Tài liệu Báo cáo khoa học: Association of mammalian sterile twenty kinases, Mst1 and Mst2, with hSalvador via C-terminal coiled-coil domains, leads to its stabilization and phosphorylation doc

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

types has been reported to induce cell death in a stress
activated protein kinase (SAPK) dependent pathway
[11–13]. In apoptotic cells, activated caspases can
cleave Mst1 and Mst2 C-terminal to the kinase domain
Keywords
coiled-coil domain; dimerization; Mst kinase;
phosphorylation; Salvador
Correspondence
B. A. Callus, Department of Biochemistry,
La Trobe University, Plenty Road, Bundoora,
VIC 3086, Australia
Fax: +613 9479 2467
Tel: +613 9479 1669
E-mail:
*Present address
Department of Biochemistry, La Trobe Uni-
versity, Plenty Road, Bundoora, VIC 3086,
Australia
(Received 21 May 2006, accepted 18 July
2006)
doi:10.1111/j.1742-4658.2006.05427.x
Genetic screens in Drosophila have revealed that the serine ⁄ threonine kinase
Hippo (Hpo) and the scaffold protein Salvador participate in a pathway
that controls cell proliferation and apoptosis. Hpo most closely resembles
the pro-apoptotic mammalian sterile20 kinases 1 and 2 (Mst1 and 2), and
Salvador (Sav) has a human orthologue hSav (also called hWW45). Here
we show that Mst and hSav heterodimerize in an interaction requiring the
conserved C-terminal coiled-coil domains of both proteins. hSav was also
able to homodimerize, but this did not require its coiled-coil domain. Coex-
pression of Mst and hSav led to phosphorylation of hSav and also increased

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

response to several cytokines nor affected by serum
withdrawal or addition [16]. While cleavage of Mst1
has been observed in cells following CD95 ⁄ Fas cross-
linking or IL-2 withdrawal, this effect is apparently
independent of activation of full-length Mst1 [11,12],
and may be a late consequence of caspase activation.
Recently, several reports have revealed a role in Dro-
sophila for the Mst1 and Mst2 homologue Hippo. The
salvador gene (also known as shar-pei) was identified in
flies in a screen to identify genes that imparted signifi-
cant growth advantages in mutant versus normal tissue
[17,18]. The Salvador protein has domains that permit
protein–protein interaction, including a WW domain
and a predicted coiled-coil motif in its C-terminus, sug-
gesting it might function as a scaffold in a multimeric
complex. Subsequently, the serine ⁄ threonine kinase
Hippo (Hpo) was identified as a binding partner of
Salvador [19–23]. Mutation of hpo and salvador yield
identical phenotypes, characterized by increased cell
proliferation and impaired apoptosis. These effects can
be at least partly explained by the elevated levels of the
cell cycle regulator, cyclin E, and the Drosophila inhib-
itor of apoptosis protein, dIAP1, in mutant tissue. The
hpo and salvador mutant phenotypes also resemble
those due to mutation of another serine ⁄ threonine
kinase, warts (wts). Indeed, Wts can bind to the WW
domains of Salvador [17], and was subsequently shown
to complex with and be activated by Hpo in a Salvador
dependent manner [19,20,23]. Furthermore, Wts was
recently shown to phosphorylate and subsequently

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

Raf1 ⁄ Rassf1 ⁄ Nore1 or to Salvador, but not both at
the same time. Furthermore, there appear to be no
known orthologues of Nore1 and Rassf1 in flies, thus
raising the possibility that complexes of Sav and Mst
might not occur in mammals.
Here we report that hSalvador can tightly interact
with the kinases Mst1 and Mst2, just as their counter-
parts, Salvador and Hpo interact in Drosophila.
Results
Mst kinase interacts with, and stabilizes,
hSalvador protein
To determine whether hSalvador (referred to hereafter
as Sav) can interact with Mst kinases, we generated
isogenic stable cell lines that could inducibly express
flag epitope-tagged Sav. Following treatment with
doxycycline, two independent clones of cells efficiently
expressed flag-Sav (Fig. 1A). Moreover, endogenous
Mst1 was easily detected in antiflag immune complexes
in cells that expressed flag-Sav. As a positive control
for this experiment, in a separate cell line, induced
myc-tagged Mst1 was also precipitated, albeit less effi-
ciently, with antibody raised against Mst1. Interest-
ingly, induction of Mst1 in these cells resulted in the
appearance of two smaller proteins that corresponded
in size to the caspase-cleaved forms of myc-tagged and
endogenous Mst1. In contrast to Mst1, endogenous
Mst2 was not detectable in these cells (data not
shown).
To confirm and extend this observation, flag-Sav
was coexpressed with myc-Mst1 or myc-Mst2 in 293T

