Association of mammalian sterile twenty kinases, Mst1
and Mst2, with hSalvador via C-terminal coiled-coil
domains, leads to its stabilization and phosphorylation
Bernard A. Callus
1,
*, Anne M. Verhagen
1
and David L. Vaux
1,
*
1 The Walter and Eliza Hall Institute, Parkville, VC, Australia
The mammalian serine ⁄ threonine kinases, Mst1 and
Mst2, were originally identified by their similarity to
yeast Sterile Twenty (Ste20) kinase [1,2]. Mst3 and
Mst4 were subsequently identified the same way [3–5].
The four Mst kinases belong to a subfamily of Ste20-
like germinal center kinases (GCKs) that is character-
ized by an N-terminal kinase domain (reviewed in [6]).
Based on their similarity with each other, the Mst kin-
ases can be further subdivided into two groups, Mst1
and Mst2 (GCKII) and Mst3 and Mst4 (GCKIII).
Mst1 has been widely studied and is the best charac-
terized member of the family. In addition to its kinase
domain, Mst1 contains an inhibitory domain, deletion
of which results in increased kinase activity, and a pre-
dicted coiled-coil domain at the C-terminus that is
essential for the formation of Mst1 dimers (multimers)
[7]. Full-length Mst1 is mainly cytoplasmic, but can
shuttle continuously between the cytoplasm and nuc-
leus in a phosphorylation dependent manner [8–10].
Ectopic expression of Mst1 and Mst2 in certain cells

its abundance. In vitro phosphorylation experiments indicate that the phos-
phorylation of Sav by Mst is direct. The stabilizing effect of Mst was much
greater on N-terminally truncated hSav mutants, as long as they retained
the ability to bind Mst. Mst mutants that lacked the C-terminal coiled-coil
domain and were unable to bind to hSav, also failed to stabilize or phos-
phorylate hSav, whereas catalytically inactive Mst mutants that retained the
ability to bind to hSav were still able to increase its abundance, although
they were no longer able to phosphorylate hSav. Together these results
show that hSav can bind to, and be phosphorylated by, Mst, and that the
stabilizing effect of Mst on hSav requires its interaction with hSav but is
probably not due to phosphorylation of hSav by Mst.
Abbreviations
dIAP1, Drosophila inhibitor of apoptosis I; GCK, germinal center kinase; HA, hemagglutinin; Hpo, serine/threonine kinase Hippo; IL-2,
interleukin-2; Lats, large tumour suppressor; MBP, myelin basic protein; Mst, mammalian sterile20 kinase; Nore1, novel Ras effector 1;
PBST, NaCl ⁄ Pi-Tween 20; PPIA, peptidyl-prolyl cis-trans isomerase A; Rassf1, Ras suppressor factor 1; Sav, Salvador; SAPK, stress
activated protein kinase; Ste20, Sterile Twenty; Wts, warts kinase; Yki, Yorkie.
4264 FEBS Journal 273 (2006) 4264–4276 ª 2006 The Authors Journal compilation ª 2006 FEBS
[11–13]. The proteolytic fragment encompassing the
kinase domain accumulates in the nucleus and can
phosphorylate histone H2B at Ser14, possibly trigger-
ing chromosomal condensation [9,11,14], in a positive
feedback loop in cells undergoing apoptosis.
The physiological signals leading to activation of
Mst1 and Mst2 are poorly understood. Mst1 has been
reported to become activated if recruited to or artifici-
ally targeted to the plasma membrane [15,16] as well
as in response to specific nonphysiological stress stim-
uli such as staurosporine, sodium arsenite, hyper-
osmotic concentrations of sucrose, and heat-shock
[11,13,16], but Mst1 was not activated in HeLa cells in

