The Journal of Cell Biology, Volume 164, Number 7, March 29, 2004 1009–1020
The Rockefeller University Press, 0021-9525/2004/03/1009/12 $8.00
YSK1 is activated by the Golgi matrix protein GM130
and plays a role in cell migration through
its substrate 14-3-3
and Francis A. Barr
Veerle De Corte,
Max-Planck-Institute of Biochemistry, Martinsried, 82152 Germany
Department of Biochemistry and Molecular Biology, Faculty of Medicine and Health Sciences, Ghent University/Flanders
Interuniversity Institute for Biotechnology (VIB09), B-9000 Ghent, Belgium
Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital (1P7),
B-9000 Ghent, Belgium
he Golgi apparatus has long been suggested to be
important for directing secretion to specific sites on
the plasma membrane in response to extracellular
signaling events. However, the mechanisms by which
signaling events are coordinated with Golgi apparatus
function remain poorly understood. Here, we identify a
scaffolding function for the Golgi matrix protein GM130
that sheds light on how such signaling events may be
regulated. We show that the mammalian Ste20 kinases
YSK1 and MST4 target to the Golgi apparatus via the Golgi
matrix protein GM130. In addition, GM130 binding activates
these kinases by promoting autophosphorylation of a
conserved threonine within the T-loop. Interference with
YSK1 function perturbs perinuclear Golgi organization, cell
migration, and invasion into type I collagen. A biochemical
screen identifies 14-3-3
as a specific substrate for YSK1
that localizes to the Golgi apparatus, and potentially links
YSK1 signaling at the Golgi apparatus with protein transport
events, cell adhesion, and polarity complexes important
for cell migration.
Recent evidence suggests that the Golgi apparatus, in addition
to its function in protein secretion, may be the site of signaling
events important for diverse cellular processes (for review see
Ferri and Kroemer, 2001; Bivona and Philips, 2003; Rios
and Bornens, 2003). Thus, it has been proposed that the
Golgi apparatus may act as a signaling platform, sensing and
integrating signals from growth factor signaling pathways,
and thereby participating in the regulation of downstream
events (Bivona and Philips, 2003). This raises the question
of which cellular processes require coordination of signaling
at the cell surface and the various functions associated with
the Golgi apparatus. The position of the Golgi apparatus
within the cell responds to extracellular signals, rapidly re-
orienting in cells at experimental wound edges, migrating
and polarizing cells, and in activated cytotoxic T lympho-
cytes toward the site of killing (Kupfer et al., 1982; Kupfer
and Dennert, 1984). These processes involve a complex series
of signaling events coordinated with reorientation of the
microtubule cytoskeleton and changes in actin dynamics
dependent on dynein, Rho family GTPases, and the Arp2/3
complex (Ridley et al., 2003), and it is possible that membrane
traffic and signaling via the Golgi apparatus are significant in
this context. However, to date it is unknown how this in-
tegration between Golgi apparatus–associated signaling
molecules and Golgi apparatus function could be achieved.
Parallels between the organizational functions of Golgi matrix
proteins and signaling scaffolds can be drawn (Barr and
Short, 2003; Gillingham and Munro, 2003), and it is possible
that Golgi matrix proteins might organize and thereby control
signaling complexes on Golgi membranes. This would also
provide a means for coordinating signaling events with
Golgi apparatus integrity and function.
C. Preisinger, B. Short, and V. De Corte contributed equally to this paper.
The online version of this article contains supplemental material.
Address correspondence to Francis Barr, Max-Planck-Institute of Bio-
chemistry, Am Klopferspitz 18, Martinsried, 82152 Germany. Tel.:
49-89-8578-3135. Fax: 49-89-8578-3102. email: firstname.lastname@example.org
Key words: Ste20 kinases; cell migration; polarity; collagen invasion; scaffold
Abbreviations used in this paper: MBP, myelin basic protein; MST,
mammalian Ste20; siRNA, small interfering RNA; YSK1, yeast Sps1/
Ste20-related kinase 1.
The Journal of Cell Biology
1010 The Journal of Cell Biology
Volume 164, Number 7, 2004
The Ste20 family of serine/threonine protein kinases is
implicated in a variety of signaling pathways including those
involved in the control of cell migration and polarity (Dan
et al., 2001). In mammals, over 30 Ste20 kinases exist classi-
fied into two subgroups. These are the p21-activated kinases
and the germinal center kinases (Dan et al., 2001), and it
is this latter group that is of interest here. Germinal cen-
ter kinases possess an NH
-terminal kinase domain and a
COOH-terminal regulatory domain and can be further sub-
divided into eight groups based on sequence homologies
(Dan et al., 2001). Subgroups II and III contain the mam-
malian Ste20 (MST) kinases, MST1 (Creasy et al., 1996),
MST2 (Creasy and Chernoff, 1995), MST3 (Schinkmann
and Blenis, 1997), MST4 (Lin et al., 2001; Qian et al.,
2001), and yeast Sps1/Ste20-related kinase 1 (YSK1; Pombo
et al., 1996; Osada et al., 1997). MST1 and MST2 are
cleaved during apoptosis by caspase-3 and translocate into
the nucleus (Lee et al., 2001; Ura et al., 2001) where they
function in proapoptotic signaling (De Souza et al., 2002;
Lin et al., 2002). Less is known about the other MST ki-
nases. YSK1, also known as Ste20/oxidant stress response ki-
nase 1, is weakly activated by reactive oxygen intermediates
but not by any other environmental stresses, or by growth
factors (Pombo et al., 1996). However, this kinase does not
participate in any of the known MAPK pathways (Pombo et
al., 1996; Osada et al., 1997) and a physiological function
for YSK1 remains unknown. Like YSK1, MST4 overexpres-
sion fails to activate the JNK and p38 MAPK pathways, al-
though it promotes anchorage-independent growth and tu-
mor formation, and has been implicated in prostate cancer
progression (Qian et al., 2001; Sung et al., 2003). Here, we
investigate the MST family kinases YSK1 and MST4 and
uncover a signaling function linked with the Golgi matrix
protein GM130 that may play a role in the control of cell
migration and polarization.
