Review

Mammalian Sterile 20Like Kinases in Tumor Suppression:

An Emerging Pathway
1

Eric E. ONeill, David Matallanas, and Walter Kolch

1,2

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

Abstract
Emerging evidence suggests that the proapoptotic kinase
mammalian sterile 20like kinase 2 (MST2) acts in a novel
tumor suppression pathway. Recently, we showed that Raf-1
kinase sequesters and inhibits MST2 and that this event is
critical for Raf-mediated cell survival. In this review, we
summarize Raf control of MST2 and we outline a novel
pathway involving the downstream effector proteins Salvador
and Warts/Lats that may act to limit the positive effects of
Rafmitogen-activated protein kinase signaling in cancer
cells. (Cancer Res 2005; 65(13): 5485-7)

The Mammalian Sterile 20Like Kinases

The kinase mammalian sterile 20 (Ste20)like kinase 2 (MST2,
Krs1) and its close homologue MST1 (Krs2) are members of the
germinal center kinase group II (GCK II) family of mitogen-activated
protein kinase (MAPK)related kinases that includes the more
distantly related kinases MST3, MST4, LOK, SOK, and SLK (1). This
GCK II family also shares homology to the yeast Ste20 kinase in the
kinase domain. GCK II kinases seem to be mainly involved in
apoptotic signaling in response to stress signals, including Ste20
itself (2). Both MST1 and MST2 have been described as MAPK kinase
kinase kinases that lead to c-Jun-NH2-kinase (JNK) activation in
response to stress and Fas signaling, apparently through a MEKK1/
MKK7 route (3). Activation of JNK and subsequent activation of
caspases seems to explain the original description of MST1 and
MST2 as proapoptotic kinases. Once activated, caspase-3 removes
the carboxyl-terminal portion of MST2; this also occurs on MST1
together with a distinct caspase-6/7mediated cleavage event (4).
The carboxyl-terminal region contains an inhibitory region together
with the Salvador and RASSF homology (SARAH) protein-protein
interaction domain, dimerization domain, and a nuclear export
signal (4). Caspase cleavage results in a constitutively active kinase
domain residing in the nucleus that phosphorylates histone 2B and
potentiates the DNA condensation characteristic of apoptosis (5).
The yeast homologue, Ste20, has been shown recently to function in
a similar manner to promote an apoptotic-like response in yeast,
which do not have caspase-like activity or Ste20 cleavage (2).
Caspase regulation of MST1/2 may have arose later in evolution as
the full-length kinases seem to be able to be activated in cells by
caspase-independent mechanisms. However, activation of the fulllength kinase is not readily observed, in agreement with the idea that
only a small pool of MST1/2 is active at one time (4). Activation of fulllength MST1/2 occurs in response to stress conditions or treatment
with staurosporine or okadaic acid (6), requiring a functional

Requests for reprints: Walter Kolch, Beatson Insitute for Cancer Research,
Garscube Estate, Switchback Road, Glasgow, Scotland G61 1BD, United Kingdom.
Phone: 44-141-330-3983; Fax: 44-141-942-6521; E-mail: wkolch@beatson.gla.ac.uk.
I2005 American Association for Cancer Research.

www.aacrjournals.org

dimerization domain that is thought to mediate a trans-autophosphorylation event that is essential for kinase activity (4). At later times,
usually several hours after initial activation, ensuing caspase activity
severs the carboxyl-terminal regulatory domain from the kinase
domain, further enhancing MST1/2 catalytic activity.

Raf Regulates MST2 Signaling

Raf-MAPK signaling mediates diverse cellular fates, including
proliferation, differentiation, and survival. Activation of Raf occurs
as a result of GTP loading of Ras in response to mitogens, which
allows a cascade of activation through Raf and MEK kinases to the
MAP kinases, also known as the extracellular signal-regulated
kinases (ERK; ref. 7). The MAPK pathway does provide mechanisms
to generate specific biological responses that can be imparted
through the overall strength and duration of the ERK signal, allowing
sustained versus transient activation of ERK substrates. The
existence of multiple isoforms at each step in the pathway allows
further specificity through distinct binding partners and, hence,
integration of additional signals that could modulate the overall
response (7). The kinase cascade consists of three Raf isoforms, Raf1, B-Raf, and A-Raf, which are able to phosphorylate both isoforms of
MEK (MEK1/2), which in turn can activate either ERK1 or ERK2 (7).
Ras activates a group of pathways, but is the only known signal that
activates the Raf kinases (7) and the importance of this step in
cellular events has been established through the identification of
mutation in Ras and the B-Raf kinase, which lead to increased ERK
signaling in human cancers (8). Genetic ablation of the Raf isoforms
in mice allowed us to separate some unique aspects of individual Raf
isoform signaling. Most notably, mice in which the raf1 gene was
deleted died in utero due to an overwhelming level of apoptosis,
which occurred despite normal expression of the alternate Raf
isoforms and undisturbed ERK activation (9).
The fact that ERK activation was unaffected in cells from Raf -1 /
mice pointed to a survival signal, other than Ras-induced ERK
signaling, that is defective and that led to the apoptotic phenotype of
these mice (10, 11). A survival function for Raf-1 that is independent
of ERK-promoting activity was confirmed by phenotypic rescue of
the Raf-1 null mice with a Raf-1-YY340/341FF derivative that is not
activated by mitogens and that has low basal kinase activity (10).
Mouse embryo fibroblasts derived from the Raf-1 / mice also
exhibited a hyperapoptotic phenotype; furthermore, the sensitivity
was specific to certain stimuli (i.e., serum starvation and engagement
of the death receptor Fas). Using this system, we identified the
pathway that leads to the proapoptotic phenotype, defining MST1
and MST2 in proteomic screens for potential pathways through
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
by mutant Ras, growth factors, or proapoptotic stimuli, only the
latter of which led to MST2 activation, however (12, 13). Ras and
growth factors also contribute to cell survival by collateral signaling

