1 Department of Molecular Genetics and Microbiology, University of Massachusetts
Medical School, Worcester, MA 01605, USA
2 Department of Biochemistry and Molecular Biology, University of Miami School
of Medicine, Miami, FL 33101, USA
* Author for correspondence (e-mail: dannel.mccollum{at}umassmed.edu)
Accepted 2 October 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cell polarity, Cell cycle, Orb6p, Mob1p, Schizosaccharomyces pombe
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The fission yeast Schizosaccharomyces pombe is an attractive model
organism for studying cell polarity (Chang,
2001; Mata and Nurse,
1998
; Nurse, 1994
;
Snell and Nurse, 1994
;
Verde, 1998
). Fission yeast
cells are cylindrical, grow by linear extension at their ends and divide by
medial fission. Moreover, they undergo morphological transitions that are
tightly coupled with progression through the cell cycle
(Mitchison and Nurse, 1985
;
Nurse, 1975
). During G1 and S
phases, cells grow only from the old cell end (the one that was present in the
previous cell cycle). Early in G2 phase, after completion of DNA replication
and attainment of a critical cell length, cells switch to bipolar growth,
growing from both the old end and the new end formed by the previous cell
division. This transition from unipolar to bipolar growth is known as
new end take off (NETO)
(Mitchison and Nurse, 1985
).
At the onset of mitosis, polarized cell growth ceases and is only
reestablished after cytokinesis. The sites of cell growth and division are
reflected by the distribution of the actin cytoskeleton. For example, actin
patches are always observed at the growing end(s) of the cell during
interphase (Marks et al.,
1986
; Marks and Hyams,
1985
). When cell elongation ceases at mitosis, actin disappears
from the cell ends and relocates to the division site.
Genetic studies show that the protein kinase Orb6p is important for
maintaining cell polarity and for coordinating cell morphogenesis with cell
cycle progression (Verde et al.,
1998). A number of Orb6p-related kinases have been identified in
other organisms, including Saccharomyces cerevisiae Cbk1p
(Bidlingmaier et al., 2001
;
Racki et al., 2000
),
Neurospora crassa Cot-1 (Yarden
et al., 1992
), Ustilago maydis Ukc1
(Durrenberger and Kronstad,
1999
), Caenorhabditis elegans LET-502 and SAX-1
(Wissmann et al., 1997
;
Zallen et al., 2000
) and
Drosophila Warts/Lats and Trc
(Geng et al., 2000
;
Justice et al., 1995
;
Xu et al., 1995
). Functional
studies of these kinases suggest that they are involved in regulation of cell
polarity.
By contrast, S. pombe Sid2p, another Orb6p-related kinase, is
required for cytokinesis (Sparks et al.,
1999), and its S. cerevisiae homolog Dbf2p is required
for the exit from mitosis and the onset of cytokinesis
(Frenz et al., 2000
;
Toyn and Johnston, 1994
). Both
Sid2p and Dbf2p associate with Mob1p (Hou
et al., 2000
; Komarnitsky et
al., 1998
; Salimova et al.,
2000
), a member of an emerging conserved protein family
(Hou et al., 2000
;
Luca and Winey, 1998
;
Salimova et al., 2000
). Mob1p
appears to be important for both the localization and the kinase activity of
both Sid2p (Hou et al., 2000
;
Salimova et al., 2000
)
(M.-C.H. and D.M., unpublished) and Dbf2p
(Frenz et al., 2000
;
Lee et al., 2001
;
Mah et al., 2001
). Both the
S. pombe and S. cerevisiae genomes contain a second
Mob1-related protein called Mob2p (Hou et
al., 2000
; Luca and Winey,
1998
; Salimova et al.,
2000
). Recent studies have shown that S. cerevisiae Mob2p
is not essential for growth but interacts with Cbk1p to promote polarized cell
growth and to induce asymmetric cell fate by activating daughter-specific gene
expression (Colman-Lerner et al.,
2001
; Weiss et al.,
2002
).
In this study, we report the characterization of fission yeast Mob2p. Mob2p is an essential protein that functions in polarized cell growth and in regulating the onset of mitosis. Mob2p associates directly with Orb6p. We propose that Mob2p and Orb6p form a kinase complex that coordinates polarized cell growth with cell cycle progression.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Construction of the mob2-null mutant
A strain containing the mob2-null mutation (mob2)
was constructed in diploid cells (YDM634) as previously described
(Bähler et al., 1998
) by
replacing one copy of mob2 with a fragment generated by PCR using
plasmid pKS-ura4 (pDM155) as a template. The two primers contained
the sequences corresponding to the regions immediately upstream and downstream
of the mob2 start and stop codons. The PCR fragment was gel-purified
and transformed into the diploid strain using the lithium acetate method. The
strain bearing the mob2 deletion (YDM 679) was identified by PCR. A
mob2
haploid strain bearing an episomal copy of mob2
(YDM1032) was obtained by plating a spore preparation from YDM679 containing
pREP3X-mob2 (see below) and selecting for Leu+
Ura+ haploid colonies.