inactivate Yorkie (Yki), a Drosophila orthologue of the
mammalian transcriptional coactivator Yes-associated
protein, in a Hpo ⁄ Sav dependent manner [24]. Yki can
transcriptionally up-regulate the genes for cyclin E and
dIAP1. Therefore the failure to inactivate Yki in hpo ⁄
sav ⁄ wts mutant tissue accounts for the elevated levels
of cyclin E and dIAP1. Thus the Hpo ⁄ Salvador ⁄ Wts
complex defines a novel pathway regulating cell growth
and apoptosis in Drosophila in vivo, primarily through
the regulation of Yki activity.
Hpo is a Drosophila orthologue of the Ste20-like
kinases, and is most similar to mammalian Mst2. Mst1
and Mst2 have been shown to interact with a number
or proteins, including the novel Ras effector 1, Nore1
[15,16], the putative tumour suppressor (Ras suppres-
sor factor 1; Rassf1) [15,16,25], and most recently,
Raf1 (with Mst2) [26]. The association of Mst with
Nore1 or Rassf1 leads to an inhibition of Mst kinase
activity, yet these complexes appear to mediate the
pro-apoptotic activity of active Ras [15,25]. Raf1, on
the other hand, directly inhibits Mst2 activation,
thereby preventing apoptosis in cells following serum
starvation [26]. These studies provide a possible link
from Ras or Raf signalling to apoptosis through regu-
lation of Mst activity. These findings are also consis-
tent with the apoptotic effects of Hpo in flies and
potentially link Mst ⁄ Hpo activity to upstream signal-
ling events. Interestingly however, Salvador, also called
WW45 in mammals [27], was not found in these
complexes of Mst, suggesting that Mst may bind to

B. A. Callus et al. Mst kinases bind, stabilize and phosphorylate hSav
FEBS Journal 273 (2006) 4264–4276 ª 2006 The Authors Journal compilation ª 2006 FEBS 4265
cells, and complexes were isolated by coimmunoprecip-
itation. As seen in Fig. 1B, both Mst1 and Mst2 were
efficiently coimmunoprecipitated with flag-Sav. The
efficiency of this coprecipitation was similar to that of
the direct immunoprecipitation of Mst1 and Mst2 with
anti-myc IgG, suggesting that most of the Mst kinases
were in association with Sav. Interestingly, the coex-
pression of Mst kinases, especially Mst2, appeared to
increase the abundance of Sav (Fig. 1C). To confirm
this, we repeated the experiment, and again found that
the presence of either Mst1 or Mst2 appeared to
increase the abundance of Sav (Fig. 1D). Once again,
despite similar expression levels themselves, Mst2 con-
sistently had a greater stabilizing effect on Sav than
Mst1. This effect was not due to differences in trans-
fection efficiency because coexpression of Sav with
green fluorescent protein or another protein that does
not bind Sav (peptidyl-prolyl cis-trans isomerase A;
PPIA) (see below), had no effect on Sav abundance
(Fig. 1E).
Mst kinase and hSalvador interact via
their C-terminal coiled-coil domains
Mst1, Mst2, and Sav all contain C-terminal coiled-coil
domains (Fig. 2). Because coiled-coil domains mediate
protein interactions, and Mst1 has previously been
shown to homodimerize via its C-terminal coiled-coil
IP:
Blot: α-Mst1

37
26
62
79
48
37
26
Lysates:
Re-blot: α-flag
Lysates:
Blot: α-Mst1
Lysates:
Re-blot: α-β-actin
IP:
α-Mst1
myc-Mst1
Sav
Sav
β-actin
endog. Mst1
myc-Mst1
endog. Mst1
cleaved Mst1
Mst
62
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37
24

24
62
110
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37
24
Sav
Mst
myc-Mst2
-+-
-
flag-Sav
++-+
myc-Mst1
+
GFP
+-
pcDNA3
+
Lysates
Blot: α-flag
Lysates
Blot: α-myc
-+
++-+
+
myc-Mst2
flag-Sav
myc-Mst1

62
37
48
79
37
26
19
15
37
48
26
19
62
37
48
79
62
37
48
79
26
Lysates
Re-blot: α-GFP
Lysates
Re-blot: α-HA
PPIA
GFP
GFP
Mst
HA-PPIA