YSK1 and MST4 localize to the Golgi apparatus
Antibodies raised against the recombinant human YSK1 ki-
nase domain detected a protein of the expected size in total
HeLa cell extract, rat liver cytosolic extract, and purified rat
liver Golgi membranes (Fig. 1 A). This reaction was com-
peted by preincubation of the antibody with the purified an-
tigen before Western blotting (Fig. 1 A). Due to the high
degree of sequence similarity between the MST family ki-
nases these antibodies were further tested on lysates of cells
transfected with GFP-tagged MST3, MST4, and YSK1.
This revealed that polyclonal antibodies raised to YSK1 also
react to a lesser extent with MST4, but not with MST3. An
antibody raised to a unique sequence in the first 19 aa of
YSK1 recognized only YSK1 (Fig. 1 A). When used in im-
munofluorescence studies of HeLa cells these antibodies
gave a cytosolic and Golgi apparatus staining pattern most
similar to the cis-Golgi protein GM130 and the medial-
Golgi protein GRASP55, and adjacent to but not overlap-
ping with the trans-Golgi marker golgin97 (Fig. 1 B). A sim-
ilar staining pattern was observed in two other human cell
lines, MelJuSo and HS68 (Fig. S1, available at http://
lished data). Endogenous YSK1/MST4 are therefore Golgi
proteins based on their localization in human cell lines and
presence in purified Golgi membrane fractions.
YSK1 and MST4 interact with the Golgi matrix
To identify interaction partners for these kinases that
could explain their localization to the Golgi apparatus, a
yeast two-hybrid screen of 2.2 million clones from a hu-
man testis cDNA library was performed. Only two clones
interacted specifically with YSK1, and both contained the
full-length ORF for the human Golgi matrix protein
containing 25 ?g of protein from insect cells expressing His6-tagged
human YSK1 (up shifted due to the presence of the tag), HeLa cells,
rat liver cytosol, or 10 ?g of rat liver Golgi membranes were analyzed
by Western blotting with 1 ?g/ml of affinity-purified rabbit antibody
4257 to human YSK1. For antibody competition, 10 ?g/ml of the
antigen was preincubated with the antibody for 1 h before use.
Samples containing 20 ?g of protein from HeLa cells transfected
with constructs for GFP-tagged YSK1, MST3, and MST4 for 18 h
were blotted with 1 ?g/ml of affinity-purified rabbit antibody 4257
to YSK1, a peptide antibody raised against the unique first 19 aa
of YSK1 (N19), or a sheep antibody to GFP. (B) HeLa cells were
costained with rabbit antibodies to YSK1 and sheep antibodies to
GM130, GRASP55, or golgin97. Bar, 10 ?m.
YSK1 localizes to the Golgi apparatus. (A) Samples
GM130 functions as a kinase scaffold |
Preisinger et al. 1011
GM130 (Fig. 2 A). A directed two-hybrid analysis was
then used to map the interaction sites for YSK1 on rat
GM130 (Fig. 2 A). The YSK1 binding site of GM130
maps to amino acids 75–271, a region predicted to adopt
a coiled-coil structure. This is discrete from the binding
site for the vesicle docking protein p115 that lies in the
first 75 aa (Fig. 2 A), and the COOH-terminal region
containing the binding sites for GRASP65 and Rab GTP-
ases (Barr et al., 1998). Binding assays using purified re-
combinant proteins confirmed that GM130
directly to YSK1 (Fig. 2 B), whereas the NH
domain of another Golgi matrix protein, golgin45, and
GST did not (Fig. 2 B). Furthermore, YSK1 failed to lo-
calize to the Golgi apparatus in cells depleted of GM130
using small interfering RNA (siRNA) duplexes (Fig. 2 C).
Therefore, YSK1 binds to a specific domain of the Golgi
matrix protein GM130 and shows GM130-dependent lo-
calization in cells, thus providing a mechanism for its lo-
calization to the Golgi apparatus.
Other MST kinases were then tested for interaction
with GM130 and localization to the Golgi apparatus. A
directed two-hybrid analysis revealed that MST4 inter-
acted with full-length GM130 and the first 271 aa of
GM130, but failed to interact with the empty vector con-
trol or golgin45 (Fig. 3 A). MST3 showed no interactions
with any of the proteins tested (Fig. 3 A). Binding assays
confirmed that MST4 bound directly to GM130
not to the NH
-terminal domain of golgin45 (Fig. 3 B).
Consistent with these results MST4 targeted to the Golgi
apparatus in HeLa cells (Fig. 3 C), whereas MST3 showed
a cytoplasmic distribution (Fig. 3 D). YSK1 displays sig-
nificant homology with MST3 and MST4, to a high de-
protein GM130 as a binding partner for
YSK1. (A) Bait constructs comprising the
full-length human GM130 splice variant
rescued from the library screen, full-length
rat GM130 or the indicated deletion
constructs of rat GM130 were tested
against full-length human YSK1 for the
ability to grow on selective medium
(QDO), compared with nonselective
medium (?LW) in the yeast two-hybrid
system. Lighter colony color on QDO is
an indicator of a strong signal. Boxed
regions indicate the p115 binding site
(red) and predicted coiled-coil domains
(green). (B) His-tagged YSK1 was incu-
bated with GST-tagged fragments of rat
GM130, golgin45, or GST and recovered
on glutathione-agarose. Recovered com-
plexes were analyzed by Western blotting.
(C) HeLa cells depleted for lamin A or
GM130 for 67 h using siRNA were stained
and Western blotted for GM130, YSK1,
and ?-tubulin. Bar, 10 ?m.