Tagging of Mob2p and Orb6p
Mob2p was tagged at its C-terminus with GFP or 13 tandem copies of the Myc
epitope (13 Myc) by direct chromosomal integration into strain YDM105 of
fragments produced by PCR using plasmids pFA6a-GFP(S65T)-kanMX6 and
pFA6a-13Myc-kanMX6 as templates as previously described
(Bähler et al., 1998). The
resultant strains (YDM927 and YDM928) were confirmed by PCR. These strains
were then crossed with a variety of mutant strains, producing other strains
listed in Table 1.
To express an HA-tagged Orb6p from its own promoter, one promoterless copy of the orb6 gene, tagged at the C-terminus with three copies of the HA epitope (3HA) obtained by PCR (XhoI and EcoRI sites included in the primers), was inserted into an integrative plasmid containing the sup3-5 marker. The resultant plasmid was then transformed into ade6-704 leu1-32 ura4-D18 (PN567) cells and selected for colonies capable of growing in the absence of adenine. The correct integration was confirmed by PCR. Three strains (YDM1077) were used to test the expression of Orb6p-3HA by western blotting. In all cases, a single band of the right size was recognized by the mouse anti-HA antibody.
Construction of mob2- and orb6-containing
plasmids
To express mob2 under the control of the thiamine-repressible
nmt1 promoter, the multicopy plasmid pREP3X-mob2 was
constructed by cloning a 750 bp BamHI fragment containing the entire
mob2 cDNA obtained by PCR (BamHI sites included in the
primers) into the BamHI site of the vector pREP3X
(Maundrell, 1990).
pREP41MH-mob2 expresses mob2 from an attenuated
nmt1 promoter with an N-terminal tag consisting of six histidine
residues followed by two copies of the Myc epitope (MH); it was constructed by
cloning a 750 bp AseI-BamHI fragment containing the entire
mob2 cDNA obtained by PCR (AseI and BamHI sites
included in the primers) into the NdeI- and BamHI-cut vector
pREP41MH (Craven et al., 1998
).
Triple HA-tagged orb6 (pREP41HA-orb6) was constructed by
cloning a
1.4 kb NdeI-BamHI fragment containing the
entire orb6 cDNA (NdeI and BamHI sites included in
the primers) into the NdeI and BamHI-cut vector pREP41HA
(Craven et al., 1998
).
Two-hybrid screen for proteins interacting with Orb6p
S. cerevisiae strain Y190 (MATa gal4 gal80 his3 trp1-901
ade2-101 ura3-52 leu2-3-112, URA3::GALlacZ, LYS2::GAL(UAS)
HIS3
cyhr) (provided by S. Elledge, Baylor College of Medicine) was
used as the host for the two-hybrid interaction experiments. Strain Y187
(MAT
gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3-112
met-URA3::GAL
lacZ) was used for mating experiments
(Durfee et al., 1993
).
orb6 was fused to the DNA-binding domain of GAL4 in a
plasmid (pAS1) carrying the TRP1 marker (pAS1-orb6) and to
the GAL4 activation domain in a plasmid (pACT2) carrying the
LEU2 marker (pACT2-orb6). Plasmids pAS1, pACT2, pSE1111
(pACT2-SNF4), pSE1112 (pAS1-SNF1) were kindly provided by S.
Elledge. Cells were cultured in YEPD or selective SC at 30°C. The test for
cell growth on medium lacking histidine (and containing 100 mM 3-aminotriazole
(3-AT), the ß-galactosidase activation assay, and the mating test to
detect interaction specificity were performed as described previously
(Durfee et al., 1993
).
The S. pombe library prepared in phage vectors (a gift
from S. Elledge) was used in a two-hybrid screen to identify proteins that
interact with Orb6p. After screening 2x106 transformants, one
of the interactions was found to be Mob2p. The specificity of this interaction
was controlled by co-transformation of pAS1-orb6 and
pACT-mob2 independently with a number of control plasmids.
pAS1-orb6 was unable to activate the GAL4 promoter when
co-expressed with Snf4p (pACT2-SNF4), with Pak1p
(pACT2-pak1) fused to the GAL4 activation domain or with
Tea1p (pACT2-tea1) fused to the GAL4 activation domain (data
not shown). Similarly, pACT2-mob2 did not activate the GAL4
promoter when co-expressed with Snf1p (pAS1-SNF1), with Cdt1p
(pAS1-cdt1) fused to the GAL4 DNA-binding domain or with p53
protein (pAS1-p53) fused to the GAL4 DNA-binding domain (data not
shown).
Microscopy
Cells were prepared for immunofluorescence staining by methanol fixation as
described previously (Balasubramanian et
al., 1997). The actin antibody (Amersham; Arlington Heights, IL)
was used at a 1:200 dilution. The tubulin antibody TAT-1
(Woods et al., 1989
) was a
gift from K. Gull (University of Manchester, UK) and used at a 1:30 dilution.
Primary antibodies were detected with anti-mouse Alexa 488 or Alexa 594
(Molecular Probes; Eugene, OR) at a 1:200 dilution. Calcofluor staining was
performed as described previously
(Balasubramanian et al., 1997
).