Mst kinases via these domains. To test this, we engin-
eered C-terminally truncated mutants of Mst1 and
Mst2 that lacked the coiled-coil domain, and deter-
mined whether they were capable of interacting with
wild-type Sav. Consistent with earlier experiments, the
full-length Mst kinases efficiently coprecipitated flag-
Sav, but the truncated mutants of Mst1 and Mst2 did
not. Similarly, in the reciprocal coimmunoprecipita-
tions, flag-Sav was able to bring down full-length Mst1
and Mst2 but not Mst proteins that lacked their
coiled-coil domains (Fig. 3A).
To confirm that the C-terminal coiled-coil domain
of Sav was also required for binding to Mst kinases,
we generated a series of C-terminally truncated Sav
mutants (Fig. 2), and examined their ability to interact
with Mst1 and Mst2. As seen in Fig. 3B, full-length
Mst1 and Mst2 were able to coprecipitate versions of
flag-Sav that bore the coiled-coil domain, namely flag-
Sav WT and D374, but not the smaller proteins, D344
and D321, that lacked the domain. Again, in the recip-
rocal coimmunoprecipitations, flag-Sav and D374 were
able to efficiently bring down full-length Mst1 and
Mst2. Thus the C-terminal coiled-coil domains of both
Sav and the Mst kinases are essential for their interac-
tion.
Once again we noted that in lysates from cells that
coexpressed full-length Mst1 or Mst2 together with
Sav, levels of Sav were elevated compared to extracts
that expressed Sav alone (Fig. 3A). However, when the
truncated versions of Mst1 and Mst2 that could not

efficiency than the full-length protein. If so, this could
be because other parts of Sav reduce access to the
coiled-coil domain or that regions, such as the WW
domain, interact with other proteins that exclude the
interaction of Mst.
Mst:
Salvador:
Δ433
WT
Δ374
Δ344
Δ321
Δ268
Δ199
WT
199-383
268-383
321-383
kinase domain
inhibitory
domain
coiled-coil
domain
WW domains
coiled-coil
domain
Fig. 2. The structure of Mst kinase and
hSalvador. Schematic illustrations of Mst
kinase and hSalvador primary structures
show the relative positions of their func-

myc-Mst2 Δ437
+
Mst
Mst
Mst
*
Mst
62
79
48
37
62
79
48
37
*
Sav
Sav
Sav
Sav
Sav
Lysates
Blot: α-flag
Lysates
Re-blot: α-myc
IP: α-flag
Blot: α-myc
IP: α-flag
Re-blot: α-flag
IP: α-myc

+
-
flag-Sav Δ344
-+
-+-
-
flag-Sav Δ321
+-

+
myc-Mst1

+++
+
myc-Mst2
++ ++

-
Mst
Mst
Mst
Mst
*
*
*
*
Sav
Sav
Sav
Sa

79
48
37
26
62
79
48
37
26
pcDNA3
myc-Mst2 WT
flag-Sav WT
flag-Sav 199-383
flag-Sav 268-383
flag-Sav 321-383
-
+
+
-
-
-
-
+
-
+
-
-
+
-
-

+
-
-
+
-
-
-
-
+
β-actin
Mst2
Mst2
62
48
37
79
26
19
15
6
62
48
37
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26
19
15
6
Mst2
Sav

Lysates
Re-blot: α-β-actin
IP: α-flag
Blot: α-flag
IP: α-flag
Re-blot: α-myc
Lysates
Re-blot: α-myc
19
15
6
62
48
37
79
110
19
15
6
62
48
37
79
110
Lysates
Blot: α-myc
Lysates
Blot: α-flag
IP: α-flag
Blot: α-myc

myc-Mst2 Δ437
-

-
-
+
+
++++
-
+
Mst kinases bind, stabilize and phosphorylate hSav B. A. Callus et al.
4268 FEBS Journal 273 (2006) 4264–4276 ª 2006 The Authors Journal compilation ª 2006 FEBS
Expression of Sav(321–383) was only detectable
when coexpressed with either WT Mst1 or Mst2, but
not with mutants of Mst that lacked their coiled-coil
domain (Fig. 3D, bottom). Consistent with the earlier
results, Sav(321–383) coprecipitated Mst2 but not the
mutants that lacked their coiled-coil domains and
unexpectedly, also failed to coprecipitate Mst1
(Fig. 3D, top). It is possible that the interaction
between the Sav coiled-coil domain and Mst1 and
Mst2 inside cells is sufficient to stabilize its abundance,
but that Sav’s interaction with Mst1 is significantly
weaker than with Mst2, such that its interaction with
Mst1 is disrupted upon cell lysis.
hSalvador can homodimerize
⁄
multimerize
independently of its coiled-coil domain
Based on the above findings, and earlier observations