Identification of the Golgi
1012 The Journal of Cell Biology
Volume 164, Number 7, 2004
gree within the kinase domain and GM130 binding re-
gion and to a lesser but significant extent in the COOH
terminus (Fig. S2, available at http://www.jcb.org/cgi/
content/full/jcb.200310061/DC1). MST4 like YSK1
may target to the Golgi apparatus via an interaction with
GM130, and therefore have a common regulatory mecha-
nism. Interestingly, MST3 although highly homologous
to YSK1 and MST4 does not localize to the Golgi appara-
tus or bind to GM130, suggesting it has different regula-
YSK1 shows activity-dependent targeting
to the Golgi apparatus
To investigate the targeting of YSK1 to the Golgi appara-
tus further, the features of YSK1 needed for its interaction
with GM130 were mapped. Amino acids 20–302, com-
prising the kinase domain and a short region of charged
amino acids distal to it, was the smallest fragment tested
that interacted with GM130 (Fig. 4 A). The NH
COOH-terminal sequences before amino acid 20 and after
amino acid 302, respectively, were not essential for the in-
teraction with GM130 (Fig. 4 A). These constructs were
then tested for their ability to target to the Golgi apparatus
in transfection assays. Full-length YSK1 and, to a lesser ex-
tent, the first 302 aa comprising the GM130 binding re-
gion were Golgi apparatus localized (Fig. 4 B). Decreased
Golgi apparatus targeting of the first 302 aa of YSK1 com-
pared with the wild-type protein may be explained by the
observation that the COOH terminus is important for
YSK1 homodimerization (Fig. S3, available at http://
kinase activity-deficient mutants YSK1
the kinase domain alone, and a COOH-terminal construct
lacking the kinase domain were unable to target to the
Golgi apparatus (Fig. 4 B). Therefore, the targeting of
YSK1 to the Golgi apparatus requires an NH
gion from amino acids 20 to 302 that binds to GM130,
and includes the kinase domain. Kinase activity is also re-
quired for Golgi apparatus targeting and stable GM130
binding in vivo because kinase-dead mutants do not local-
ize to the Golgi apparatus.
GM130 is an activator of YSK1 and MST4
Because GM130 appears to function as a Golgi apparatus lo-
calized scaffold protein for YSK1, the relationship between
YSK1 kinase activity and GM130 binding was investigated.
A preliminary analysis of recombinant YSK1 purified from
insect cells found that the pure kinase showed no activity to-
ward the Golgi proteins GM130, GRASP65, and p115.
From a variety of model substrates tested, only myelin basic
protein (MBP) was phosphorylated (unpublished data), and
this was therefore used for subsequent experiments. Titra-
tion of the GM130 NH
-terminal domain into YSK1 kinase
assays up to a 20-fold molar excess resulted in a 25-fold
stimulation of YSK1 activity above the basal level of activity
(Fig. 5 A). Under the same conditions, the NH
domain of golgin45 maximally activated YSK1 3.5-fold,
whereas the protein storage buffer did not stimulate YSK1
(Fig. 5 B). Analysis of activated YSK1 revealed phosphoryla-
tion of threonine 174, a modification absent from the non-
activated kinase (Fig. 5 C and Fig. S4, available at http://
observation could be confirmed with pT174, an antibody
directed against phosphorylated threonine 174, which de-
tected YSK1 only after activation by GM130 (Fig. 5 D,
peptide), and was competed by the corresponding phospho-
peptide (Fig. 5 D,
peptide). Using the pT174 antibody,
phosphorylated YSK1 could only be detected in Golgi mem-
branes but not cytosol, even though like p115, another
GM130 partner, a pool of YSK1 was present in cytosol (Fig.
(A) Full-length YSK1, MST3, and MST4 were tested against empty
vector (control), full-length GM130, GM1301-271, and golgin45 for
the ability to grow on selective medium (QDO), compared with
nonselective medium (?LW) in the yeast two-hybrid system. Lighter
colony color on QDO is an indicator of a strong signal. (B) His-tagged
MST4 was incubated with GST-tagged fragments of rat GM130,
golgin45, or GST and recovered on glutathione-agarose. Recovered
complexes were analyzed by Western blotting. (C and D) HeLa cells
transfected with myc-tagged MST3 and MST4 constructs for 18 h
were costained with the 9E10 mAb to the myc epitope (green) and
a sheep antibody to GM130 (red). Bar, 10 ?m.
The YSK1-related kinase MST4 is Golgi apparatus localized.
GM130 functions as a kinase scaffold |
Preisinger et al. 1013
5 E). This supports the model that a pool of activated YSK1
exists at the Golgi apparatus.
Phosphorylation at the equivalent position of the T-loop
in many other kinases is an important determinant for their
activation (Russo et al., 1996). Mutation of the T-loop thre-
onine to alanine to give YSK1
kinase that showed no activity toward MBP in the presence
or absence of GM130, and was comparable to the kinase-
dead ATP binding site mutant YSK1
results to those obtained for YSK1 were also found with
MST4. MST4 is specifically activated by binding to GM130
and is autophosphorylated as a consequence on threonine
178 (Fig. 5 F; unpublished data). Therefore, activation of
YSK1 and MST4 involves autophosphorylation at threonine
174 (178 in MST4), possibly as a consequence of dimeriza-
tion stabilized by binding to GM130 at the Golgi apparatus.
resulted in a form of the
(Fig. 5 F). Similar
In addition to its function as part of the Golgi apparatus lo-
calized tethering complex required for vesicle docking and
stacking of Golgi cisternae, GM130 has been proposed to
form a structural landmark important for establishment of a
polarized Golgi structure (Pfeffer, 2001). One aspect of po-
larized Golgi structure is the cis- to trans-polarity of the
stacked cisternae, and the other is its asymmetric distribu-
tion within the cell. Association of the Ste20 family kinases
YSK1 and MST4 with GM130 suggest this complex might
act as a landmark in a transduction event important for sig-
naling Golgi apparatus integrity and position within the
cell, and controlling Golgi apparatus function. The effects
of expressing wild-type YSK1, and YSK1
behave as a dominant-negative mutant form of YSK1 un-
able to be activated by GM130, were then compared (Fig.
6, A and B). At high expression levels, YSK1 saturated the
available binding sites at the Golgi apparatus and accumu-
lated in the cytoplasm without causing any obvious change
in the two Golgi markers GM130 and p115 (Fig. 6 A). Ex-
pression of YSK1
resulted in the dispersal of the peri-
nuclear ribbon-like Golgi apparatus pattern of GM130 and
p115 typical of HeLa cells (Fig. 6 B). This effect was spe-
cific to the Golgi apparatus because the perinuclear late-
endosomal and lysosomal compartments defined by LAMP1
showed no obvious differences in YSK1 and YSK1
pressing cells (Fig. 6 C). Golgi apparatus dispersal was
75% of cells expressing YSK
with the dominant-negative MST4
and MST4 constructs (Fig. 6 D). Preliminary investiga-
tion revealed that transport of the transmembrane glycopro-
tein of vesicular stomatitis virus was not compromised in
expressing cells, indicating they do not have a
general defect in secretion (unpublished data). To obtain
supporting evidence for a function of endogenous MST ki-
nases at the Golgi apparatus in HeLa cells, depletion of
YSK1 and MST4 was performed using siRNA and the cells
stained for the Golgi marker GM130. In cells depleted of
YSK1 and MST4 the Golgi apparatus was dispersed into the
cell periphery, whereas in control cells depleted for lamin A
the typical perinuclear Golgi apparatus morphology was
preserved (Fig. 6 E). Interfering with YSK1 and MST4
function, therefore, disturbs the ordered localization of the
Golgi apparatus in the perinuclear region.