Images were captured using a Nikon Eclipse E 600 microscope with a cooled CCD
camera (Orca-ER; Hamamatsu Photonics, Japan) and IPLab Spectrum software
(Scanalytics; Fairfax, VA).
Immunoprecipitation and western blotting
Cell pellets were obtained from 4-5x108 (20-25 total OD
units at 595 nm) exponentially growing cells, which were collected by
centrifugation and frozen at -80°C if not used immediately. All subsequent
manipulations were carried out at 4°C or on ice. Cells were lysed in NP-40
buffer {1% NP-40, 150 mM NaCl, 2 mM EDTA, 6 mM Na2HPO4, 4 mM NaH2PO4,
100µg/ml phenylmethylsulfonyl fluoride (PMSF), 80 mg/ml benzamidine, 1
mg/ml pepstatin, 10 mg/ml leupeptin, and 120 mg/ml
[4-(2-aminoethyl)benzenesulfonyl floride, HCl] (AEBSF, Hydrochloride)} by
votexing vigorously with acid-washed glass beads (Sigma; St Lious, MO) for 1
minute. Immunoprecipitations were performed by adding either 1 µl of
anti-Myc mouse monoclonal IgG (9E10, 5.5 mg/ml; a gift from K. Gould,
Vanderbilt University) or 1 µl of anti-HA mouse monoclonal IgG (12CA5,
5mg/ml; a gift from K. Gould, Vanderbilt University) to the NP-40 cell
lysates, followed by incubation on a rocker for 1 hour at 4°C. Immune
complexes were purified by adding 40 µl of a 1:1 slurry of protein
G-sepharose beads (Sigma), followed by incubation on a rocker for 1 hour at
4°C and centrifugation in a microfuge for 1 minute. The beads were washed
then three times with 1 ml of NP-40 buffer. For detection of Myc-tagged,
HA-tagged proteins and Cdc2p, the cell lysates or the immune complex beads
were subjected to SDS-PAGE (7.5%), transferred onto Immobilon P nylon
(Millipore; Bedford, MA) using a tank transfer system (BioRad; Hercules, CA).
The blotted membranes were probed with the anti-Myc antibody (1:2000
dilution), anti-HA antibody (1:1000 dilution) or Cdc2p mouse monclonal
antibody (anti-PSTAIRE, 1:5000 dilution; Sigma) and developed using an
alkaline phosphatase chemiluminescent system (BioRad).
In vitro binding assay
An MH-tagged mob2 cDNA was amplified from pREP41MH-mob2
(see above) by PCR using the 5' primer
5'-CTAGTAATACGACTCACTATAGGGCGTCGACCCCATGGGTAGCAGCCAC-3',
which contained the T7 promoter (underlined) and a sequence homologous to the
MH tag, and the 3' primer
5'-GGGGGGATCCTTAAATATTTCCTTGATTTTC-3', which contained sequence
homologous to the C-terminus of mob2. Similarly, an HA-tagged
orb6 cDNA was amplified from pREP41HA-orb6 by PCR using the
5' primer
5'-CCCTAATACGACTCACTATAGGGACTCGAGCCCATGGCATACCCTTAC-3'
and the 3' primer 5'-GCAGGATCCTTACAATGCTCCTTTCATCGTTAA-3',
which contained the orb6 stop codon. The PCR products were
gel-purified and concentrated to 30 µl with Qiagen columns, and 2 µl of
DNA was then used in a 25-µl coupled in vitro transcription/translation
reaction system according to the manufacturer's instruction (TNT®,
Promega; Madison, WI). Each reaction contained 12.5 µl of TNT® rabbit
reticulocyte lysate, 1 µl of TNT® reaction buffer, 0.5 µl of
TNT® T7 RNA polymerase, 0.5 µl of 1 mM amino acid mixture (minus
methionine), 1 µl of Redivue L-[35S]methionine (>1000 Ci/mmol
at 10 mCi/ml; Amersham Pharmacia Biotech), 0.5 µl (2 units) of RNasin
(Promega) and 7.0 µl of nuclease-free water. Luciferase DNA (TNT®,
Promega) was used as a control reaction. Reactions were incubated at 30°C
for 90 minutes. To examine the amounts of each protein in each reaction, 5
µl was taken and boiled for 2 minutes in 20 µl of 2x SDS sample
buffer, and the proteins were resolved by SDS-PAGE. For the in vitro binding
assay, the remaining 20 µl aliquots of each specific TNT reaction were
mixed and incubated at 30°C for another 30 minutes, and the mixtures were
then diluted to 500 µl with NP-40 buffer. The reaction mixtures were then
precleared with 40 µl of protein G-sepharose beads for 1 hour prior to
immunoprecipitation using anti-Myc mouse monoclonal IgG as described above and
analysis by SDS-PAGE. Gels were processed for fluorography using Amplify
(Amersham Pharmacia Biotech) according to the manufacturer's instructions.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To further analyze Mob2p function, the phenotype of a mob2
strain containing a mob2 shut-off plasmid (pREP3X-mob2) was
examined. In the absence of thiamine, cells were viable and showed a normal
cell morphology with normal organization of the actin and microtubule
cytoskeletons (Fig. 1B,C). By
contrast, changes in cell morphology and reorganization of the actin
cytoskeleton and microtubules were observed when mob2 expression was
repressed by thiamine addition. After 15 hours, cells became shorter at cell
division (Table 2) and
exhibited actin patches at only one end (80% versus 28% in a wild-type strain)
(Fig. 1D), but microtubule
organization appeared to be normal (Fig.