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

IP:
Re-blot: α-flag
62
79
48
37
26
Dox
-+ -+- +
62
110
79
48
37
flag-
SavB
flag-
SavC
myc-
Mst1
IP:
α-flag
62
110
79
48
37
62
79
48

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
110
79
48
37
24

172
-+ +
+++
+
myc-Mst2
flag-Sav
myc-Mst1
+-
*
*
Sav
Mst
IP:
α-flag
IP:
α-myc
Blot: α-myc
Re-blot: α-flag
62
110
79
48
37
24
172
62
110
79
48
37

24
62
110
79
48
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

pcDNA3
++
62
48
37
48
37
48
37
Sav
Sav
Mst
Sav
β-actin
Lysates
Blot: α-flag
Lysates
Re-blot: α-myc
Lysates
Re-blot: α-β-actin
Sav
Sav
Mst
β-actin
Lysates
Blot: α-flag
Lysates
Re-blot: α-myc
Lysates
Re-blot: α-β-actin

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

+
GFP
+
myc-Mst2
-+
flag-Sav
+++++
myc-Mst1

+
-
-
pcDNA3
-+- - -
ACE
D
B
Fig. 1. Mst kinase interacts with hSalvador and increases its abundance. (A) Independent clones of Flp-In T-REx-293 cells (flag-SavB, flag-
SavC and myc-Mst1) were cultured overnight with or without doxycycline as indicated. Cell lysates and immune complexes were separated
by SDS ⁄ PAGE, transferred to membrane and sequentially immunoblotted as indicated on the left. (B–E) Flag-tagged Sav cDNA was cotrans-
fected with or without myc-Mst1, myc-Mst2, HA-PPIA or green floiurescent protein cDNAs into 293T cells as indicated. Two days after
transfection cell lysates were prepared. Immune complexes (B) or total cell lysates (C) were separated by SDS ⁄ PAGE, transferred to mem-
brane and sequentially immunoblotted as indicated on the left. The position of relevant bands is indicated with arrows. The heavy and light
immunoglobulin chains in antimyc immune complexes (B, bottom) are indicated (*). The panels shown in (C) are from identical duplicate gels
of the same lysates. The blots shown in (D) are from a separate experiment to that shown in (B) and (C). In this experiment total cell lysates
were separated on 10% denaturing gels prior to transfer and immunoblotting. Each experiment was performed at least twice with similar
results.
Mst kinases bind, stabilize and phosphorylate hSav B. A. Callus et al.
4266 FEBS Journal 273 (2006) 4264–4276 ª 2006 The Authors Journal compilation ª 2006 FEBS
domain [7], we hypothesized that Sav interacted with

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

bind to Sav were coexpressed, levels of Sav were un-
affected. Therefore it appears that the interaction of
Mst with Sav is required, and might be sufficient, for
it to increase levels of Sav.
The levels of N-terminally truncated mutants of
Sav that retained the C-terminal coiled-coil domain,
and thus were able to bind Mst, were increased even
more dramatically than WT Sav. As shown in Fig. 3C
(top), successive deletions of the Sav N-terminus
strongly destabilized these proteins to such an extent
that the Sav(268–383) and (321–383) constructs were
expressed at or below the limit of detection in this
system. Indeed, several attempts to detect Sav(321–
383) when expressed alone were unsuccessful. How-
ever, coexpression of Mst2 with these truncation
mutants dramatically enhanced their abundance, par-
ticularly Sav(268–383) and (321–383), such that they
were readily detected (Fig. 3C, top). As expected these
N-terminal mutants were all able to bind Mst2, as
demonstrated by the presence of Mst2 in antiflag
immune complexes (Fig. 3C, bottom). Notably, the
Sav(321–383) fragment was able to efficiently copre-
cipitate Mst2, indicating that the Sav coiled-coil
domain is not only essential but is also sufficient for
binding. Furthermore, despite their greatly different
abundances, the three Sav mutants were able to co-
precipitate similar amounts of Mst2 compared to WT
Sav, suggesting that in this system Mst is limiting.
Alternatively, it is possible that the coiled-coil domain
on its own is able to interact with Mst with higher