, which should
or other YSK1
to the Golgi apparatus. (A) Bait constructs comprising
full-length YSK1, or the indicated deletion constructs
were tested against full-length rat GM130 or an empty
bait plasmid (control) for the ability to grow on selective
medium (QDO), compared with nonselective medium
(?LW) in the yeast two-hybrid system. Lighter colony
color on QDO is an indicator of a strong interaction.
Boxed regions indicate the canonical serine/threonine
kinase domain (green), and the extension to this
necessary for binding to GM130 (red). (B) HeLa cells
transfected with myc-tagged YSK1 constructs for 18 h
were costained with the 9E10 mAb to the myc epitope
and a sheep antibody to GM130. Bar, 10 ?m.
YSK1 shows activity-dependent targeting
1014 The Journal of Cell Biology
Volume 164, Number 7, 2004
GM130 triggers autophosphorylation and activation
of YSK1. (A) Purified YSK1, 0.8 pmoles, was incubated
in the absence or presence of 0.7, 3.3, 6.7, and 16.7
pmoles of His-tagged GM13075-271 or (B) His-tagged
golgin451-122 at 37?C for 30 min. After this preincubation,
samples were analyzed for kinase activity toward the
model substrate MBP using ?-[32P]ATP, and by Western
blotting for YSK1 and GM130 or golgin45 to control
for gel loading. (C) Fragmentation of the phosphorylated
NTFVGTPFWMAPEVIK peptide (Fig. S4) by tandem
mass spectrometry gives daughter ions derived from
the NH2 and COOH termini of the peptide indicating
that threonine 174 (pT), shown in red, is the phosphor-
ylated residue. (D) Activated and mock-activated
YSK1 was Western blotted with affinity-purified antibody
N19 to YSK1 and the pT174 antibody to the phosphor-
ylated T-loop sequence IKRNpTFVGT, in the presence
and absence of 5 ?g of blocking peptide. (E) 25 ?g of
rat liver cytosol and 10 ?g of Golgi membranes were
Western blotted with antibodies to GM130, p115, YSK1
4256, and pT174 to phosphorylated YSK1. (F) Purified
YSK1, YSK1K49R, YSK1T174A, MST4, and MST4D162A, 0.8
pmoles, were incubated in the absence or presence of
16.7 pmoles of His-tagged GM13075-271 at 37?C for 30
min. After this preincubation, samples were analyzed
for kinase activity toward the model substrate MBP
using ?-[32P]ATP, and by Western blotting for YSK1
MST4 and GM130 to control for gel loading.
Binding to the NH2-terminal domain of
constructs for the wild-type myc-tagged (A) YSK1 or (B) YSK1T174A for 44 h and then stained with mouse antibodies to the myc epitope and
sheep antibodies to either GM130 or p115. (C) HeLa cells were transfected with expression constructs for the wild-type myc-tagged YSK1 or
YSK1T174A for 44 h and then stained with rabbit antibodies to the myc epitope and mouse antibodies to LAMP1. (D) Golgi apparatus dispersal
was scored after 48 h in HeLa cells transfected with YSK1 and MST constructs indicated in the legend. Mean values are plotted (n ? 3) with
at least 300 cells counted per experiment for each condition. (E) HeLa cells were transfected with siRNA duplexes for YSK1 and MST4 for
112 h then stained with antibodies to GM130 (green) and YSK1/MST4 (red), and with DAPI for DNA (blue). Exposure times of 1 s were used
for all images. Bars, 10 ?m.
YSK1/MST4 are required for Golgi apparatus localization in the perinuclear region. (A) HeLa cells were transfected with expression
GM130 functions as a kinase scaffold |
Preisinger et al. 1015
A biochemical screen for YSK1 targets identifies 14-3-3
One explanation for the differences seen with the dominant-
negative forms of YSK1 and MST4 is that they have different
substrates. To test this hypothesis, a biochemical screening ap-
proach for the kinase substrate tracking and elucidation
(KESTREL) was used (Knebel et al., 2001). Application of
this method to HeLa cell extracts fractionated by size exclusion
chromatography revealed a number of phosphorylation events
(Fig. 7 A). Strong autophosphorylation of both kinases hin-
dered the identification of substrates in the 50–60-kD region.
Significantly, a protein of apparent molecular mass 32 kD in
fraction 17 was phosphorylated in YSK1 treated samples (Fig.
7 A), but was not in MST4, kinase inactive YSK1
buffer-treated samples (Fig. 7, A and B). Further analysis using
mass spectrometry and Western blotting revealed that this
phosphoprotein corresponds to 14-3-3
Serine 58 (Fig. 7 A; unpublished data). Confirming these ob-
servations, recombinant 14-3-3
but not MST4 (Fig. 7 C). Furthermore, an mAb against 14-3-
(Leffers et al., 1993) stained the Golgi apparatus and
showed considerable overlap with the YSK1 activator GM130
(Fig. 7 D). Therefore, 14-3-3
fulfills the criteria of a specific
target for YSK1 at the Golgi apparatus.
was phosphorylated by YSK1
invasion and cell migration
It has recently been reported that in mammalian cells there
is a link between the oncogenic Ras-signaling pathway and
cell invasion induced by the actin severing protein gelsolin
(De Corte et al., 2002). Together with other recent findings
that Ras-signaling also occurs at the surface of the Golgi ap-
paratus (Chiu et al., 2002; Bivona et al., 2003), these obser-
vations hint that YSK1 and MST4 may signal some aspect
of cell migration involving the Golgi apparatus. The effects
of expressing wild-type and various mutant forms of YSK1
and MST4 on gelsolin-induced invasion were therefore ex-
plored, using an established assay (Braecke et al., 2001). Co-
expression of GFP, YSK1, and the kinase-dead YSK1
mutant had no significant effect on gelsolin-induced inva-
sion (Fig. 8 A), whereas coexpression of dominant-negative
abrogated collagen invasion. If 14-3-3
YSK1 substrate mediating these effects, then expressing 14-
should give the same result as dominant-negative
, whereas 14-3-3
and this was indeed the case (Fig. 8 B). Gelsolin-induced in-
vasion was abrogated by 14-3-3
, whereas 14-3-3
in the absence of gelsolin, and this was not overcome by the
expression of YSK1
(Fig. 8 B). Therefore, 14-3-3
act downstream of both gelsolin and YSK1 in collagen inva-
sion, and be the key substrate mediating the effects of YSK1.
