; Fax: 44-141-942-6521; E-mail: [email protected]. latter of which led to MST2 activation, however (12, 13). Ras and I2005 American Association for Cancer Research. growth factors also contribute to cell survival by collateral signaling www.aacrjournals.org 5485 Cancer Res 2005; 65: (13). July 1, 2005

Figure 1. Small GTP-binding proteins such as Ras and Rap1 (collectively, RAS*) become loaded with GTP upon growth factor stimulation, allowing association with effectors. RA domains on RASSF and Raf-1 allow GTP-bound Ras proteins to promote MST activity and apoptosis through RASSF and simultaneous removal of negative regulation by Raf-1. This leads to an activation of the Lats kinases via the Salvador adapter protein Sav1, resulting in regulation of cyclins and antiapoptotic IAPs. RASSF is observed to control the cell cycle indirectly, but may also control the ability of MST kinase to regulate downstream tumor suppressors. pathways involving ERK and Akt (13); it is currently unknown of MST2 in the knockout cells reversed their elevated apoptotic whether these pathways may also impinge on MST2 activation. sensitivity, whereas overexpression of exogenous MST2 increased Significantly, the interaction with Raf-1 suppressed MST2 phos- this sensitivity. Similar experiments in Raf-1+/+ control fibroblasts phorylation and kinase activity, and a Raf-1 mutant devoid of kinase indicated that MST2 had to be overexpressed to an extent where its activity was equally active as observed in reconstitution experi- levels exceeded the levels of Raf-1 before an apoptotic sensitivity ments of Raf-1À/À cells. Thus, the kinase activity of Raf-1 is manifested, suggesting that MST2 is quantitatively sequestered by dispensable for MST2 control, strongly suggesting a different Raf-1 (12). In human cell lines, down-regulation of Raf-1 and MST2 structural mechanism. Indeed, Raf-1 binding to MST2 sequesters by small interfering RNA confirmed that Raf-1/MST2 act as MST2 monomers and prevents the dimerization required for trans- ubiquitous mediators of apoptotic sensitivity. Notably, B-Raf does autophosphorylation and activation. Additionally, Raf-1 recruits not associate with MST1 or MST2 and hence has no direct control a phosphatase that dephosphorylates MST2 preserving the over MST induced apoptosis like Raf-1 (12). inactive state (12). This phosphatase is presumably protein phosphatase 2A (PP2A), which has been previously shown to A Novel Tumor Suppressor Pathway that Includes associate with Raf-1 and remove an inhibitory phosphorylation MST2, Salvador, and Warts/Lats from Ser259 during Raf-1 activation (7). Thus, PP2A could coordinate Genetic analyses conducted in Drosophila suggest that the MST2 the activation of Raf-1 with the concomitant deactivation of MST2, homologue Hippo functions in a conserved tumor suppressor path- possibly explaining why growth factor stimulation can dissociate way that includes the Salvador adapter protein and the Warts/Lats the Raf-1/MST2 complex without inducing MST2 activation. This kinase(s). As mentioned above, MST2 kinase activity functions to double control of MST2 by Raf-1 seems to ensure that the release of promote apoptosis via JNK and caspase activation (3). However, the MST2 only results in MST2 activation and proapoptotic activity if recently described MST2/Hippo pathway has been implicated in MST2 phosphorylation is permitted at the same time. both apoptosis and cell cycle inhibition in Drosophila (14). Intri- The apoptotic sensitivity of the Raf-1À/À mouse embryo guingly, this pathway seems to be conserved in mammals. Hippo fibroblasts in response to Fas signaling and serum starvation indeed (dMST) has been proposed to phosphorylate and activate the AGC- correlates with an enhancement of MST2 kinase activity. Ablation like kinase Warts, facilitated by the scaffolding protein Salvador,