linear gels, suggestive of phosphorylation, was seen,
but only in lanes that coexpressed Mst1 or Mst2
pcDNA3
AB
flag-Sav WT
flag-Sav Δ374
flag-Sav Δ344
flag-Sav Δ321
HA-Sav WT
HA-Sav
HA-Sav
HA-Sav
HA-Sav
flag-Sav
flag-Sav
62
79
37
48
62
79
37
48
62
79
37
48
62
79
37

26
19
15
62
48
37
26
19
HA-Sav
HA-Sav
PPIA
flag-Sav
flag-Sav
*
*
123456
flag-Sav WT
flag-Sav Δ199
HA-Sav WT
flag-CrmA-DQMD
flag-Sav Δ268
flag-Sav Δ321
HA-PPIA
Fig. 4. hSalvador can homo-multimerize
independently of its C-terminal coiled-coil
domain. (A,B) HA-tagged Sav cDNA was co-
transfected with either WT or C-terminally
truncated mutants of flag-Sav cDNA or with
HA-PPIA or flag-CrmA-DQMD cDNAs into
293T cells as indicated. Two days after

62
79
48
37
26
Sav
62
79
48
37
26
Lysates
Blot: α-flag
Lysates
Re-blot: α-myc
123456
flag-Sav WT
myc-Mst1
myc-Mst2
flag-Sav Δ374
+
+
-
-
+
-
+
-
+
-

flag-Sav
++ +++
-
myc-Mst1 WT
+-
-
myc-Mst1 Δ433
-+
-
myc-Mst2 WT
-+-
-
myc-Mst2 Δ437
+
-
Sav
48
37
Sav
Lysates
Blot: α-flag
Lysates
Re-blot: α-myc
62
79
48
37
pMst2
pSav
flag-Sav

26
19
110
*
*
Mst2
Sav
Re-blot: α-myc
62
79
37
48
26
19
110
*
*
12345678910
pMst2
pSav
Autoradiograph
62
79
37
48
26
19
15
110
pMBP

P]ATP[cP] either alone, or with purified flag-Sav or MBP as indicated. Reactions were terminated and separated by
SDS ⁄ PAGE, transferred to membrane, dried and exposed to film. Following autoradiography, membranes were sequentially immunoblotted
as indicated on the left. The position of Mst and Sav is marked with arrows while the position of immunoglobulin chains from antimyc
immune complexes in lanes 1–8 is indicated with an asterisk (*). Lanes 3, 6 and 9 are blank lanes. Each experiment was performed at least
twice with similar results except that shown in (C) which was performed once.
Mst kinases bind, stabilize and phosphorylate hSav B. A. Callus et al.
4270 FEBS Journal 273 (2006) 4264–4276 ª 2006 The Authors Journal compilation ª 2006 FEBS
To determine whether Mst kinase had to bind to
Sav to induce this mobility shift, we coexpressed WT
Sav with Mst1, Mst2 or Mst truncation mutants that
are unable to interact with Sav (Fig. 3). As seen in
Fig. 5B, the coexpression of Sav with WT Mst1 or
Mst2 altered the mobility of Sav, as well as increasing
its abundance. In contrast, coexpression of Sav with
the truncated mutants of Mst that were unable to bind
to Sav failed to induce a mobility shift. These results
indicate that the mobility shift of Sav is not simply
due to overexpressing Mst kinases, but is likely to be a
direct consequence of Mst kinase interacting with, and
directly phosphorylating, Sav.
To confirm that Mst phosphorylates Sav in vivo,
we first generated catalytically inactive ‘kinase-dead’
mutants of both Mst1 and Mst2. The mutation of
K59R in the ATP-binding region of the Mst1 kinase
domain renders the kinase inactive [7,11,12,16], and by
homology with Mst1 the analogous mutation of K56R
in Mst2 should also render the kinase inactive.
Flag-Sav was coexpressed with WT Mst2 and the
K56R mutant in cells, and labelled in vivo with
32