To analyze the effects of YSK1 on the polarization of
the Golgi apparatus and centrosome a second assay for cell
migration was used, in which confluent monolayers of
cells are wounded and then cells along the wound edge
microinjected with the constructs of interest (Etienne-
is the key
should oppose YSK1
triggered collagen invasion
, but not by other forms
YSK1 targets identifies 14-3-3?. (A) A
modified KESTREL approach was used to
identify substrates for YSK1 and MST4 as
described in the Materials and methods.
Phosphorylations with YSK1 and MST4
of fractions 13–21 from a fractionation
of HeLa S3 cell extract by gel filtration
on Superose-6 together with a Western
blot for 14-3-3? are shown. Fraction 17
is shown as a cut out to the right, with
the position of 14-3-3? indicated by a
closed arrowhead and the respective
kinases indicated by open arrowheads.
(B) Superose-6 column fraction 17
was treated with buffer, YSK1, or kinase-
dead YSK1K49R using the KESTREL method,
and analyzed by SDS-PAGE and auto-
radiography. Closed and open arrow-
heads indicate phosphorylation of 14-3-3?
and YSK1 autophosphorylation, respec-
tively. (C) Kinase assays were performed
for 60 min at 37?C in KESTREL assay buffer
with 2 ?g of recombinant His-tagged
14-3-3?, and 500 ng of preactivated
YSK1 or MST4. An autoradiograph of
14-3-3? phosphorylations by YSK1 and
MST4 is shown in the top panel, and
the corresponding region of a Coomassie
blue stained gel in the bottom panel.
Closed arrowheads indicate 14-3-3?.
(D) HS68 cells were costained with a
sheep antibody to GM130 and the mouse
mAb 22-II-D8 to 14-3-3?. Bar, 10 ?m.
A biochemical screen for
1016 The Journal of Cell Biology | Volume 164, Number 7, 2004
Manneville and Hall, 2001). Consistent with the collagen
invasion defect YSK1T174A expressing cells were unable to
migrate into the wound in this assay (Fig. 9 A). Further-
more, cells expressing YSK1T174A failed to show polariza-
tion of the Golgi apparatus and centrosome in the direc-
tion of migration toward the wound edge (Fig. 9 B).
Interestingly, YSK1T174A expression, although causing dis-
persal of the Golgi apparatus in some cells consistent with
the phenotype seen in HeLa cells (Fig. 6 B), resulted in
the displacement of essentially intact Golgi apparatus
away from the perinuclear region in others (Fig. 9 C, ar-
rowhead). Expression of wild-type YSK1 had little effect
on either cell migration or Golgi apparatus and cen-
trosome polarization (Fig. 9, A–D). Signaling via YSK1
appears to be required for cell migration and invasion
into collagen because dominant-negative YSK1T174A blocks
these processes. Intriguingly, the wild-type MST4 kinase
abrogated gelsolin-induced invasion into collagen, whereas ki-
nase-dead MST4D162A and dominant-negative MST4T178A
had no effect (Fig. 8 A), suggesting that MST4 opposes
the signaling pathway leading to invasion into collagen.
Therefore, YSK1 and MST4 may function in a signaling
pathway at the surface of Golgi membranes required for
cell migration and polarization.
MST4. (A) Cotransfection of YSK1T174A but not other forms of YSK1
abrogates gelsolin-induced invasion of collagen type I by HEK293T
cells. HEK293T cells were left untransfected, or cotransfected with
plasmids encoding gelsolin and constructs for wild-type YSK1 and
MST4, or point mutants of these kinases, as indicated in the figure
(n ? 3). (B) Effects of 14-3-3? phosphorylation mutants on invasion
into collagen type I. HEK293T cells were left untransfected, or
cotransfected with plasmids encoding wild-type and point mutant
forms of 14-3-3?, gelsolin, YSK1, YSK1K49R, and YSK1T174A, as indicated
in the figure (n ? 3). Collagen invasion assays were performed and
quantitated as described in the Materials and methods. The invasive
index is the percentage of cells invading the collagen gel over the
total number of cells.
Cell migration and invasion is modulated by YSK1 and
by YSK1T174A. (A and B) Cells were microinjected with plasmids
encoding myc-tagged YSK1 and YSK1T174A, fixed after 16 h, and stained
with antibodies to GM130 or c-Nap1, and 9E10 monoclonal to the
myc epitope. Bar graphs show the percentage of YSK1 or YSK1T174A
expressing cells migrating at the wound edge or into the wound
(n ? 12), and the percentage of cells with Golgi apparatus and centro-
somes (MTOC) polarized toward the wound edge (n ? 4). (C and D)
Images of cells expressing YSK1 or YSK1T174A (green) and stained for
GM130 or c-Nap1 (red) are shown. The position of the wound corre-
sponds to the bottom of the figure. Arrowheads indicate the position
of the Golgi apparatus and centrosome. Bars, 10 ?m.
Golgi apparatus and centrosome polarization is inhibited
GM130 functions as a kinase scaffold | Preisinger et al. 1017
GM130: a scaffold and activator for MST kinases
We have investigated the MST family of Ste20 kinases and
identified two members, YSK1 and MST4, as Golgi appara-
tus–associated kinases. These two kinases bind to the Golgi
matrix protein GM130 and thereby target to the Golgi ap-
paratus, whereas a third and the most closely related family
member MST3 is not associated with the Golgi apparatus
and fails to bind to GM130. A similar mechanism has been
observed for the MAPK ERK1 localized on the surface
of endosomes, which involves a small scaffolding protein
termed p14 (Wunderlich et al., 2001; Teis et al., 2002).