1E). After 22 hours, cells became spherical or oval
(Table 2), and the actin
cytoskeleton was dispersed around the cell cortex
(Fig. 1F) whereas microtubules
became depolarized with a criss-cross pattern
(Fig. 1G).
|
A possible explanation for the high frequency of cells with actin patches
at one end after 15 hours in thiamine is that Mob2p may have a role in the
activation of bipolar growth. To address this possibility, we measured new end
growth by examining the distance between the new end and the most recent birth
scar, the site where the previous cell division occurred. Only cells with
septa were analyzed to ensure that all cells examined were at the end of their
growth phase. 15 hours after thiamine addition, the new ends of 80% of
mob2 shut-off cells showed no growth
(Fig. 2Ab,d), whereas the new
ends of wild-type cells grew normally (Fig.
2Aa,c). Thus, a reduction in Mob2p protein level leads to a defect
in activation of new end growth. However, our results also showed that a
decrease in Mob2p level resulted in entry into mitosis at a reduced size
(Table 2). It is possible that
cells go through mitosis before bipolar growth is initiated, thereby leading
to accumulation of monopolar cells. If this is the case, then monopolar cells
would not be observed if they had sufficient G2 phase to grow. To test this
possibility, mob2 shut-off cells carrying the cdc25-22
mutation (YDM1456) were grown in the presence of thiamine at permissive
temperature (25°C) for 12 hours then arrested at G2 phase by shifting to
36°C for an additional 4 hours. These cells displayed
30% of total
cell population with monopolar growth (Fig.
2Ba,d). By contrast, both strain YDM1456 without thiamine addition
and a cdc25-22 single mutant strain YDM151 showed that
10% of
cells are monopolar and
90% are bipolar
(Fig. 2Ba-c). This result
showed that a decrease in Mob2p level results in a bipolar growth defect even
after a prolonged delay in G2. Interestingly, the cdc25-22 mob2
shut-off double mutant after elimination of mob2 expression did not
elongate to the same extent as the double mutant before elimination of
mob2 expression or the cdc25-22 single mutant, suggesting
that Mob2p is important for general polarized growth
(Fig. 2Bb,c,d).
|
Mob2p functions in regulation of the onset of mitosis
As noted above, reduced expression of mob2 caused cells to become
shorter at division, although the cell width was not significantly changed
(Table 2; 12-15 hours). By
contrast, mob2 overexpression in wild-type cells resulted in an
increase in cell length at division owing to a delay in G2 phase
(Table 3; data not shown).
These data suggested that Mob2p plays a role in regulating the onset of
mitosis. Control of entry into mitosis is regulated temporally by the timing
of activation of Cdc2p. The protein kinase Wee1p inhibits the activation of
Cdc2p and is a key regulator of cell size at mitosis. Thus, if Wee1p is
required for the delay of the onset of mitosis caused by mob2
overexpression, then a wee1 mutation should compromise this effect.
To test this possibility, mob2 was overexpressed in the
wee1-50 temperature-sensitive strain at the restrictive temperature.
In this case, mob2 overexpression failed to increase cell length at
division, and the cells were the same size as wee1-50 cells with a
control plasmid (Table 3). The
same result was also obtained in a wee1 deletion mutant
overexpressing mob2 (M.-C.H. and D.M., unpublished). Thus, the
regulation of the onset of mitosis by Mob2p may require Wee1p activity.
Interestingly, the same phenotype was observed upon overexpression of Orb6p
(Verde et al., 1998).
|
Localization of Mob2p to the cell periphery/cytoplasm and the
division site
To investigate the localization of Mob2p, we constructed a strain (YDM1203)
expressing a C-terminally GFP-tagged Mob2p fusion protein from its chromosomal
locus under the control of its own promoter (see Materials and Methods).
YDM1203 cells showed normal morphology and had no significant defect in growth
rate. Mob2p-GFP localization was observed in living cells, since in fixed
cells the GFP fluorescence of Mob2p-GFP was much fainter and the localization
not perfectly maintained (M.-C.H. and D.M., unpublished). During interphase,
Mob2p-GFP localized faintly at the cell periphery and the cytoplasm
(Fig. 3A, cells 1 and 2; also
see Fig. 4 and
Fig. 6, wild-type cells). The
signal at the cell periphery was slightly enriched at the cell tips but seemed
to be present all around the cell cortex. Some faint punctate signals were
observed in the cytoplasm as well as around the nucleus, particularly at high
temperatures, that seem to be caused by autofluorescence as it was observed in
cells not expressing Mob2p-GFP (M.-C.H. and D.M., unpublished). During septum
formation, Mob2p-GFP was clearly localized to the division site, where it
remained during septation and cytokinesis
(Fig. 3A, cells 3-5). Following
cell separation, Mob2p-GFP relocalized evenly at the cell periphery and the
cytoplasm (Fig. 3A, cell
6).
|
|
|
Because the Mob2p-GFP signal was more intense at the division site during cytokinesis than at the cell periphery and the cytoplasm during interphase, we examined whether Mob2p levels varied during the cell cycle. A strain (YDM933) that expressed a C-terminally 13 Myc-tagged Mob2p fusion protein from its chromosomal locus under the control of its own promoter (see Materials and Methods) was synchronized by cdc25-22 block and release, and Mob2p-13Myc levels were determined at 15 minute intervals. It appeared that Mob2p-13Myc levels did not change significantly as cells progressed through the cell cycle (Fig. 3B).