bottom). Because myelin basic protein (MBP) can
serve as a pseudosubstrate for Mst1 [16], we reasoned
that this is probably also the case for Mst2, and it
would serve as a positive control for this assay.
Indeed, MBP is well phosphorylated by WT Mst2 but
not by the mutant kinase. Similarly, the incubation of
flag-Sav with WT Mst2, but not the kinase-dead Mst2,
resulted in the phosphorylation of a protein that was
superimposable with that of flag-Sav (Fig. 5D, top and
middle, lane 4). The incubation of flag-Sav alone in
this assay yielded no radiolabelled proteins (Fig. 5D,
lane 10) indicating that the phosphorylation of Sav is
due to the addition of purified WT Mst2 rather than
some other protein that had copurified with flag-Sav.
This result provides strong evidence that Sav is directly
phosphorylated by Mst2 kinase.
Having confirmed the ability of Mst to phosphory-
late Sav, we then examined what role, if any, that
phosphorylation played in the ability of Mst to
increase the abundance of Sav. To do this we coex-
pressed both WT and kinase-dead mutants of Mst1
and Mst2 with flag-Sav, and determined the effect of
these mutant Mst kinases on Sav stability. In contrast
to WT Mst1 and Mst2, the kinase-dead mutants failed
to induce a mobility shift in Sav (Fig. 6A). The failure
of the kinase mutants to phosphorylate Sav was not
due to an inability to bind to it, because both mutant
kinases could be coimmunoprecipitated with flag-Sav
pcDNA3
flag-Sav

-
-
-
-
62
48
37
Mst
Sav
Mst
β-actin
Sav
Sav
62
48
37
62
48
37
Lysates
Blot: α-flag
Lysates
A
B
Re-blot: α-myc
Lysates
Re-blot: α-β-actin
Mst
62
79

phorylation by Mst, but rather the association of Mst
with Sav is required for stabilization of Sav.
Discussion
These results demonstrate that the mammalian scaffold
protein, hSav (hWW45), can bind to the mammalian
orthologues of Sterile Twenty kinase, Mst1 and Mst2.
Recently, it was shown in yeast two-hybrid analyses
that the C-terminal halves of the Mst2 and Sav pro-
teins were required for interaction, but this study failed
to define the region of interaction [28]. As we demon-
strate here, this association is absolutely dependent
upon their respective C-terminal coiled-coil domains
(Fig. 3). A slightly larger region of similarity ( 50
amino acids) between Sav and Mst harbouring most of
the coiled-coil domain, dubbed the Sarah domain, was
previously predicted to be essential for interaction
between the two proteins [29]. Consistent with this
finding, the truncation mutants Mst1 D433, Mst2 D437
and Sav D321, that all lacked this region of similarity,
all failed to heterodimerize. Furthermore, deletion of
just the coiled-coil domain of Sav (Sav D344) was suffi-
cient to abolish heterodimerization (Fig. 3B), indica-
ting this domain is essential for interaction with Mst
kinases. That the coiled-coil domain of Sav alone was
sufficient to coprecipitate Mst2 (Fig. 3C,D) demon-
strates that this domain is both necessary and sufficient
to bind Mst kinase. These findings are consistent with
studies in Drosophila that revealed the C-terminal
coiled-coil domains of Sav and Hpo were also crucial
and ⁄ or sufficient for their interaction [19,21,23]. These

quences. First, the abundance of Sav was increased in
the presence of Mst, and second, Sav was phosphoryl-
P
Upstream signals
Mst activation
Sav
“Inactive”
Sav phosphorylation
dependent
Sav phosphorylation
independent
“Active
Sav/Mst Complex”
Sav
P
X
Downstream effects
P
X
Sav Sav
“Less stable”
“Active”
Mst
Fig. 7. Possible activation model for the hSalvador ⁄ Mst kinase
pathway. In the inactive state, Mst and Sav can coexist as function-
ally inactive homo- or heterodimers. Sav on its own is less stable
than Sav bound to Mst. If activated by upstream signals active Mst
kinase can interact with Sav via their coiled-coil domains. Alternat-
ively, Mst might become activated while bound to Sav. The associ-
ation of active Mst with Sav in itself induces a conformational