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-

tional domains. The structure of Mst1 is
given representatively for the very similar
Mst1 and Mst2 kinases and shows the posi-
tion of the kinase domain (light grey box),
the inhibitory domain (medium grey box),
C-terminal coiled-coil ⁄ dimerization domain
(black box) and the caspase cleavage site
(arrowhead). The Mst1 mutant lacking the
coiled-coil domain, D433, analogous to that
of Mst2 D437, is also shown. The structures
of wild-type and mutant Sav constructs
used in this study are shown indicating the
location of the coiled-coil domain and the
proline binding WW domains (dark grey
box).
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 4267
+
++ +++
+-
-+
-+-
pcDNA3
A
CD
B
flag-Sav
myc-Mst1 WT
myc-Mst1 Δ433
myc-Mst2 WT

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

Blot: α-flag
IP: α-myc
Re-blot: α-myc
62
79
48
37
62
79
48
37
62
79
48
37
62
79
48
37
62
79
48
37
26
flag-Sav WT
+-
+
-
flag-Sav Δ374
-+

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

+
myc-Mst1

+++
+
myc-Mst2
++ ++

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

v
Sav
62
79
48
37
26
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
Blot: α-flag
IP: α-myc
Re-blot: α-myc
26
62
79
48
37
26
62
79
48
37
62

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
79
26
19
15
6
Mst2
Sav

Sav
Sav
62
48
37
79
26
19
15
6
Sav
62
48
37
79
26
19
15
6
62
48
37
79
26
19
15
6
Sav
Lysates
Blot: α-flag

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

IP: α-flag
Blot: α-flag
Mst
Mst
Sa
v
Sa
v
pcDNA3
flag-Sav 321-383
-
+
-
-
-
-
+
-
-
+
-
-
-
-
-
-
-
myc-Mst1 WT
myc-Mst1 Δ433
myc-Mst2 WT

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

that Mst1 can multimerize (dimerize) via its C-terminal
coiled-coil domain [7], we hypothesized that Sav also
homo-multimerized via its coiled-coil domain in a sim-
ilar way. To test this we coexpressed full-length
hemagglutinin (HA) tagged Sav together with C-ter-
minally truncated Sav mutants tagged with the flag
epitope. As predicted, full-length HA-Sav was indeed
capable of coprecipitating full-length flag-Sav
(Fig. 4A). Unexpectedly, however, HA-Sav was also
efficiently coimmunoprecipitated with flag-Sav D344
and D321, two mutants that lacked the coiled-coil
domain. These results indicate that Sav can homo-
dimerize ⁄ multimerize, but it does not require its C-ter-
minal coiled-coil domain to do so.
Next we attempted to identify the region(s) that medi-
ate Sav homo-multimerization. To do this we
coexpressed C-terminally truncated flag-Sav mutants
with full-length HA-Sav. As seen in Fig. 4B, all mutants
we examined were able to coprecipitate full-length
HA-Sav at levels comparable with that of WT flag-Sav.
Importantly, this multimerization was specific to Sav
because when Sav was coexpressed with two unrelated
proteins, HA-PPIA and flag-cytokine response modifier
A-DQMD mutant (CrmA-DQMD), they failed to
coimmunoprecipitate with Sav (Fig. 4B, lanes 5 and 6).
hSalvador is phosphorylated by Mst kinase
To determine whether Sav was a phosphorylation sub-
strate of Mst1 or Mst2, we coexpressed them with Sav
and separated the lysates on a 10% linear gel. A
mobility shift of WT flag-Sav that is only apparent on

