Báo cáo khoa học: Liên kết của mammalian sterile twenty kinases Mst1 và Mst2 với hSalvador qua C-terminal coiled-coil domains dẫn đến sự ổn định và phosphoryl hóa

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

*, Anne M. Verhagen

and David L. Vaux

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: b.callus@latrobe.edu.au

*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 ª2006 The Authors Journal compilation ª2006 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 ª2006 The Authors Journal compilation ª2006 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

Dox -+ -+- +

110

flag-

SavB

flag-

SavC

myc-

Mst1

IP:

α-flag

110

Lysates:

Re-blot: α-flag

Lysates:

Blot: α-Mst1

Lysates:

Re-blot: α-β-actin

IP:

α-Mst1

myc-Mst1

Sav

β-actin

endog. Mst1

myc-Mst1

endog. Mst1

cleaved Mst1

Mst

110

172 -+--+

+++--

+--

myc-Mst2

flag-Sav

myc-Mst1 +-

Sav

Mst

IP:

α-flag

IP:

α-myc

Blot: α-myc

Re-blot: α-flag 62

110

172

110

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 --++

Sav

Mst

Sav

β-actin

Lysates

Blot: α-flag

Lysates

Re-blot: α-myc

Lysates

Re-blot: α-β-actin

Sav

Mst

β-actin

Lysates

Blot: α-flag

Lysates

Re-blot: α-myc

Lysates

Re-blot: α-β-actin

Lysates

Re-blot: α-GFP

Lysates

Re-blot: α-HA PPIA

GFP

Mst

HA-PPIA -- - -+

GFP ----+

myc-Mst2 --- -+

flag-Sav ++ + ++

myc-Mst1 --+--

pcDNA3 -+- - -

ACE

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 ª2006 The Authors Journal compilation ª2006 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:

∆433

∆374

∆344

∆321

∆268

∆199

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 ª2006 The Authors Journal compilation ª2006 FEBS 4267

-- --+

++ +++

+- ---

-+ ---

-- -+-

pcDNA3

flag-Sav

myc-Mst1 WT

myc-Mst1 ∆433

myc-Mst2 WT

myc-Mst2 ∆437 -- +--

Mst

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

flag-Sav WT +- --+---

flag-Sav ∆374 -+ ----+-

flag-Sav ∆344 -- -+-+--

flag-Sav ∆321 -- +----+

myc-Mst1 -- --++++

myc-Mst2 ++ ++----

Mst

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

pcDNA3

myc-Mst2 WT

flag-Sav WT

flag-Sav 199-383

flag-Sav 268-383

flag-Sav 321-383

β-actin

Mst2

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

110

Lysates

Blot: α-myc

Lysates

Blot: α-flag

IP: α-flag

Blot: α-myc

IP: α-flag

Blot: α-flag

Mst

pcDNA3

flag-Sav 321-383

myc-Mst1 WT

myc-Mst1 ∆433

myc-Mst2 WT

myc-Mst2 ∆437

++++-

Mst kinases bind, stabilize and phosphorylate hSav B. A. Callus et al.

4268 FEBS Journal 273 (2006) 4264–4276 ª2006 The Authors Journal compilation ª2006 FEBS

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

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