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. MST Kinases in Tumor Suppression which presents separate interaction domains for Hippo and Warts As the RASSF proteins do not have any enzymatic activity (14). Warts signaling reduces the transcription and stability of the themselves, it has been suggested that these proteins provide a inhibitor of apoptosis protein dIAP, thereby priming cells for apop- scaffold function for the MST kinases (4). This may explain the tosis. It further blocks cell cycle progression by diminishing the limited activation of MST kinase activity that is observed via levels of cyclin E (14). Whereas there is conjecture over the tumor physiologic stimulation, a scaffold may simply target the restricted suppressor status of human Salvador homologue, hSav1, the human activity to a specific region of the cell when required. Through RA homologues of warts, Lats1 and Lats2, seem to be bona fide tumor domain association, RASSF family members have been shown to suppressors and regulators of cell cycle progression and apoptosis. variably associate with a variety of GTP-binding proteins, including Lats1 ablation promotes tumorigenesis in mice, highlighting its Ki-Ras, Ha-Ras, R-Ras, M-Ras, and Rap1/2 (Fig. 1; ref. 4). Rap1B status as a tumor suppressor (15). Recently, biochemical evidence binding to RASSF5b/Nore/RapL has been shown to play a role in has been provided for the activation of human Lats1 by MST2 (16). lymphocyte migration and adhesion along endothelial vessel walls, However, no binding between MST2 and Lats1 was observed, sug- indicating additional functions of RASSF proteins in addition to gesting that in humans the activation mechanism may be different apoptosis and cell cycle control (23). The different associations than in Drosophila. Nevertheless, the skeleton of the pathway seems through the RA domain show a potential for spatial targeting within to be conserved, and it will be interesting to map the further the cell of the different isoforms. Further support for this idea elements that connect the MSTactivation of Lats to the regulation of comes from the presence of a diacylglycerol binding domain that cyclin E and IAP levels. In human cells, Lats1/2 have been shown to is specific to the amino terminal of the RASSF1a isoform and is affect cyclin E and cyclins A/B, inducing cell cycle arrest at G1-S absent from the 1c isoform that remains expressed in tumors (4). or G2-M accordingly (17, 18). Failure to maintain correct cyclin Spatial localization of MST kinases may also allow access to expression patterns benefits tumor growth, whereas overexpression activation signals that would be required for full activity. When of IAP proteins is commonly observed to aid tumor survival (19). Raf-1 control of MST2 is removed by serum or active Ras, MST2 The upstream regulation of MST2 is just beginning to be still requires an activation step that could potentially be through a elucidated. The removal of the negative regulation by Raf-1 is not RASSF interaction. Some evidence for this has been provided by the the sole means of control over MST2 activation. Interestingly, potentiation of MST1-induced apoptosis via CNK1 binding (24). MST1/2 each have been shown to be associated with members of Once Raf-1 control is relieved and MST is activated, the RASSF the RASSF tumor suppressor family through the SARAH protein- proteins may also serve to deliver the kinase activity to potential protein interaction domain (20). The RASSF family consists of six substrates, such as apoptotic targets or components of the cell members that each have numerous splice variants. Methylation cycle, e.g., the anaphase promoting complex (APC/C; ref. 25). silencing of a RASSF1-specific splice form promoter was observed in Elucidation of how the RASSF-MST interaction is regulated by the 3p tumor suppressor locus of lung, breast, and ovarian tumors Raf-1 and the RA domain of RASSF will help explain the acti- (21, 22). Further studies showed a much wider penetrance of epi- vation and/or targeting of the MST kinase activities. Moreover, genetic silencing in the majority of primary tumors analyzed. This exactly how MST activity is affected by variation in RASSF splice silencing—mainly of the RASSF1a splice isoform—is linked to forms is likely to provide an important insight into the tumor severity of disease and/or poor prognosis. Whereas variation bet- relevance of the RASSF1a silencing. ween splice forms arises from independent promoters, producing exclusive amino-terminal regions, commonality exists in a con- Acknowledgments served Ras association (RA) domain that defines the family and a Received 4/27/2005; accepted 4/27/2005. SARAH domain that is responsible for the interaction with MST. Grant support: European Union and Cancer Research UK.

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Eric E. O'Neill, David Matallanas and Walter Kolch

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