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

48
Lysates
Blot: α-HA
Lysates
Re-blot: α-flag
IP: α-flag
Blot: α-HA
IP: α-flag
Re-blot: α-flag
62
48
37
26
79
Lysates
Re-blot: α-flag
Lysates
Blot: α-HA
IP: α-flag
Blot: α-HA
IP: α-flag
Re-blot: α-flag
62
48
37
26
19
62
48
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

transfection the cells were washed and
lysed. Immune complexes and total cell ly-
sates were separated on denaturing gels,
transferred to membrane and sequentially
immunoblotted as indicated on the left. The
migration of HA-Sav and PPIA (B) is indica-
ted with arrows while the position of flag-
Sav is marked with arrowheads. (B) The
position of CrmA-DQMD (lane 6) is marked
with an asterisk (*). Each experiment was
performed at least twice with similar
results.
Fig. 3. Mst kinase and hSalvador interact via their C-terminal coiled-coil domains. (A–D) WT, C-terminally or N-terminally truncated mutants
of flag-Sav cDNA were cotransfected with or without WT or C-terminally truncated mutants of myc-tagged Mst1 or Mst2 cDNAs into 293T
cells as indicated. Two days after transfection cell lysates were prepared. Immune complexes and total cell lysates were separated by
SDS ⁄ PAGE, transferred to membrane and sequentially immunoblotted as indicated on the left. The migration of Mst and Sav are marked
with arrows and arrowheads, respectively. The position of immunoglobulin heavy chain in antimyc immune complexes is indicated with an
asterisk (*). Each experiment was performed at least twice with similar results.
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 4269
(Fig. 5A, top). An equivalent mobility shift was also
detected with flag-Sav D374 in the presence of Mst.
This suggests that Sav itself might be phosphorylated
by Mst kinase. Again, consistent with earlier results,
coexpression of Mst resulted in an increased abun-
dance of Sav WT and D374 proteins. This increase was
more apparent when the same samples were separated
on gradient gels (Fig. 5A, middle).
Mst
Sav

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
+
+
-
-
+
-
+
-
+
-

-
-
-
+
-
+
-
-
+
+
-
-
-
+
62
48
37
Sav
AC
BD
Lysates
Blot: α-flag
123456
Mst
62
48
37
pcDNA3
+
+

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

myc-Mst2 WT
myc-Mst2 K56R
+
-
+
+
+
-
Sav
Mst2
Sav
IP: α-flag
Blot: α-flag
IP: α-flag
Re-blot: α-myc
IP: α-flag
Autoradiograph
62
48
37
62
79
48
37
Sav
Blot: α-flag
62
79
37
48

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

myc-Mst2 WT
myc-Mst2 K56R
-
+
+
-
-
+
+
-
-
+
+
-
Sav
alone +Sav +MBP
Fig. 5. Mst kinase phosphorylates hSalvador. (A) Myc-tagged Mst1 or Mst2 cDNAs were cotransfected with WT or D374 flag-Sav cDNA into
293T cells as indicated. (B) WT flag-Sav cDNA was cotransfected with either WT or mutant Mst1 or Mst2 cDNAs into 293T cells as indica-
ted. Two days after transfection total cell lysates were prepared and separated by SDS ⁄ PAGE, transferred and sequentially immunoblotted
as indicated on the left. The same cell lysates in (A) were separated either on a 10% linear gel (top) or on a 4–20% gradient gel (middle and
bottom) prior to transfer. The migration of Mst and Sav is marked with arrows while the position of the slower migrating form of Sav is indi-
cated with an arrowhead. (C) Flag-Sav cDNA was cotransfected with either WT or kinase-dead Mst2 (K56R) cDNAs into 293T cells as indica-
ted. After two days cells were labelled in vivo with
32
P-orthophosphate. Immune complexes were separated on a 10% denaturing gel,
transferred to membrane, dried and exposed to film. Following autoradiography, membranes were sequentially immunoblotted as indicated
on the left. (D) WT or kinase-dead Mst2 (K56R) cDNAs were transfected into 293T cells. After two days cells were lysed and Mst kinases
were immunoprecipitated. Antimyc immune complexes were washed, equally divided three ways and incubated for 30 min at 30 °C in kin-
ase buffer with [
32

