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

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 Correspon

and Mst2, with hSalvador via C-terminal coiled-coil

domains, leads to its stabilization and phosphorylation

Bernard A Callus1,*, Anne M Verhagen1and David L Vaux1,*

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.

[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-sophilafor the Mst1 and Mst2 homologue Hippo The

salvadorgene (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

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

37

26

Dox - + - + - +

62

110

79

48

flag-SavB SavCflag-

myc-IP:

α-flag

62

110

79

48

62

37

26

62

79

48

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

24

172 - - + - +

+ +

-+

-myc-Mst2

flag-Sav

-*

*

Sav Mst

IP:

α-flag α-mycIP:

Blot: α-myc

Re-blot: α-flag 62

110

79

48

24

172

62

110 79 48 24

62

110 79 48 24

Sav

Mst

myc-Mst2 - - - +

flag-Sav - + + +

myc-Mst1 - - +

-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 15 37 48 26

62 37 79

62 37 48 79

26

Lysates Re-blot: α-GFP

Lysates

GFP

GFP Mst

HA-PPIA - - - - +

GFP + - - -

-myc-Mst2 - - - +

-flag-Sav + + + + +

myc-Mst1 - - + -

-pcDNA3 - + - -

D

B

Fig 1 Mst kinase interacts with hSalvador and increases its abundance (A) Independent clones of Flp-In T-REx-293 cells (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.

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

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).

- - - -+

+ + + + +

+ - -

+ -

- +

-pcDNA3

A

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 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

Sav

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 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

37 79

26 15 6

62 37 79

26 15 6

Mst2

Sav

Sav

Sav

62 37 79

26 15 6

Sav

62 37 79

26 15 6

62 37 79

26 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 6

62 37 79 110

19 6

62 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 Sav

Sav

pcDNA3

flag-Sav 321-383

-+

-+

-+

-myc-Mst1 WT myc-Mst1 Δ433 myc-Mst2 WT myc-Mst2 Δ437

-+

+ + + + +

-+

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

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 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 37 26 62 37 26 15 62 37 26

HA-Sav

HA-Sav

PPIA flag-Sav

flag-Sav

*

*

1 2 3 4 5 6

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.

(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

flag-Sav WT

myc-Mst1

myc-Mst2

flag-SavΔ374 +

+ -+ -+ -+

-+ -+ -+ + -+ 62

48 37

Sav

Lysates

Blot: α-flag

Mst 62

48 37

pcDNA3 + + - - -

-flag-Sav - + + + + +

myc-Mst1 WT - - + - -

-myc-Mst1Δ433 - - - + -

-myc-Mst2 WT - - - - +

-myc-Mst2Δ437 - - - +

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 37 26 19

110

*

*

Mst2 Sav Re-blot: α-myc 62

37 26 19

110

*

*

pMst2 pSav Autoradiograph

62 37 26 19 15

110

pMBP

myc-Mst2 WT myc-Mst2 K56R +

-+

-+

+

-+

+

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.

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 with32

P-or-thophosphate As shown in Fig 5C,32P-labelled Sav is

clearly detected in antiflag immune complexes from

cells expressing Sav and WT Mst2 However, the

amount of 32P-labelled protein was much less when

Sav was coexpressed with the mutant kinase

Consis-tent with this result, the amount of 32P-labelled Mst2

was also reduced in cells coexpressing the mutant

kin-ase 32P-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.

(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

“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 conformassoci-ational 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.

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]