To further investigate whether the localization of Mob2p at the division
site requires the actomyosin ring, Mob2p-GFP localization was examined in
living temperature-sensitive mutant cells of cdc3 (encoding profilin)
(Balasubramanian et al., 1994)
and cdc8 (encoding tropomyosin)
(Balasubramanian et al., 1992
).
In these mutants, actomyosin rings are not formed and the septal material is
disorganized at the restrictive temperature. At the permissive temperature,
Mob2p-GFP localization in cdc3-124 (YDM1205) and cdc8-110
(YDM1206) cells appeared normal (Fig.
4A). However, after incubation at the restrictive temperature for
2.5 hours, when the majority of cells of both mutant strains had two separate
nuclei but no actomyosin ring or septa
(Balasubramanian et al., 1992
;
Balasubramanian et al., 1994
),
Mob2p-GFP did not localize to the cell middle in either mutant but localized
normally in wild-type cells (Fig.
4A, bottom panels), suggesting that Mob2p requires actomyosin ring
assembly to localize to the division site. However, it is likely that septum
formation affects Mob2p-GFP localization to the actomyosin ring. Mob2p-GFP
localization to the actomyosin ring was examined in living cdc7-24
and sid2-250 temperature-sensitive mutant cells. Both Cdc7p and Sid2p
are serine/threonine protein kinases and components of sepation initiation
network (SIN) (Fankhauser and Simanis,
1994
; Sparks et al.,
1999
). In these mutants, no septum formation occurs, and thus
cells become multinuclear at the restrictive temperature. At the permissive
temperature, Mob2p-GFP localization in cdc7-24 (YDM1252) and
sid2-250 (YDM1253) cells appeared normal
(Fig. 4B). However, after
incubation at the restrictive temperature for 2.5 hours, when the great
majority of cells of both mutant strains had two separate nuclei but no septa,
Mob2p-GFP did not localize to the cell middle in either mutant
(Fig. 4B) but localized
normally in wild-type cells (Fig.
4A).
Mob2p interacts with Orb6p kinase directly
Given that Orb6p is a Sid2p-related kinase and orb6 mutants have
the same phenotype as the mob2 mutant
(Verde et al., 1998
), it
seemed likely that Mob2p may interact with Orb6p. In fact, Mob2p was
identified independently in a two-hybrid screen for Orb6p-binding partners
(see Materials and Methods). Mob2p and Orb6p interacted specifically with each
other but not with negative controls (Fig.
5A). To further confirm the interaction between Mob2p and Orb6p in
vivo, co-immunoprecipitation was performed. Using a strain (YDM1151)
expressing chromosomal Mob2p-13Myc and Orb6p-3HA, we found that the two
proteins could be co-immunoprecipitated
(Fig. 5B). Mob2p and Orb6p were
also found to interact by a binding assay using in vitro translated proteins
(Fig. 5C), suggesting that
their interaction is direct. Because the Mob2p-related protein Mob1p interacts
with the Orb6p-related kinase Sid2p, we also tested for interaction between
Mob1p and Orb6p or between Mob2p and Sid2p by co-immunoprecipitation. No
interactions were detected between them (M.-C.H. and D.M., unpublished). These
results indicate that Mob2p interacts with Orb6p specifically in vivo.
|
Relationship between Mob2p and Orb6p localization
Since Mob2p associates with Orb6p, we tested whether Orb6p was important
for Mob2p localization. In an orb6-25 temperature-sensitive mutant
strain (YDM1204) at the permissive temperature, Mob2p-GFP was localized the
same as in wild-type cells (Fig.
6A). By contrast, at the restrictive temperature, Mob2p-GFP was no
longer observed at the cell cortex or the division site
(Fig. 6A), even though the
expression level of Mob2p remained unchanged
(Fig. 6B), indicating that the
absence of normal Mob2p-GFP localization is not due to degradation. Thus,
Orb6p appears to be required for proper Mob2p localization.
We then examined the localization Orb6p-3HA localization in a mob2 shut-off strain (YDM1080). In the absence of thiamine, Orb6p-3HA was localized to the cell tips and the medial region, but under repressing conditions, no signal was observed (data not shown). To explore why no Orb6p signal was observed, we examined the Orb6-3HA protein levels in the mob2 shut-off strain and found that a decrease in Mob2p protein levels resulted in a reduction in Orb6p-3HA protein levels (Fig. 6C). Thus, we could not determine if Mob2p is required for Orb6p localization, but it does seem that Mob2p is required for the stability of Orb6p-3HA.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mob2p localization to the division site appears to depend on actomyosin ring assembly. The question of how the actomyosin ring assembly promotes Mob2p localization to the division site remains to be answered. One possibility is that Mob2p is directly recruited by components of the actomyosin ring to the division site. However, Mob2p localization corresponds more to the septum and not the actomyosin ring; thus, the failure of Mob2p to localize in the actomyosin ring mutants may be due to an indirect effect of actomyosin ring failure on septum formation. Indeed, Mob2p is no longer localized to the medial region in SIN mutants, which make actomyosin rings, but fail to make septa.