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

P-or-
thophosphate. As shown in Fig. 5C,
32
P-labelled Sav is
clearly detected in antiflag immune complexes from
cells expressing Sav and WT Mst2. However, the
amount of
32
P-labelled protein was much less when
Sav was coexpressed with the mutant kinase. Consis-
tent with this result, the amount of
32
P-labelled Mst2
was also reduced in cells coexpressing the mutant kin-
ase.
32
P-labelling of Sav and Mst2-K56R in this sam-
ple was presumably due to endogenous kinases, most
likely endogenous Mst. Thus, these results show that
Sav is phosphorylated as a result of coexpression with
Mst kinase.
To confirm these results and to address whether
phosphorylation of Sav by Mst is direct, we performed
an in vitro phosphorylation assay using purified Mst
kinases and flag-Sav as substrate (see below). Incuba-
tion of WT Mst2 alone (without substrate) resulted in
robust autophosphorylation of the kinase (Fig. 5D,
top). In contrast, kinase-dead Mst2 was unable to
autophosphorylate despite identical amounts of
immunoprecipitated kinase being present (Fig. 5D,

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

myc-Mst1 WT
myc-Mst1 K59R
myc-Mst2 WT
myc-Mst2 K56R
-
+
-
+
-
-
-
+
-
-
+
-
-
+
-
-
-
+
-
+
+
-
-
-
+
+

-
-
-
-
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

48
37
Mst
Sav
IP: α-flag
Blot: α-myc
IP: α-flag
Re-blot: α-flag
62
79
48
37
Fig. 6. The stabilization of hSalvador is independent of its phos-
phorylation by Mst kinase. (A,B) WT flag-Sav cDNA was
cotransfected with either WT or kinase-dead Mst1 (K59R) or Mst2
(K56R) cDNAs into 293T cells as indicated. Two days after transfec-
tion cell lysates were prepared. Total cell lysates (A) or immune
complexes (B) were separated by SDS ⁄ PAGE, transferred and
sequentially immunoblotted as indicated on the left. Samples in (A)
were separated on 9% a denaturing gel. The position of relevant
bands is indicated with arrows except for the slower migrating
form of Sav, which is marked with an arrowhead. Each experiment
was performed at least twice with similar results.
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 4271
(Fig. 6B). Despite being unable to phosphorylate Sav,
both kinase mutants, particularly mutant Mst2, were
nevertheless able to increase levels of Sav (Fig. 6A).
Together, these results indicate that the stabilizing
effect of Mst kinase on Sav is independent of its phos-

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

results indicate that the coiled-coil interaction between
Mst and Sav has been evolutionarily conserved
between flies and man.
The C-terminal coiled-coil domain in Mst1 is also
required for it to form homodimers (multimers) [7].
Based on this finding, it seemed reasonable to predict
that Sav might also homodimerize via its coiled-coil
domain. We found that Sav could indeed specifically
homodimerize (multimerize) but, surprisingly, this did
not require its coiled-coil domain (Fig. 4). Because
Salvador bears a WW domain, a motif that allows
interaction with proline residues, we considered the
possibility that this region might allow multimerization
independently of the coiled-coil domain. Such a WW
domain ⁄ proline interaction might explain why full-
length HA-Sav, with its intact WW domains, was still
able to interact with flag-Sav D199. Unfortunately, it
was not possible to test this hypothesis, because the
HA-Sav D199 construct, as well as a flag-tagged WW
domain (residues 200–267) construct, was unstable.
Therefore, although it is clear that Sav can homo-
multimerize independently of its C-terminal coiled-coil
domain, the region(s) involved remain to be deter-
mined.
It therefore seems probable that Sav, Mst1 and
Mst2 exist in a state of equilibrium (competition)
between Sav and Mst homodimers and the formation
of Sav ⁄ Mst heterodimers, in an association dependent
on their C-terminal coiled-coil domains (Fig. 7).
The coexpression of Mst and Sav had two conse-