There is a second consequence of the interaction of YSK1
and MST4 with GM130. These kinases, when purified from
insect cells, show only low levels of activity toward the
model substrate MBP, and incubation with GM130 causes a
?25-fold increase in their activity. Analysis of activated
YSK1 and MST4 reveals that they become autophosphory-
lated on T-loop residue threonine 174 (T178 in MST4) as a
consequence of incubation with GM130, and that mutation
of this residue in YSK1 abolishes kinase activity. Supporting
evidence for the view that this is a key regulatory modifica-
tion comes from studies on MST1 that identified phosphor-
ylation of the equivalent residue in the T-loop as an activat-
ing event (Glantschnig et al., 2002). Other studies on
MST1 have shown that the region COOH-terminal to the
kinase domain also contributes to MST1 regulation, possi-
bly via an autoinhibitory mechanism because truncation or
cleavage of this domain during apoptosis results in kinase ac-
tivation (Creasy et al., 1996; Graves et al., 1998; Ura et al.,
2001). We have found that YSK1 and MST4 associate with
GM130 via the kinase domain and a small region immedi-
ately COOH-terminal to it (Fig. 4 A). Together, with the
fact that GM130 is a dimer, this suggests that two molecules
of YSK1 or MST4 may associate with a single molecule of
GM130 and autophosphorylate in trans, thus providing a
mechanism for the activation process and suggesting that
Golgi apparatus–associated pools of these kinases are active.
Functions for Golgi apparatus–associated
Directed cell motility is a highly complex process involving
many discrete events such as polarization of the cytoskele-
ton, signal transduction events, and regulation of cell adhe-
sion complexes (Ridley et al., 2003). In particular, cytoskele-
tal rearrangements and the secretion of proteins and lipids to
specific subdomains of the plasma membrane accompany
changes in cell polarity, and Golgi apparatus–localized ki-
nases such as YSK1 and MST4 are in an ideal location to
regulate these processes by modulating Golgi apparatus
function. The association with GM130, and high degree of
similarity between, YSK1 and MST4 might indicate that
they have similar functions and downstream effectors. How-
ever, this appears not to be the case. Dominant-negative
YSK1 but not MST4 causes dispersal of the Golgi apparatus
and blocks cell migration, whereas wild-type MST4 but not
YSK1 blocks cell migration without having any visible ef-
fects on the Golgi apparatus. One possibility is that compe-
tition for GM130 controls the relative levels of YSK1 and
MST4 activity, although how this would be achieved is un-
clear at present. Together with their differential effects on
cell migration they would therefore be predicted to act on
different downstream pathways. The identification of 14-3-
3? as a specific Golgi apparatus localized substrate for YSK1
but not MST4 supports this idea, and gives some clues as to
how YSK1 signaling may control Golgi apparatus function
and cell migration. The 14-3-3 proteins are dimeric adaptors
typically binding to phosphorylated acceptor sites on their
targets, thereby regulating a wide variety of cellular processes
(Tzivion et al., 2001). Furthermore, 14-3-3 proteins are
themselves regulated by phosphorylation and dimerization
(Tzivion et al., 2001). In the context of this work, three par-
ticular functions reported for these proteins may be of rele-
vance (depicted schematically in Fig. 10). First, 14-3-3?
binds to phosphorylated Raf in the Ras-signaling pathway
and stimulates its activity (Fantl et al., 1994; Freed et al.,
1994). Because a pool of activated Ras is generated at the
Golgi apparatus in response to growth factor receptor activa-
tion, it is possible that YSK1 via 14-3-3? can modulate this
pathway. Second, 14-3-3 proteins have been found associ-
ated with the cytoplasmic domains of specific integrin com-
plexes (Han et al., 2001; Bialkowska et al., 2003; Santoro et
al., 2003), and with Par3/Baz, one of a number of proteins
important for control of cell polarity and cell asymmetry
during development (Benton et al., 2002; Hurd et al.,
2003). Overexpression of 14-3-3 proteins blocks cell migra-
tion, and this could be exerted via integrins and their func-
tion in cell adhesion (Han et al., 2001; Santoro et al., 2003).
An obvious way for YSK1 to control cell migration would
therefore be via the 14-3-3?–dependent modulation of cell
adhesion. Finally, 14-3-3 proteins have been reported to act
in the quality control pathway regulating assembly and
transport of multimeric membrane protein complexes from
the ER to the Golgi apparatus (O’Kelly et al., 2002; Yuan et
al., 2003). This provides another point of control for YSK1
that may be relevant for regulation of Golgi apparatus func-
tion. Exactly which if any of these potential mechanisms is
relevant for YSK1 will require further characterization of 14-
3-3? binding partners.
cell migration? Together with the vesicle tethering factor p115,
GM130 is part of a landmark complex on the Golgi apparatus
important for protein transport and Golgi structure. Binding to
GM130 activates YSK1 and MST4. Activated YSK1 phosphorylates
14-3-3? and potentially other downstream targets needed for normal
cell migration, whereas MST4 acts via an uncharacterized pathway.
Known targets of 14-3-3? important for regulating cell migration
and polarity are depicted.
MST kinases: linking Golgi apparatus function with
1018 The Journal of Cell Biology | Volume 164, Number 7, 2004
Until recently, the range of signaling events occurring at
the Golgi apparatus was unsuspected, and now includes spe-
cific aspects of Ras and growth factor signaling (Chiu et al.,
2002; Bivona et al., 2003), apoptotic signaling (Lane et al.,
2002), and evidence that the Golgi apparatus acts as a sensor
controlling mitotic entry (Sutterlin et al., 2002). To these
we now add signaling events important for cell migration.
Materials and methods
Reagents and antibodies
Reagents were obtained from Sigma-Aldrich unless specified otherwise.