Functional relationship of Mob2p and Orb6p
Our results show that Mob2p and Orb6p form a complex to regulate cell
polarity and cell cycle progression. Orb6p has been suggested to function
downstream of the Cdc42-activated Pak1p kinase
(Verde et al., 1998).
Overexpression of Orb6p partially suppressed the phenotype of pak1
temperature-sensitive mutants, and Pak1p-dependent establishment of cell
polarity is required for Orb6p to localize correctly. However, overexpression
of Mob2p cannot complement the phenotypes of pak1 and orb6
temperature-sensitive mutants (M.-C.H. and D.M., unpublished). One plausible
explanation is that Orb6p kinase activity is important for Mob2p function, and
overexpression of Mob2p is unable to bypass the requirement for Orb6p kinase.
Elucidation of how Orb6p kinase is regulated will be helpful to resolve this
question.
At present, nothing is known about whether Mob2p is important for Orb6p
kinase activity. Previous studies have shown that Mob1p associates with Dbf2p
and Sid2p in S. cerevisiae and S. pombe, respectively
(Hou et al., 2000;
Komarnitsky et al., 1998
;
Salimova et al., 2000
). In
both yeasts, Mob1p plays an essential role in kinase activity. In S.
cerevisiae, Dbf2p kinase activity is eliminated in mob1
temperature-sensitive mutants (Lee et al.,
2001
; Mah et al.,
2001
), and in S. pombe, Sid2p kinase activity is
abolished in mob1 temperature-sensitive mutants (M.-C.H. and D.M.,
unpublished). Thus, it is possible that Mob2p is essential for Orb6p kinase
activity. Unfortunately, we have been unable to detect strong Orb6p kinase
activity in vitro using artificial substrates, and thus were unable to assess
whether Mob2p is required for the Orb6p kinase activity. Resolution of this
issue may have to wait until in vivo substrates of Orb6p are identified.
Genetic studies suggest that Mob2p and Orb6p play a role in regulation of
the onset of mitosis, which may require the activity of Wee1p kinase, an
inhibitor of p34cdc2 mitotic kinase activity. Overexpression of
either Mob2p or Orb6p resulted in a delay in entry into mitosis, whereas a
decrease in their levels caused cells to divide at a reduced size
(Verde et al., 1998). In
addition, overexpression of Mob2p or Orb6p in wee1 mutants showed no
delay in the onset of mitosis (Verde et
al., 1998
). Thus, it is possible that Mob2p-Orb6p kinase complex
functions directly in activating Wee1p kinase or inactivating its
inhibitor(s), thereby activating Wee1p kinase. However, the onset of mitosis
is triggered by simultaneous activation of Cdc25 phosphatase and inactivation
of Wee1 kinase (Ohi and Gould,
1999
). Overexpression of Mob2p or Orb6p in cdc2 mutants
that are able to bypass the requirement for Cdc25 phosphatase exhibited a
delay in the onset in mitosis, but the effect was not as dramatic as that in
wild-type cells (M.-C.H., F.V. and D.M., unpublished). Thus, at this moment we
cannot exclusively rule out the possibility that Mob2p-Orb6p regulates the
onset of mitosis through inhibition of Cdc25p phosphatase activity.
Interestingly, it was recently found that the human tumor suppressor LATS1,
which shares sequence similarity with Orb6p, complexes with and inhibits Cdc2p
(Tao et al., 1999
) and causes
G2/M arrest when overexpressed (Xia et
al., 2002
; Yang et al.,
2001
). This raises an alternative possibility that Mob2p-Orb6p
complexes with and inactivates Cdc2p kinase directly. Further studies will be
needed to distinguish between these models.
Finally, we propose that the interaction between the Mob1p/Mob2p protein family and Sid2p/Orb6p protein kinase family are likely to be evolutionarily conserved. Along with the known Orb6p-related kinases (see Introduction), there are at least four Mob1p/Mob2p-related proteins in animal cells (M.-C.H. and D.M., unpublished), suggesting that the specific interaction between these two protein families may be evolutionarily conserved in higher eukaryotes. Thus, it will be quite interesting in future studies to determine if all Mob1p/Mob2p-related proteins function together with Sid2p/Orb6p family protein kinases.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bähler, J., Wu, J. Q., Longtine, M. S., Shah, N. G., McKenzie, A. R., Steever, A. B., Wach, A., Philippsen, P. and Pringle, J. R. (1998). Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14,943 -951.[CrossRef][Medline]
Balasubramanian, M. K., Helfman, D. M. and Hemmingsen, S. M. (1992). A new tropomyosin essential for cytokinesis in the fission yeast S. pombe. Nature 360, 84-87.[CrossRef][Medline]
Balasubramanian, M. K., Hirani, B. R., Burke, J. D. and Gould, K. L. (1994). The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis. J. Cell Biol. 125,1289 -1301.[Abstract]
Balasubramanian, M. K., McCollum, D. and Gould, K. L. (1997). Cytokinesis in fission yeast Schizosaccharomyces pombe. Methods Enzymol. 283,494 -506.[Medline]
Bidlingmaier, S., Weiss, E. L., Seidel, C., Drubin, D. G. and
Snyder, M. (2001). The Cbk1p pathway is important for
polarized cell growth and cell separation in Saccharomyces cerevisiae.Mol. Cell. Biol. 21,2449
-2462.