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

change in Sav or leads to a phosphorylation-induced conformational
change in Sav facilitating recruitment of unknown substrates (X) to
the complex. Phosphorylation of substrates by Mst results in their
activation or inactivation and subsequent downstream effects. Sta-
bilization of Sav by Mst would enhance this process by potentially
providing more scaffold ‘sites’ with which to recruit more sub-
strates into the complex, thus strengthening the potential down-
stream effects.
Mst kinases bind, stabilize and phosphorylate hSav B. A. Callus et al.
4272 FEBS Journal 273 (2006) 4264–4276 ª 2006 The Authors Journal compilation ª 2006 FEBS
ated by Mst (Figs 1, 3, 5 and 6). The effect of Mst2 on
Sav phosphorylation and stability was almost always
greater than that of Mst1, suggesting that Sav is a pre-
ferred partner ⁄ substrate of Mst2 compared to Mst1,
which may reflect the fact that Drosophila Hpo is
slightly more similar to Mst2 than to Mst1. Further-
more, the observation that Mst2 but not Mst1 could
be coprecipitated with the Sav coiled-coil domain
alone suggests that Sav binds Mst2 with a higher affin-
ity than with Mst1 (Fig. 3D).
Both phosphorylation of Sav and its increase in
abundance were dependent of the ability of Sav and
Mst to interact. Deletion of the coiled-coil domain,
and thus the ability of the two proteins to dimerize,
abolished both the phosphorylation of Sav and the sta-
bilizing effect of Mst on Sav abundance. We consid-
ered the possibility that the phosphorylation of Sav by
Mst might account for the increased stability of Sav.
However, while it is still possible that phosphorylation
of Sav by Mst might further enhance its stability, the

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

be recruited to the Mst⁄ Sav complex, and in turn be
phosphorylated by Mst (Fig. 7). Alternatively, phos-
phorylation of Sav might induce a conformational
change in the protein that facilitates recruitment of
substrates to the complex. The observation that Hpo
can also phosphorylate Sav in S2 cells [19,20] suggests
that this modification might be an important regula-
tory aspect of this complex.
In flies, the phenotypes of hpo and salvador mutants
overlap with the phenotype of flies mutant for the ser-
ine ⁄ threonine kinase, warts [20–23]. Consistent with
this, Hpo and Sav are capable of forming a complex
with Wts that leads to its activation [20,22,23]. The
WW domains of Sav mediate this interaction by bind-
ing to PPXY motifs in Wts [17]. It was shown recently
that Mst2 could phosphorylate and activate the human
orthologues of Wts, large tumour suppressor-1 and -2
(Lats1 and Lats2), both in vitro and in cells [28]. How-
ever, unlike the Hpo ⁄ Sav ⁄ Wts complex in flies, Lats1
was not detectable in a complex with Mst2 and Sav
either in cells or using in vitro translated proteins, sug-
gesting that the complex is either very unstable or that
the activation of Lats by Mst2 might be indirect.
Indeed, we also failed to detect endogenous Lats kin-
ase in Sav ⁄ Mst immune complexes using a proteomics
approach (data not shown), adding further support to
the notion that Mst might indirectly activate Lats kin-
ases in mammalian systems.
Mst1 and Mst2 are known to interact with several
proteins. The growth inhibitory proteins, Rassf1 and