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Cancer Research

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

whether these pathways may also impinge on MST2 activation.
Significantly, the interaction with Raf-1 suppressed MST2 phosphorylation and kinase activity, and a Raf-1 mutant devoid of kinase
activity was equally active as observed in reconstitution experiments of Raf-1 / cells. Thus, the kinase activity of Raf-1 is
dispensable for MST2 control, strongly suggesting a different
structural mechanism. Indeed, Raf-1 binding to MST2 sequesters
MST2 monomers and prevents the dimerization required for transautophosphorylation and activation. Additionally, Raf-1 recruits
a phosphatase that dephosphorylates MST2 preserving the
inactive state (12). This phosphatase is presumably protein
phosphatase 2A (PP2A), which has been previously shown to
associate with Raf-1 and remove an inhibitory phosphorylation
from Ser259 during Raf-1 activation (7). Thus, PP2A could coordinate
the activation of Raf-1 with the concomitant deactivation of MST2,
possibly explaining why growth factor stimulation can dissociate
the Raf-1/MST2 complex without inducing MST2 activation. This
double control of MST2 by Raf-1 seems to ensure that the release of
MST2 only results in MST2 activation and proapoptotic activity if
MST2 phosphorylation is permitted at the same time.
The apoptotic sensitivity of the Raf-1 / mouse embryo
fibroblasts in response to Fas signaling and serum starvation indeed
correlates with an enhancement of MST2 kinase activity. Ablation

Cancer Res 2005; 65: (13). July 1, 2005

of MST2 in the knockout cells reversed their elevated apoptotic

sensitivity, whereas overexpression of exogenous MST2 increased
this sensitivity. Similar experiments in Raf-1+/+ control fibroblasts
indicated that MST2 had to be overexpressed to an extent where its
levels exceeded the levels of Raf-1 before an apoptotic sensitivity
manifested, suggesting that MST2 is quantitatively sequestered by
Raf-1 (12). In human cell lines, down-regulation of Raf-1 and MST2
by small interfering RNA confirmed that Raf-1/MST2 act as
ubiquitous mediators of apoptotic sensitivity. Notably, B-Raf does
not associate with MST1 or MST2 and hence has no direct control
over MST induced apoptosis like Raf-1 (12).

A Novel Tumor Suppressor Pathway that Includes

MST2, Salvador, and Warts/Lats
Genetic analyses conducted in Drosophila suggest that the MST2
homologue Hippo functions in a conserved tumor suppressor pathway that includes the Salvador adapter protein and the Warts/Lats
kinase(s). As mentioned above, MST2 kinase activity functions to
promote apoptosis via JNK and caspase activation (3). However, the
recently described MST2/Hippo pathway has been implicated in
both apoptosis and cell cycle inhibition in Drosophila (14). Intriguingly, this pathway seems to be conserved in mammals. Hippo
(dMST) has been proposed to phosphorylate and activate the AGClike kinase Warts, facilitated by the scaffolding protein Salvador,

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MST Kinases in Tumor Suppression

which presents separate interaction domains for Hippo and Warts

(14). Warts signaling reduces the transcription and stability of the
inhibitor of apoptosis protein dIAP, thereby priming cells for apoptosis. It further blocks cell cycle progression by diminishing the
levels of cyclin E (14). Whereas there is conjecture over the tumor
suppressor status of human Salvador homologue, hSav1, the human
homologues of warts, Lats1 and Lats2, seem to be bona fide tumor
suppressors and regulators of cell cycle progression and apoptosis.
Lats1 ablation promotes tumorigenesis in mice, highlighting its
status as a tumor suppressor (15). Recently, biochemical evidence
has been provided for the activation of human Lats1 by MST2 (16).
However, no binding between MST2 and Lats1 was observed, suggesting that in humans the activation mechanism may be different
than in Drosophila. Nevertheless, the skeleton of the pathway seems
to be conserved, and it will be interesting to map the further
elements that connect the MST activation of Lats to the regulation of
cyclin E and IAP levels. In human cells, Lats1/2 have been shown to
affect cyclin E and cyclins A/B, inducing cell cycle arrest at G1-S
or G2-M accordingly (17, 18). Failure to maintain correct cyclin
expression patterns benefits tumor growth, whereas overexpression
of IAP proteins is commonly observed to aid tumor survival (19).
The upstream regulation of MST2 is just beginning to be
elucidated. The removal of the negative regulation by Raf-1 is not
the sole means of control over MST2 activation. Interestingly,
MST1/2 each have been shown to be associated with members of
the RASSF tumor suppressor family through the SARAH proteinprotein interaction domain (20). The RASSF family consists of six
members that each have numerous splice variants. Methylation
silencing of a RASSF1-specific splice form promoter was observed in
the 3p tumor suppressor locus of lung, breast, and ovarian tumors
(21, 22). Further studies showed a much wider penetrance of epigenetic silencing in the majority of primary tumors analyzed. This
silencingmainly of the RASSF1a splice isoformis linked to
severity of disease and/or poor prognosis. Whereas variation between splice forms arises from independent promoters, producing
exclusive amino-terminal regions, commonality exists in a conserved Ras association (RA) domain that defines the family and a
SARAH domain that is responsible for the interaction with MST.

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Acknowledgments
Received 4/27/2005; accepted 4/27/2005.
Grant support: European Union and Cancer Research UK.

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