results in Fig. 6 show that association of kinase-dead
mutants of Mst with Sav is sufficient to significantly
enhance Sav abundance. A similar effect on Sav stabil-
ity was also seen when a kinase-dead mutant of Hpo
was coexpressed with Sav in Drosophila S2 cells [19],
indicating the stabilizing effect of Mst on Sav expres-
sion is also conserved. Interestingly, N-terminal dele-
tions of Sav rendered the mutant proteins less stable
than the wild-type protein (Fig. 3C), however, when
coexpressed with Mst2, a dramatic stabilizing effect
was seen on the abundance of the smaller of these trun-
cated proteins, namely Sav(268–383) and (321–383).
Indeed, we have only ever been able to detect
Sav(321–383) when coexpressed with either Mst1 or
Mst2 (Fig. 3D). Sav ⁄ Mst heterodimers might be more
stable than Sav homodimers because of conforma-
tional changes in Sav bound to Mst that render the
protein more stable, or because Mst itself masks degra-
dative signals in Sav. Alternatively, it may be that in
its unbound state, the coiled-coil domain has a desta-
bilizing influence on Sav. Thus, it seems that stability
of Sav protein is increased by the presence of its
N-terminal region as well its C-terminal coiled-coil
domain due to its ability to bind Mst.
Phosphorylation substrates of Mst kinases have not
been well characterized. Here we have provided strong
evidence that Sav is indeed phosphorylated by Mst
and that the phosphorylation is likely to be direct
(Figs 5 and 6). The phosphorylation of Sav by Mst
provides an additional means by which proteins may

Nore1 can form complexes with and inhibit Mst1
activity in an interaction involving their conserved
C-termini [15,25]. Interestingly, while Nore1 and Rassf1
maintain Mst1 activity at low or basal levels it has
been shown that Mst1 in complex with either Nore1 or
with Rassf1 bound to the scaffold protein, connector
enhancer of KSR1, CNK1, mediates the pro-apoptotic
effects of a constitutively active Ras [15,25]. Further-
more, Nore1 appears to direct recruitment of Mst1 to
Ras complexes following serum stimulation and the
observation that artificially targeting Mst1 to the
plasma membrane augments its pro-apoptotic activity
has led to speculation that Nore1 and Rassf1 might
direct Mst1 to sites of activation [15,16]. It is worth
noting that endogenous Sav was not identified in these
Mst-containing complexes. Moreover, in a proteomic
screen using flag-Sav as bait we failed to detect the
presence of endogenous Rassf-1 or Nore-1 in immune
complexes (data not shown). Together these observa-
tions question the existence of proposed complexes
such as Mst ⁄ Sav ⁄ Rassf1 [29]. Alternatively, the
Mst ⁄ Sav interaction may be strong enough to prevent
binding of other proteins to Mst, particularly when the
interaction between Mst and Nore1 ⁄ Rassf1 occurs
through the same coiled-coil domain of Mst that we
have shown binds Sav [15,16,25].
B. A. Callus et al. Mst kinases bind, stabilize and phosphorylate hSav
FEBS Journal 273 (2006) 4264–4276 ª 2006 The Authors Journal compilation ª 2006 FEBS 4273
It was recently reported that in serum starved cells,
Mst2 was sequestered into a complex with Raf1 that

clonal antimyc (9B11) and rabbit polyclonal antibodies
to Mst1 (#3682) and Mst2 (#3952) were purchased from
Cell Signaling Technology (Genesearch, Arundel, QLD,
Australia).
Plasmids and cDNAs
The mammalian expression plasmid, pcDNA3 (Invitrogen,
Melbourne, VIC, Australia), containing N-terminally flag-
tagged human Salvador (hSav) cDNA was a kind gift from
D Haber (MGH Cancer Center, Charlestown, MA, USA).
J Chernoff (Fox Chase Cancer Center, Philadelphia, PA,
USA) generously provided the N-terminally myc-tagged
cDNAs for mammalian sterile20 kinases (Mst1 and Mst2).
Flag-CrmA-DQMD was described previously [30]. Pept-
idyl-prolyl cis-trans isomerase A cDNA was cloned into
pcDNA3 with a C-terminal HA tag (HA-PPIA). Mst1 and
Mst2 were subcloned into pcDNA3 prior to use in expres-
sion studies. All mutant cDNA constructs were generated
by PCR using Pfu DNA polymerase and subcloned into
either pcDNA3 or pcDNA5 FRT ⁄ TO (Invitrogen) expres-
sion plasmids. All constructs were sequenced for authenti-
city and purified using Qiagen Maxi prep kits (Qiagen,
Clifton Hill, VIC, Australia).
Cell culture
293T cells were grown continuously in Dulbecco’s Modified
Eagle medium supplemented with 10% (v ⁄ v) foetal bovine
serum (Gibco, Melbourne, VIC, Australia), penicillin G
(50 UÆmL
)1
), streptomycin (50 lgÆmL
)1