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

suppressed its activation and thereby prevented apop-
tosis from occurring [26]. Again, Sav was not detected
in these Raf1 immune complexes, suggesting that com-
plexes of Mst2 ⁄ Sav and Mst2 ⁄ Raf1 may also be mutu-
ally exclusive. For example, it may be that following
mitogenic stimulation, Mst2 dissociates from Raf1,
and is then free to bind Sav to induce its downstream
effects. Nevertheless, more experiments will be required
to exclude the possibility that Sav bound to Mst is also
recruited into some of these regulatory complexes.
Recent studies in Drosophila support a pro-apoptotic
role for the Hpo ⁄ Sav ⁄ Wts pathway. Clearly the regula-
tion of the Mst1 and Mst2 in mammals is more com-
plex especially as orthologues of Nore1 and Rassf1 do
not appear to exist in flies. How the Sav ⁄ Mst pathway
contributes to apoptosis and ⁄ or coordinates with other
regulatory pathways of Mst, if at all, remains to be
determined.
Experimental procedures
Antibodies
Mouse monoclonal antiflag (M2) and agarose-conjugated
antiflag (M2) beads were obtained from Sigma (Castle
Hill, NSW, Australia). For immunoblotting, high affinity,
rat monoclonal anti-HA (3F10) was purchased from
Roche (Kew, VIC, Australia) and for immunoprecipita-
tions, rabbit polyclonal anti-HA (HA.11) antisera was
purchased from Covance (Berkeley, CA, USA). Mouse
monoclonal antimyc (9E10) antibody was obtained from
the monoclonal antibody facility, The Walter and Eliza
Hall Institute (Bundoora, VIC, Australia). Mouse mono-

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

) and l-glutamine
(2 mm) in a humidified atmosphere of 10% CO
2
at 37 °C.
Cells were seeded at 10–15% confluency the day prior to
transfection of cDNA constructs (1 lg plasmid DNA total
per10 cm dish) using Effectene (Qiagen) according to the
manufacturer’s specifications. Flp-In
TM
T-REx
TM
-293 cells
(Invitrogen) were maintained as above but in the presence
of blasticidin (9 lgÆmL
)1
) and hygromycin (90 lgÆmL
)1
).
Isogenic, stable inducible cell lines were generated accord-
ing to the manufacturer’s guidelines. Protein expression was
induced in these cells by overnight treatment with doxy-
cycline (25 ngÆmL
)1
).
Cell lysis, immunoprecipitation and
immunoblotting
Cells were washed with phosphate-buffered saline
(NaCl ⁄ P
i
) and incubated for 1 h on ice in DISC lysis buf-

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

32
P-orthophosphate
Two days after transfection, cells were incubated at 37 °C
in phosphate-free media [Dulbecco’s Modified Eagle with-
out Na-phosphate (Gibco) supplemented with 10% (v ⁄ v)
dialysed foetal bovine serum (Gibco), 1 mm Na-pyruvate
and l-glutamine (2 mm)]. After 2 h, 2.5 mCi
32
P-orthophos-
phate (Amersham) was added per 10 cm dish and incubated
for a further 2 h at 37 °C. Cells were washed twice with
ice-cold NaCl ⁄ P
i
before being lysed in DISC lysis buffer
and incubated for 1 h on ice. Immune complexes were pre-
pared as described above and separated by SDS ⁄ PAGE
before being transferred to poly(vinylidene diflouride) mem-
brane, dried and exposed to film at )80 °C. Following
autoradiography membranes were blocked and immuno-
blotted as necessary.
Purification of flag-Sav for in vitro
phosphorylation
In our hands a bacterially expressed GST-Sav construct
was insoluble. Therefore to generate sufficient quantity of
Sav to use as a substrate in vitro we transiently transfec-
ted ten 15 cm plates of 293T cells with flag-Sav cDNA.
Two days after transfection, cells were lysed and flag-Sav
immunoprecipitated with antiflag beads as described above.
Immune complexes were eluted with 100 mm glycine
pH 3.0, neutralized with 1 m Tris pH 8.0 and dialysed

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-

blotted as necessary.
Acknowledgements
We sincerely thank Jonathon Chernoff and Dan Haber
for generously providing cDNAs used in this study.
We thank members of the Vaux lab for helpful discus-
sions. DLV is an ARC Federation Fellow. AMV is an
ARC Queen Elizabeth II Research Fellow. This work
was supported by funds from NHMRC Program
Grant (257502) and Leukemia and Lymphoma Society
Center Grant.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. hSalvador can homo-multimerise indepen-
dently of its C-terminal coiled-coil domain. Data not
shown from Fig. 4.
This material is available as part of the online article
from
Mst kinases bind, stabilize and phosphorylate hSav B. A. Callus et al.
4276 FEBS Journal 273 (2006) 4264–4276 ª 2006 The Authors Journal compilation ª 2006 FEBS