Chang, F. (2001). Establishment of a cellular axis in fission yeast. Trends Genet. 17,273 -278.[CrossRef][Medline]
Colman-Lerner, A., Chin, T. E. and Brent, R. (2001). Yeast Cbk1 and Mob2 activate daughter-specific genetic programs to induce asymmetric cell fates. Cell 107,739 -750.[Medline]
Craven, R. A., Griffiths, D. J., Sheldrick, K. S., Randall, R. E., Hagan, I. M. and Carr, A. M. (1998). Vectors for the expression of tagged proteins in Schizosaccharomyces pombe.Gene 221,59 -68.[CrossRef][Medline]
Drubin, D. G. and Nelson, W. J. (1996). Origins of cell polarity. Cell 84,335 -344.[Medline]
Durfee, T., Becherer, K., Chen, P. L., Yeh, S. H., Yang, Y., Kilburn, A. E., Lee, W. H. and Elledge, S. J. (1993). The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 7, 555-569.[Abstract]
Durrenberger, F. and Kronstad, J. (1999). The ukc1 gene encodes a protein kinase involved in morphogenesis, pathogenicity and pigment formation in Ustilago maydis. Mol. Gen. Genet. 261,281 -289.[CrossRef][Medline]
Fankhauser, C. and Simanis, V. (1994). The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast. EMBO J. 13,3011 -3019.[Abstract]
Frenz, L. M., Lee, S. E., Fesquet, D. and Johnston, L. H.
(2000). The budding yeast Dbf2 protein kinase localises to the
centrosome and moves to the bud neck in late mitosis. J. Cell
Sci. 113,3399
-3408.
Geng, W., He, B., Wang, M. and Adler, P. N.
(2000). The tricornered gene, which is required for the integrity
of epidermal cell extensions, encodes the Drosophila nuclear
DBF2-related kinase. Genetics
156,1817
-1828.
Hoffman, C. S. and Winston, F. (1987). A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57,267 -272.[CrossRef][Medline]
Hou, M. C., Salek, J. and McCollum, D. (2000). Mob1p interacts with the Sid2p kinase and is required for cytokinesis in fission yeast. Curr. Biol. 10,619 -622.[CrossRef][Medline]
Justice, R. W., Zilian, O., Woods, D. F., Noll, M. and Bryant, P. J. (1995). The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation. Genes Dev. 9,534 -546.[Abstract]
Keeney, J. B. and Boeke, J. D. (1994).
Efficient targeted integration at leul-32 and ura4-294 in
Schizosaccharomyces pombe. Genetics
136,849
-856.
Komarnitsky, S. I., Chiang, Y. C., Luca, F. C., Chen, J., Toyn,
J. H., Winey, M., Johnston, L. H. and Denis, C. L. (1998).
DBF2 protein kinase binds to and acts through the cell cycle-regulated MOB1
protein. Mol. Cell. Biol.
18,2100
-2107.
Lee, S. E., Frenz, L. M., Wells, N. J., Johnson, A. L. and Johnston, L. H. (2001). Order of function of the budding-yeast mitotic exit-network proteins Tem1, Cdc15, Mob1, Dbf2, and Cdc5. Curr. Biol. 11,784 -788.[CrossRef][Medline]
Leupold, U. (1970). Genetical methods for Schizosaccharomyces pombe. Methods Cell Physiol. 4, 169-177.
Luca, F. C. and Winey, M. (1998). MOB1, an
essential yeast gene required for completion of mitosis and maintenance of
ploidy. Mol. Biol. Cell
9, 29-46.
Mah, A. S., Jang, J. and Deshaies, R. J.
(2001). Protein kinase Cdc15 activates the Dbf2-Mob1 kinase
complex. Proc. Natl. Acad. Sci. USA
98,7325
-7330.
Marks, J. and Hyams, J. S. (1985). Localization of F-actin through the cell division cycle of Schizosaccharomyces pombe.Eur. J. Cell Biol. 39,27 -32.
Marks, J., Hagan, I. M. and Hyams, J. S. (1986). Growth polarity and cytokinesis in fission yeast: the role of the cytoskeleton. J. Cell Sci. Suppl. 5, 229-241.[Medline]
Mata, J. and Nurse, P. (1998). Discovering the poles in yeast. Trends Cell Biol. 8, 163-167.[CrossRef][Medline]
Maundrell, K. (1990). nmt1 of fission yeast. A
highly transcribed gene completely repressed by thiamine. J. Biol.