Chromatography reagents and ?-[32P]ATP (3,000 Ci/mmol and 10 mCi/ml)
were obtained from Amersham Biosciences. Antisera specific to YSK1 was
raised in rabbits 4256 and 4257 immunized with recombinant human
YSK1 by Biogenes, and then affinity purified over a column of YSK1 cou-
pled to Affigel-15 (Bio-Rad Laboratories). Peptide antibodies to YSK1 (SC-
6865, N19) and MST4 (3822) were purchased from Santa Cruz Biotech-
nology, Inc. and New England Biolabs, Inc., respectively. Mouse monoclo-
nal anti-LAMP1/CD107a was purchased from Becton Dickinson. Antibod-
ies specific to the phosphorylated YSK1/MST4 activation loop peptide
CIKRNpTFVGT were produced by Abcam Ltd., and then isolated from a
protein-A purified IgG fraction of the serum over the same peptide immo-
bilized on Sulfolink resin according to the manufacturer’s instructions
(Pierce Chemical Co.). E. Nigg (Max-Planck-Institute of Biochemistry) and
J. Celis (Institute of Cancer Biology, Copenhagen, Denmark) provided anti-
bodies to the centrosome marker c-Nap1 and 14-3-3?, respectively. Other
antibodies used in this experiment have been described and characterized
previously (Barr et al., 1997; Shorter et al., 1999; Short et al., 2001).
Molecular biology and two-hybrid screening
Full-length human YSK1, MST3, MST4, and 14-3-3? were amplified from
human testis cDNA (Becton Dickinson) using the pfu polymerase (Strat-
agene) and cloned in pCRII-TOPO (Invitrogen). Point mutants were con-
structed using the Quickchange mutagenesis protocol (Stratagene). DNA
oligonucleotides were purchased from Thermo-Hybaid and QIAGEN. All
constructs were confirmed by DNA sequencing (Medigenomix). Mamma-
lian expression constructs were made in pcDNA3.1? (Invitrogen) and
pEGFP-C2 (CLONTECH Laboratories, Inc.). For baculovirus expression,
pVL1393 or the pAcSG2 vector (Becton Dickinson) modified to include
the hexahistidine-tag from pQE32 (QIAGEN) was used. Baculoviruses
were produced and proteins expressed in Sf9 cells according the manufac-
turer’s protocols (Becton Dickinson). Bacterial expression was performed
using the His-GST expression vector pGAT2 and the His-tag expression
vector pQE32 (QIAGEN). Inserts encoding GM13075-271, golgin451-122, and
14-3-3? were inserted into pGAT2 or pQE32 and proteins expressed in
BL21(DE3) or JM109 cells, respectively. Proteins were purified over nickel-
NTA agarose (QIAGEN). Insect cell expressed kinases were desalted in
MEB (50 mM Tris-HCl, pH 7.3, 50 mM KCl, 10 mM MgCl2, 20 mM ?-glyc-
erophosphate, 15 mM EGTA), and other proteins were dialysed into PBS
and aliquots snap frozen in liquid nitrogen for storage at ?80?C. For two-
hybrid screening, a system described previously was used (James et al.,
1996). The entire coding region of the human YSK1 cDNA was inserted
into the two-hybrid bait vector pFBT9 (a version of pGBT9 [CLONTECH
Laboratories, Inc.] modified to encode kanamycin resistance), and this
plasmid transformed into the reporter strain PJ69-4A. A human testis
cDNA library (CLONTECH Laboratories, Inc.) was transformed into this
bait strain and plated on synthetic media lacking leucine, tryptophan, histi-
dine, and adenine with 2% (wt/vol) glucose as the carbon source (QDO).
Library plasmids were rescued using the ampicillin resistance marker and
retransformed into PJ69-4A together with either pFBT9 or the YSK1 bait
plasmid on synthetic medium lacking leucine and tryptophan (?LW), and
five independent colonies streaked onto QDO. Those showing strong
growth on QDO after 2 d at 30?C were taken as positive clones and the in-
serts were sequenced. Light colony color is indicative of a strong signal,
whereas dark colony color indicates a weaker signal.
GM130 binding assays
1 ?g of recombinant His-tagged YSK1 or MST4 expressed in baculovirus-
infected Sf9 cells was incubated with 5 ?g of either GST-tagged
GM13075-271, golgin451-122, or GST alone for 1 h at 4?C in HNTM buffer
(50 mM Hepes-KOH, pH 7.2, 200 mM NaCl, 0.5% [vol/vol] Triton
X-100, 5 mM MgCl2) in the presence of 15 ?l of glutathione-sepharose
and 100 ?M ATP in a total volume of 300 ?l. Beads were washed in 3?
1 ml HNTM and bound protein was eluted directly in SDS-PAGE sample
buffer. Samples were Western blotted and probed with either goat anti-
YSK1 N19 (Santa Cruz Biotechnology, Inc.) or affinity-purified rabbit
anti-YSK1/MST4. Loading controls were analyzed by SDS-PAGE and
Coomassie brilliant blue staining.
Kinase assays and substrate identification
For activation experiments with YSK1 or MST4, and comparisons of differ-
ent wild-type and mutant kinases, 0.8 pmoles of kinase in 7 ?l MEB? (MEB
containing 1 mM DTT and 2 mM ATP) were mixed with 5 ?l PBS contain-
ing the amount of His-tagged GM13075-271 or golgin451-122 indicated in the
figures and incubated for 30 min at 37?C. To this was added 8 ?l MEB con-
taining 1.5 ?g of MBP and 0.1 ?l ?-[32P]ATP. After incubation for 60 min
at 37?C, reactions were analyzed by SDS-PAGE and autoradiography. For
mass spectrometry, 40 pmoles of kinase in 20 ?l MEB? were either mock
activated or activated for 2 h at 37?C, 10 ?l of reducing sample buffer was
added and the reaction mixtures were heated for 5 min at 95?C followed
by SDS-PAGE. The corresponding bands were excised and processed for
mass spectrometry as described previously (Shevchenko et al., 1996).
Potential substrates for YSK1 and MST4 were screened for using a mod-
ified KESTREL protocol (Knebel et al., 2001). HeLa S3 cells were grown in
suspension using 1 liter of spinner flask at 37?C and 5% CO2 in DME con-
taining 10% FCS (Invitrogen). Cell pellets were lysed on ice for 30 min in
an equal volume of lysis buffer (40 mM Tris-HCl, pH 7.0, 1% [vol/vol] Tri-
ton X-100, 2.5 mM EDTA, 15 mM DTT) containing a protease inhibitor
cocktail (Roche Diagnostics). The lysate was centrifuged at 112,000 g for
30 min at 4?C. The clarified extract (typically 20 mg/ml) was aliquoted,
snap frozen in liquid nitrogen, and stored at ?80?C. Before further use ex-
tracts were thawed, desalted on Biogel P6-DG (Bio-Rad Laboratories) into
KESTREL buffer (40 mM Tris-HCl, pH 7.0, 0.1% [vol/vol] ?-mercaptoetha-
nol, 0.1 mM EGTA, 0.1% [vol/vol] Triton X-100), and incubated at 30?C
for 20 min to allow dephosphorylation. For the fractionation shown, 10 mg
of dephosphorylated cell extract was separated on a Superose-6 HR10/30
column equilibrated in KESTREL buffer containing 140 mM KCl. 1-ml frac-
tions were collected throughout and each one subjected to a kinase assay.
For kinase assays to identify substrate proteins, 10 ?l of the dephosphory-
lated extract or column fractions were adjusted to 10 mM MgCl2, 100 ?M
ATP, 5 ?Ci ?-[32P]ATP, 2 ?g YSK1 or MST4, and incubated at 37?C for 10
min. Assays were analyzed by SDS-PAGE and autoradiography. To identify
phosphorylated proteins the autoradiograph was overlaid on to a Coo-
massie blue stained gel and the corresponding region excised and pro-
cessed for mass spectrometry (Shevchenko et al., 1996).
Cell culture, RNA interference, and microscopy
HeLa cells were cultured at 37?C and 5% CO2 in DME containing 10%
FCS. Cells plated on glass coverslips were transfected 24–36 h after plating
using Fugene-6 (Roche), and left to grow for 18–24 h before fixation and
processing for immunofluorescence microscopy at RT. RNA interference
was performed on HeLa cells transfected using oligofectamine (Invitrogen)
with duplex RNA (Dharmacon Research Inc.) for 24 h, coverslips were
placed in fresh growth medium for a further 24–112 h, and then the cells
were processed for fluorescence microscopy (Elbashir et al., 2001). YSK1
and MST4 were targeted with the sequences AACACATTCGTGGGCAC-
CCCC and AATGGAATACCTGGGCGGTGG, GM130 with the sequence
AACCCTGAGACAACCACTTCT, and the lamin-A control was described
previously (Elbashir et al., 2001). Cells were fixed for 20 min in 3% (wt/
vol) PFA, quenched for 10 min with 50 mM ammonium chloride, and per-
meabilized with 0.1% (vol/vol) Triton X-100 for 5 min. All solutions were
made in PBS, and antibody staining was performed for 60 min using a
1,000-fold dilution of antiserum or purified antibody at a final concentra-
tion of 1 ?g/ml. Coverslips were mounted in 10% (wt/vol) Moviol 4-88, 1
?g/ml DAPI, 25% (wt/vol) glycerol in PBS. Images were collected using an
Axioskop-2 with a 63? Plan Apochromat oil immersion objective of NA
1.4, except for wounding assays, which were imaged with a 40? Plan
Neofluar objective of NA 0.75, standard filter sets (Carl Zeiss MicroImag-
ing, Inc.), a 1,300 by 1,030 pixel-cooled CCD camera (model CCD-1300-Y;
Princeton Instruments, Inc.) and Metavue software (Visitron Systems). Im-
ages were cropped in Adobe Photoshop 7.0, sized, and placed in figures
using Adobe Illustrator 10.0 (Adobe Systems Inc.).
Collagen invasion assays
HEK293T cells were grown in DME supplemented with 10% FBS, 2 mM
L-glutamine, 100 U/ml penicillin, and 100 ?g streptomycin (Invitrogen), and
transfected with calcium phosphate. Invasion into collagen type I was per-
formed as described previously (Braecke et al., 2001). Six-well plates were
GM130 functions as a kinase scaffold | Preisinger et al. 1019
filled with 1.25 ml of neutralized type I collagen (0.09% [wt/vol]; Upstate
Biotechnology Inc.) and incubated for at least 1 h at 37?C to allow gelifica-
tion. The cells were harvested using Moscona buffer and Trypsin/EDTA and
seeded on top of the collagen gel. The cultures were incubated for 24 h at
37?C. The depth of migration inside the gel was measured using a phase-
contrast microscope controlled by a computer program. Invasive and su-
perficial cells were counted in 12 fields of 0.157 mm2. The invasive index
is the percentage of cells invading the gel over the total number of cells.
HS68 cells were grown at 37?C and 5% CO2 in DME containing 10% FCS
on glass coverslips until a confluent monolayer was obtained, typically
3– 4 d after seeding. Wounds in the monolayers were created by scraping
a 200 ?l tip across the coverslips. Cells along the front of the wound edge
were injected (300 hPa injection pressure, 0.2 s, 60 hPa holding pressure)
with 200 ng/?l of plasmid DNA using a Femtojet microinjection system
(Eppendorf AG) mounted on an Axiovert 25 with 20? LD A-Plan objective
of NA 0.30 (Carl Zeiss MicroImaging, Inc.). After injection, the cells were
grown for 16 h under normal growth conditions and stained with the ap-
propriate antibodies. The orientations of the centrosome and Golgi appara-
tus were assessed according to a published method (Etienne-Manneville
and Hall, 2001).
Online supplemental material
Fig. S1 illustrates how YSK1/MST4 localize to the Golgi apparatus in HS68
cells. Fig. S2 shows a sequence comparison of YSK1 with MST3 and
MST4; conserved features and mutations are also marked. In Fig. S3, a
two-hybrid analysis reveals that YSK1 forms homodimers via the COOH
terminus but cannot heterodimerize with MST4. In Fig. S4, mass spectrom-
etry of activated and nonactivated YSK1 identifies the T-loop as one site of
autophosphorylation. Online supplemental material is available at http://
We thank Ulrike Grüneberg and Thomas Mayer for useful discussions and
comments on the manuscript.
The Max-Planck Society generously supports research in the group of
F.A. Barr. V. De Corte is a Postdoctoral Fellow of the Fund for Scientific
Research-Flanders (Belgium). J. Gettemans and V. De Corte greatly appre-
ciate support from Marc Mareel and Joël Vandekerckhove, and acknowl-
edge the support of the Belgian Federation against Cancer and Fortis Bank
Submitted: 14 October 2003
Accepted: 19 February 2004
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