fer [150 mm NaCl, 2 mm EDTA, 1% Triton X-100, 10%
glycerol and 20 mm Tris pH 7.5 supplemented with com-
plete protease inhibitor cocktail (Roche), 10 mm NaF,
2mm Na pyrophosphate, 1 mm Na molybdate and 5 mm
b-glycerophosphate] [31]. Total cell lysates were clarified
by centrifugation before immunoprecipitation with anti-
bodies raised against flag, Mst1, myc or HA. Anti-HA,
-Mst1 and -myc immunoprecipitations were performed in
the presence of protein G sepharose. Immune complexes
were washed three times with DISC lysis buffer before
being eluted with 100 mm glycine pH 3.0 and neutralized
with 1 m Tris pH 8.0. Unless otherwise indicated, immune
complexes or total cell lysates were separated by
SDS ⁄ PAGE on 4–20% Tris-glycine gradient gels (Bio-Rad,
Regent Park, VIC, Australia) and transferred to either
poly(vinylidene diflouride) (Millipore, North Ryde, NSW,
Australia) or Hybond C (Amersham, Castle Hill, NSW,
Australia) membrane. Membranes were blocked in 20%
horse serum (JRH Biosciences, Brooklyn, VIC, Austra-
lia) ⁄ NaCl ⁄ P
i
containing 0.05% Tween-20 (PBST) before
incubation with primary antibody. Membranes were
washed with PBST, incubated with horseradish peroxidase-
conjugated secondary antibody (Amersham) and washed
before detection with enhanced chemiluminescence (Amer-
sham).
Mst kinases bind, stabilize and phosphorylate hSav B. A. Callus et al.
4274 FEBS Journal 273 (2006) 4264–4276 ª 2006 The Authors Journal compilation ª 2006 FEBS
In vivo labelling with

against kinase buffer (see below) and stored at )80 °C. A
Coomassie stainable amount of purified flag-Sav protein
was verified by SDS ⁄ PAGE and used as substrate in subse-
quent in vitro phosphorylation assays.
In vitro phosphorylation assay
Two days after transfection, 293T cells were lysed and myc-
tagged WT or kinase-dead (K56R) Mst2 was immunopre-
cipitated with antimyc (9B11) and EZ-view Red protein A
affinity gel (Sigma) as described above. Immune complexes
were washed twice with DISC lysis buffer and once with
NaCl ⁄ P
i
. Complexes were equally divided three ways before
being pre-equilibrated in kinase buffer at 4 °C (50 mm
Hepes pH 7.4, 10 mm MgCl
2
,1mm dithiothreitol, 10%
glycerol, 1 mm EDTA, 1 mm EGTA, 100 mm NaCl, 1 mm
NaF, 5 mm b-glycerophosphate, 1 mm Na molybdate,
100 lm ATP and protease inhibitor cocktail). Five microcu-
ries of [
32
P]ATP[cP] (PerkinElmer, Rowville, VIC, Austra-
lia) was added to each sample before being incubated for
30 min at 30 °C either alone or in the presence of purified
flag-Sav (see above) or 2.5 lg of MBP (Sigma). Reactions
were terminated by the addition of 5· SDS-sample buffer
and samples separated by SDS ⁄ PAGE, transferred to mem-
brane, dried and exposed to film at )80 °C. Following
autoradiography membranes were blocked and immuno-

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