Chem. 265,10857
-10864.
Mitchison, J. M. and Nurse, P. (1985). Growth in cell length in the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 75,357 -376.[Abstract]
Moreno, S., Klar, A. and Nurse, P. (1991). Molecular genetic analysis of fission yeast, Schizosaccharomyces pombe.Methods Enzymol. 194,795 -823.[Medline]
Nurse, P. (1975). Genetic control of cell size at cell division in yeast. Nature 256,547 -551.[Medline]
Nurse, P. (1994). Fission yeast morphogenesis posing the problems. Mol. Biol. Cell 5, 613-616.[Medline]
Ohi, R. and Gould, K. L. (1999). Regulating the onset of mitosis. Curr. Opin. Cell Biol. 11,267 -273.[CrossRef][Medline]
Prentice, H. L. (1992). High efficiency transformation of Schizosaccharomyces pombe by electroporation. Nucleic Acids Res. 20,621 .[Medline]
Racki, W. J., Becam, A. M., Nasr, F. and Herbert, C. J.
(2000). Cbk1p, a protein similar to the human myotonic dystrophy
kinase, is essential for normal morphogenesis in Saccharomyces cerevisiae.EMBO J. 19,4524
-4532.
Salimova, E., Sohrmann, M., Fournier, N. and Simanis, V.
(2000). The S. pombe orthologue of the S.
cerevisiae mob1 gene is essential and functions in signalling the onset
of septum formation. J. Cell Sci.
113,1695
-1704.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Snell, V. and Nurse, P. (1994). Genetic analysis of cell morphogenesis in fission yeast a role for casein kinase II in the establishment of polarized growth. EMBO J. 13,2066 -2074.[Abstract]
Sparks, C. A., Morphew, M. and McCollum, D.
(1999). Sid2p, a spindle pole body kinase that regulates the
onset of cytokinesis. J. Cell Biol.
146,777
-790.
Tao, W., Zhang, S., Turenchalk, G. S., Stewart, R. A., St John, M. A., Chen, W. and Xu, T. (1999). Human homologue of the Drosophila melanogaster lats tumour suppressor modulates CDC2 activity. Nat. Genet. 21,177 -181.[CrossRef][Medline]
Toyn, J. H. and Johnston, L. H. (1994). The Dbf2 and Dbf20 protein kinases of budding yeast are activated after the metaphase to anaphase cell cycle transition. EMBO J. 13,1103 -1113.[Abstract]
Verde, F. (1998). On growth and form: control of cell morphogenesis in fission yeast. Curr. Opin. Microbiol. 1,712 -718.[CrossRef][Medline]
Verde, F., Wiley, D. J. and Nurse, P. (1998).
Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase
and myotonic dystrophy kinase, is required for maintenance of cell polarity
and coordinates cell morphogenesis with the cell cycle. Proc. Natl.
Acad. Sci. USA 95,7526
-7531.
Weiss, E. L., Kurischko, C., Zhang, C., Shokat, K., Drubin, D.
G. and Luca, F. C. (2002). The Saccharomyces
cerevisiae Mob2p-Cbk1p kinase complex promotes polarized growth and acts
with the mitotic exit network to facilitate daughter cell-specific
localization of Ace2p transcription factor. J. Cell
Biol. 158,885
-900.
Wissmann, A., Ingles, J., McGhee, J. D. and Mains, P. E. (1997). Caenorhabditis elegans LET-502 is related to Rho-binding kinases and human myotonic dystrophy kinase and interacts genetically with a homolog of the regulatory subunit of smooth muscle myosin phosphatase to affect cell shape. Genes Dev. 11,409 -422.[Abstract]
Woods, A., Sherwin, T., Sasse, R., MacRae, T. H., Baines, A. J. and Gull, K. (1989). Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J. Cell Sci. 93,491 -500.[Abstract]
Xia, H., Qi, H., Li, Y., Pei, J., Barton, J., Blackstad, M., Xu, T. and Tao, W. (2002). LATS1 tumor suppressor regulates G2/M transition and apoptosis. Oncogene 21,1233 -1241.[CrossRef][Medline]
Xu, T., Wang, W., Zhang, S., Stewart, R. A. and Yu, W.
(1995). Identifying tumor suppressors in genetic mosaics: the
Drosophila lats gene encodes a putative protein kinase.
Development 121,1053
-1063.
Yang, X., Li, D. M., Chen, W. and Xu, T. (2001). Human homologue of Drosophila lats, LATS1, negatively regulate growth by inducing G(2)/M arrest or apoptosis. Oncogene 20,6516 -6523.[CrossRef][Medline]
Yarden, O., Plamann, M., Ebbole, D. J. and Yanofsky, C. (1992). cot-1, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase. EMBO J. 11,2159 -2166.[Abstract]
Zallen, J. A., Peckol, E. L., Tobin, D. M. and Bargmann, C.
I. (2000). Neuronal cell shape and neurite initiation are
regulated by the Ndr kinase SAX-1, a member of the Orb6/COT-1/warts
serine/threonine kinase family. Mol. Biol. Cell
11,3177
-3190.
Related articles in JCS: