|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 15 August 2007
doi: 10.1242/dev.005181
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Molecular Microbiology and Immunology Department, University of Missouri,
Columbia, MO 65212, USA.
2 Molecular Biology Department, Nagoya University, Chikusa-ku, Nagoya 464-8602,
Japan.
Author for correspondence (e-mail:
bennettk{at}missouri.edu)
Accepted 2 June 2007
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Germline, MAPK docking site, Signalosome, Degradation, Phosphodegron, Vasa, Homeostasis
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Strome and Wood, 1983). The
four germline RNA helicases (GLHs) are constitutive P granule components
(Gruidl et al., 1996;
Kuznicki et al., 2000;
Roussell and Bennett, 1993).
GLH proteins are similar to Drosophila Vasa, a component of the polar
(germ) granules in flies (Hay et al.,
1988a; Hay et al.,
1988b; Lasko and Ashburner,
1988). Like Vasa, GLHs are putative DEAD-box RNA helicases, but
GLHs differ by the presence of multiple CCHC zinc fingers and all but GLH-3
have N-terminal, glycine-rich repeats. The loss of glh-1 by RNA
interference (RNAi) causes sterility in C. elegans when worms are
raised at the restrictive temperature of 26°C, whereas combinatorial RNAi
of glh-1 and glh-4 causes sterility even at the normally
permissive temperature of 20°C (whereas glh-2 and glh-3
have no apparent effects when knocked down by RNAi, nor as deletion mutants)
(Kuznicki et al., 2000)
(K.L.B., unpublished). glh-1/glh-4(RNAi) results in nematodes with
under-proliferated germlines that contain about one-third as many germ cells
as wild type, with no functional sperm or oocytes produced
(Kuznicki et al., 2000). A
yeast two-hybrid screen identified several GLH-interacting proteins
(Smith et al., 2002). In this
study, we report that two GLH binding partners, KGB-1 (kinase that GLHs bind)
and CSN-5 (COP9 signalosome subunit 5), associate with each other and are
likely to regulate GLH-1 levels.
The GLH binding partner KGB-1 is a MAP kinase of 390 amino acids and a
member of the Jun N-terminal kinase (JNK) subfamily
(Ip and Davis, 1998;
Robinson and Cobb, 1997).
KGB-1 has a novel, yet functional, activation site that consists of SDY rather
than TPY (Mizuno et al.,
2004); this activation site is only found in two other MAP
kinases, the C. briggsae KGB-1 homolog that is 88% identical to
KGB-1, and the C. elegans KGB-2 protein that matches KGB-1 with 86%
identity, but has no discernible phenotype when deleted
(Mizuno et al., 2004) (K.L.B.,
unpublished). Aside from this activation site, KGB-1 is very similar to the
Drosophila MAP kinase Basket (BSK) (53% identical and 67% similar)
and the human MAPK10 (52% identical and 68% similar)
(www.wormbase.org).
The mutant kgb-1(um3), which is missing more than 1.2 kb that
includes most of the kinase domain, exhibits a temperature-sensitive sterile
phenotype. At 26°C, kgb-1 worms have larger than normal gonads
containing many endomitotically replicating oocytes (EMO)
(Smith et al., 2002). As the
kgb-1(um3) mutant was used almost exclusively, when kgb-1 is
used in the text, it refers to the kgb-1(um3) allele.
The JNKs are involved in signaling cascades that regulate development in
many organisms, including Drosophila, in which JNK mutants have
disrupted embryonic dorsal closure and defects in apoptosis and wound healing
(Adachi-Yamada et al., 1999;
Bosch et al., 2005;
Igaki et al., 2002;
Jacinto et al., 2002;
Mattila et al., 2005). In
C. elegans, the JNK family members JNK-1 and KGB-1 are known to
respond to the stress of bacterial infections or exposure to heavy metals
(Huffman et al., 2004;
Koga et al., 2000;
Mizuno et al., 2004).
CSN-5 is the most highly conserved subunit of the COP9 signalosome, a
complex in plants and animals that regulates protein stability through SCF
ubiquitin ligases (for reviews, see
Chamovitz and Glickman, 2002;
Cope and Deshaies, 2003). CSN-5
can stabilize some targets and promote the degradation of others, functioning
alone or in the CSN complex. Proteins including p53 (the well-known tumor
suppressor), rat lutropin/choriogonadotropin, p27Kip1 (also known as Cdkn1b)
and the Arabidopsis auxin responsive proteins (IAAs) are degraded by
CSN-mediated ubiquitination (Bech-Otschir
et al., 2001; Li et al.,
2000; Schwechheimer et al.,
2001). By contrast, the transcriptional regulators c-Jun, Id1 and
Id3 are protected from degradation by their interactions with CSN. In C.
elegans, CSN-5 orchestrates the degradation of MEI-1, a protein that
functions after fertilization in the switch from meiosis to mitosis
(Pintard et al., 2003). CSN-5
also binds the GLHs, with loss of csn-5 mirroring
glh-1/glh-4(RNAi) (Kuznicki et
al., 2000; Smith et al.,
2002); these results are consistent with CSN-5 stabilizing GLH
proteins, as indicated for GLH-1 by this work.
In C. elegans, regulation of protein degradation has been
elegantly examined in relation to embryonic patterning and the transition from
oocyte to embryo (DeRenzo et al.,
2003; Reese et al.,
2000; Nishi and Lin,
2005; Pellettieri et al.,
2003; Shirayama et al.,
2006; Stitzel et al.,
2006). As GLH-1, GLH-4, KGB-1 and CSN-5 are each important for
adult fertility, these studies focused on the adult germline. The C.
elegans germline develops from the single germline precursor P4 cell that
divides once in the embryo to produce the germline progenitor cells Z2 and Z3,
which arrest until during the second larval stage (L2). The rate of germline
mitosis increases in the third larval stage (L3), and spermatogenesis
initiates and completes in the fourth larval stage (L4), with sperm then
stored in the spermatheca, awaiting the production of oocytes. After the
L4/adult molt, the syncytial germ cells switch to oogenesis. With the
completion of the pachytene stage of meiosis, oocytes cellularize and mature
in a single-file, assembly-line fashion. After maturation, individual oocytes
pass through the spermatheca and are fertilized, with the resulting embryos
retained by the mother while they undergo several cell divisions. Thus, the
bi-lobed gonad of the C. elegans adult contains a continuum - from
germ cell nuclei in mitosis and meiosis to mature sperm, oocytes and
developing embryos (see Fig.
2A). C. elegans become young adults within 12 hours of
the L4 stage and produce most of their 
300 progeny in the next 3 days,
with all aspects of development occurring more rapidly at 26°C than at
20°C (Epstein and Shakes,
1995).
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
-tubulin (Santa Cruz) or anti-ß-tubulin antibodies (Sigma)
were diluted 1:1000 and 1:10,000, respectively. Goat anti-rabbit or goat
anti-mouse secondary antibodies conjugated to horseradish peroxidase (ICN)
were used at 1:10,000. The C-terminal rabbit C2-2 anti-KGB-1 antibody
(Mizuno et al., 2004) was used
at 1:500. Western blots were developed using Supersignal West Pico or Dura
chemiluminescence (Pierce) and exposed to film or phosphorimaging plates.
Films were processed using Adobe Photoshop. A BioRad FX phosphorimager and
Quantity One software were used for quantification.
Northern blot analysis
Total RNA was isolated from adult N2 and kgb-1 animals grown at
either 20°C or 26°C from the L1 stage. RNA was extracted using Trizol
(Invitrogen) as described (Reinke et al.,
2000). RNA samples were run on MOPS-acrylamide gels and
transferred to nylon membranes. Probes specific to glh-1, glh-4,
kgb-1 or to eIF-4A (Roussell
and Bennett, 1993) were generated using the Prime-It II Kit
(Stratagene).
Immunocytochemistry
N2 and kgb-1(um3) or kgb-1(km21)
(Mizuno et al., 2004) animals
were grown at 20°C or 26°C; L4 worms were isolated and adults analyzed
as described (Kuznicki et al.,
2000). For GLH-1 and KGB-1 localizations, gonads were fixed for 1
hour in 3% formaldehyde/0.1 M K2HPO4 pH 7.2 at room
temperature, followed by 5 minutes in methanol at -20°C
(Jones et al., 1996). For
PGL-1, slides were fixed in methanol, then in acetone, both for 15 minutes at
-20°C (Kawasaki et al.,
1998). Anti-PGL-1 antibody was used at 1:3000, with anti-GLH-1 at
1:500-1:2000. The anti-KGB-1 antibodies used for immunocytochemistry were
generated in mice with the N-terminal KGB-1-specific peptide,
MEVDLPVHNEYDASRFHQVT, conjugated to KLH (keyhole limpet hemocyanin). Combined
sera from two positive mice were affinity purified with high salt (5 M KI)
(Olmstead, 1986); preimmune
and hyperimmune sera were diluted 1:2. Fluorescent secondary antibodies (Alexa
Fluor, Molecular Probes) were diluted 1:3000. Images were recorded with a Spot
CCD camera on a Zeiss Axioplan microscope. When comparing images, the same
exposure was used.
GST pull-down experiments
GST pull-downs were performed with baculovirus-expressed proteins in
HighFive insect cells (Invitrogen) as described
(Smith et al., 2002). Proteins
were tagged with GST or 6-His and cloned into pFastBac (Invitrogen).
Constructs were sequenced to verify that intact proteins would be produced.
For protein homogenates, N2 and kgb-1(um3) worms were grown in liquid
culture (Epstein and Shakes,
1995) to the adult stage, washed, frozen in liquid nitrogen,
thawed in PBS and then lysed by three passages through a French pressure-cell
under 900 psi. Lysates were centrifuged for 10 minutes at 10,000
g, removing insoluble materials. For experiments using
expressed proteins combined with C. elegans lysates, 1 mL insect-cell
lysate mixed with 3 mL worm lysate was incubated at 4°C for 2 hours with
500 µL 1:1 (v/v) Glutathione-Uniflow Resin:PBS (BD Biosciences Clonetech).
Beads were washed four times in 1 mL PBS containing 1% Triton X-100 and
proteins eluted and analyzed as described above.
Site-directed mutagenesis
Primers deleting the GLH-1 D site, or changing the lysine at 581 to
asparagine, and then changing the leucines at positions 588 or 589 to
tryptophan were generated using directions from the Quick Change II
Site-Directed Mutagenesis manual (Stratagene). PCR for site-directed
mutagenesis, following the manufacturer's protocol (Stratagene), was performed
on GLH-1 in the pFastBac plasmid, and clones were sequenced to verify the
mutations.
Kinase assays
Immunoprecipitations and kinase assays were performed as previously
described, using GST-tagged GLH-1 (Mizuno
et al., 2004).
Proteasome and JNK inhibitor experiments
HighFive cells (Invitrogen) were grown and treated as in GST pull-down
analysis above, except that cells were grown for 18 hours at 26°C after
infection and then treated (or not) with 1 µM MG132 (Calbiochem) for 5
hours, before harvesting. Protein homogenates were analyzed by western blot to
examine GLH-1-6-His levels using anti-His antibody (Santa Cruz Biotech)
diluted 1:2000. For the in vivo tests, young N2 adults were grown in standard
liquid culture at 20°C with either 1 µM MG132, 50 µM JNK inhibitor
(SP600125, CalBiochem) or no inhibitor added. Inhibitor was added for varying
amounts of time, but all worms were grown for a total of 6 hours and analyzed
for GLH-1 by western blot (50 worms/lane), using -tubulin (Sigma) at
1:5000 as the loading control. After Dura chemiluminescence (Pierce),
quantification employed Multigauge software (Fuji)
RNA interference
RNAi was performed for glh-1 and csn-5 as described
(Kuznicki et al., 2000;
Smith et al., 2002). For
actin, a 446 nt dsRNA that has one mismatch/primer for each of the
five C. elegans actin genes (act-1-5) was produced using
primers from Gao et al. (Gao et al.,
2006). For pan-1 (cosmid M88.6), primers
5'-GTAATACGACTCACTATAGGGCGCGAAGCTTAC-3' and
5'-GTAATACGACTCACTATAGGGCCTGGAATCGTACAG-3' were used that contain
T7 RNA polymerase sites and produce a 1742 nt full-length pan-1
product (G. Gao and K.L.B., unpublished). All dsRNAs were injected at 1
mg/mL. Fertile kgb-1; csn-5(RNAi) F1 animals, raised at
20°C, were only counted when the mother also produced sterile
F1 siblings.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
To investigate whether the increase in GLH levels observed in kgb-1 worms was due to an increase in glh transcript accumulation, total RNA from N2 and kgb-1 adults was analyzed. Little or no differences in glh-4, glh-1 or kgb-1 transcript levels were found when N2 and kgb-1 RNAs were compared, with the exception that no kgb-1 transcript was detected in the kgb-1 mutant (Fig. 1E). Therefore, the increase in GLH-1 observed in kgb-1 worms appears to be due to regulation by the KGB-1 kinase of GLH-1 protein, and not to an effect of KGB-1 on the production or stability of glh-1 or glh-4 mRNA.
kgb-1 mutants have abnormal P granules and extra germ cells
Results from immunocytochemistry of N2 and kgb-1 mutants support
the increased GLH-1 levels seen by western analysis. When wild-type and
kgb-1 worms of the same age were compared, fluorescence corresponding
to GLH-1 in the adult germline was much brighter in kgb-1 worms than
in their wild-type counterparts (Fig.
2, E versus D). In wild-type worms, GLH-1 was present in punctate
P granules, with each germ cell nucleus surrounded by multiple P granule
clusters (Fig. 2D,F). By
contrast, kgb-1 mutants were found to have greatly increased staining
for GLH-1, and instead of being localized in discrete P granules, GLH-1
protein was disorganized, completely surrounding the germ cell nuclei
(Fig. 2E,G). Also,
kgb-1 gonads were somewhat bigger than N2 gonads
(Fig. 2, C versus B) and
sterile kgb-1 hermaphrodites had 22% more germ cell nuclei than wild
type, with an average of 510 (n=6) nuclei per kgb-1 gonad
arm, compared with 419 nuclei per N2 gonad arm (n=10), with both
strains grown under the same conditions. This increase is statistically
significant (P<0.04). Although extra germ cells could result from
a back-up of healthy oocytes due to defective sperm, this is unlikely to be
the case in kgb-1 worms because mating with N2 males does not rescue
the kgb-1 sterility, as it does for the fem-1(hc17) strain
(Nelson et al., 1978;
Smith et al., 2002). In
addition, both the masculinized fem-3(q24)(gf) and the feminized
fem-1(hc17)(lf) strains have higher glh-1 mRNA levels than
wild-type worms (Roussell and Bennett,
1993); therefore, it is unlikely that the kgb-1 defect is
similar to the defects in these other strains. Thus, the kgb-1(um3)
mutation, although it may affect sperm, is most apparent in germline
proliferation and oogenesis.
PGL-1, also a constitutive component of P granules and one that depends
upon GLH-1 for its localization (Kawasaki
et al., 1998), was also elevated in kgb-1 animals (see
Fig. S1, C versus A, in the supplementary material).
|
; McCarter et al.,
1999; Pitt et al.,
2000; Strome and Wood,
1982). We found that KGB-1 is uniformly cytoplasmic in the distal
gonad, overlapping in location with GLH-1
(Fig. 3C-E) and, like GLH-1,
disperses into particles when oocytes cellularize
(Fig. 3C and see Fig. S2 in the
supplementary material); however, the GLH-1 and KGB-1 puncta were often
distinct from one another (Fig.
3D,E). Therefore, KGB-1 and GLH-1 could interact during
proliferation and meiosis and dissociate after GLH-1 leaves its peri-nuclear
location. Interestingly, in maturing oocytes, the GLH binding partner CSN-5
remains uniformly cytoplasmic and is also concentrated in a subnuclear
location, perhaps no longer bound to GLH-1
(Fig. 6C)
(Smith et al., 2002). In
contrast to KGB-1 particles, which were found in multiple oocytes
(Fig. 3C and see Fig. S2 in the
supplementary material), the activated form of the well-studied MAP kinase,
MPK-1, is most visible in the most proximal oocyte. MPK-1, like KGB-1, is
required for progression out of pachytene and additionally controls meiotic
oocyte maturation (Church et al.,
1995; Miller et al.,
2001; Page et al.,
2001; Smith et al.,
2002). However, the commercial phospho-specific MPK antibodies
(Sigma) only detect active MPK, whereas the anti-KGB-1 antibodies are likely
to detect all forms of KGB-1; therefore, it remains to be determined whether
the activated forms of these two MAPKs have similar localization patterns. In
summary, KGB-1 and GLH-1 overlap in the cytoplasm of the distal germline and
resolve into discrete particles in developing oocytes. This pattern is likely
to reflect a very dynamic relationship between GLH-1 and KGB-1. Perhaps
phosphorylation by KGB-1 signals GLH-1 to leave the nuclear membrane and be
targeted for degradation.
|
Taken together, our various in vivo studies suggest an essential interaction between KGB-1 and GLH-1. When the kinase function of KGB-1 is missing, GLH-1 protein levels increase, P granule structure appears disrupted, oogenesis is defective and the numbers of germ cell nuclei increase. Along with these relationships revealed by the kgb-1(um3) mutant, in wild-type worms KGB-1 and GLH-1 resolve into discrete sets of particles as P granules leave the nuclear membrane during oogenesis. Therefore, KGB-1, a GLH-1 binding partner, controls GLH-1 protein levels and coordinates the organization of GLH-1 in P granules.
Biochemical interactions between KGB-1 and GLH-1
In considering how KGB-1 might regulate GLH-1, we compared the predicted
protein sequences of GLH-1 and GLH-4 and found that GLH-1 contains a consensus
MAPK D (docking) site. This site is not completely conserved in GLH-4 (one
mismatch in each of two possible motifs,
Table 1). MAP kinases bind
their target substrates via D sites, characterized by an LxL motif 1-5 amino
acids downstream from several basic amino acids
(Jacobs et al., 1999)
(reviewed by Sharrocks et al.,
2000). GLH-1 also contains a consensus phosphodegron, an
additional motif used by some MAP kinases to phosphorylate their substrates
and target them for degradation. Again, neither of two potential GLH-4
phosphodegron motifs completely corresponds to consensus
(Table 1). Phosphorylation at
phosphodegron sites is responsible for the timely and rapid degradation of the
cell cycle proteins mammalian cyclin E and the yeast CDK (cyclin-dependent
kinase) inhibitor Sic1 (Orlicky et al.,
2003; Ye et al.,
2004). D sites and phosphodegrons allow for coordinated protein
recognition and degradation in response to a wide range of stimuli and
stresses. Therefore, these motifs could be important if GLH-1 levels are
controlled by KGB-1 phosphorylation that leads to degradation.
|
). We expanded upon these pull-downs by testing several
truncations of GLH-1 lacking the MAPK D site and/or the phosphodegron motif.
As summarized in Fig. 4B,
full-length GLH-1 bound KGB-1, as did an N-terminal truncation retaining the
phosphodegron and the D site, whereas a C-terminal GLH-1 truncation,
eliminating both the phosphodegron and the MAPK D site, did not bind, nor did
a truncated protein in which the D site was missing but the putative
phosphodegron remained. Next, we used site-directed mutagenesis to construct a
deletion of the D site, as well as two point-mutated sites. As judged by GST
pull-downs, neither of the point-mutated sites completely abrogated KGB-1
binding, although the K581N/L588W mutation
(Fig. 4C, lane 2) bound less
well than the wild-type protein and, perhaps surprisingly, the K581N/L589W
mutant construct (Fig. 4C, lane
1) consistently bound with higher affinity, suggesting that sequences around
the consensus might also affect binding. As predicted, the D site deletion
essentially eliminated KGB-1 binding (Fig.
4C, lane 3). Thus, these results point to the importance of the
MAPK D site in the binding of KGB-1 to GLH-1, but do not rule out possible
involvement of other interaction sites.
|
);
however, the C. elegans genome does not encode a recognizable c-Jun
homolog. To test whether KGB-1 can phosphorylate GLH-1, recombinant GST-tagged
GLH-1 was used in kinase assays with immunoprecipitated HA-tagged KGB-1 grown
in HEK293 cells that had been co-transfected with Flag-tagged MEK-1. After
incubation with [
-32P]ATP, the proteins were assayed by
autoradiography. On its own, KGB-1 was able to phosphorylate c-Jun and GLH-1,
albeit at low levels (Fig. 5A,
lane 1); however, when MEK-1 was added, phosphorylation of c-Jun and GLH-1
dramatically increased (Fig.
5A, lane 2) and when the ATP-binding site of KGB-1 was mutated
(K67N), KGB-1 was unable to phosphorylate either protein
(Fig. 5A, lane 3). Since KGB-1
can phosphorylate GLH-1, GLH-1 might be a KGB-1 substrate. To determine whether KGB-1 regulates GLH-1 in a proteosome-dependent manner, GLH-1 was expressed in insect cells alone, with GST (another negative control), or with CSN-5 or KGB-1. Cells were infected with recombinant baculovirus, grown for 18 hours, and then treated with the proteasome inhibitor MG132 for 5 hours; GLH-1 levels were then examined (Fig. 5B). When expressed alone, or with either GST or CSN-5, GLH-1 accumulated to high levels, both with and without MG132 (Fig. 5B, lanes 1-4 and 7-8). By contrast, co-expression of GLH-1 with KGB-1 resulted in greatly reduced GLH-1 levels (Fig. 5B, lane 6); however, when the proteasome was blocked, GLH-1 levels increased during the last 5 hours of infection (Fig. 5B, lane 5). These results suggest that KGB-1 targets GLH-1 for degradation by the proteasome.
To assess whether either MG132 or the JNK inhibitor SP600125 could be used in live worms, we incubated wild-type C. elegans with these chemicals at concentrations shown to be effective in vitro and then tested these worms for changes in GLH-1 levels. When live worms were exposed to MG132 for 3 hours, the GLH-1 levels increased over 3-fold, with an average increase of 3.4-fold in three trials (Fig. 5C). We also chose to test the effect of the JNK inhibitor SP600125, as it would be likely to be more specific and potentially less toxic to worms than MG132. After 3 hours incubation with SP600125, GLH-1 levels increased more than 4-fold beyond those of untreated controls, with an average increase of 4.6 in three independent experiments (Fig. 5D), indicating that blocking either the proteosome or the specific JNK activity of KGB-1 (and that of the other two C. elegans JNK proteins, JNK-1 and KGB-2) results in a relatively rapid accumulation of GLH-1. Therefore, the regulation of GLH-1 seen with the kgb-1 mutant can be mimicked with inhibitors, both in vitro and in vivo.
|
|
) (G. Gao and
K.L.B., unpublished) were also tested in the kgb-1 background; no
significant differences between N2s and kgb-1 were found for these
RNAs (Table 3). Thus,
kgb-1 is not defective in RNAi and whereas fertility is only
partially restored by glh-1(RNAi) into kgb-1
mutants (Table 2), the removal
of excess GLH-1 results in significantly more fertile animals
(P<0.05). Therefore, by reducing GLH-1 levels in kgb-1
worms, some mutant animals are rescued, perhaps indicating that elevated GLH-1
levels are detrimental to reproduction. The lack of complete rescue might
reflect the variable amount of GLH-1 removed by this technique; alternatively,
it could also imply that KGB-1 has other, as yet undetermined targets in the
C. elegans germline.
|
|
CSN-5 also regulates GLH-1 levels
CSN-5 and the COP9 signalosome complex associate with several kinases. In
mammals, protein kinase CK2 (also known as PCK2 - Human Gene Nomenclature
Database) and PKD (also known as PRKD1) and inositol 1,3,4-triphosphate 5/6
kinase associate with the CSN complex, whereas the C. elegans
interactome project reported that the MAP kinases MPK-1, PMK-1 and PMK-2 bind
CSN-5 (Li et al., 2004;
Sun et al., 2002;
Uhle et al., 2003). To
determine whether KGB-1 associates with CSN-5, we again used GST pull-down
analyses and found that KGB-1 binds CSN-5
(Fig. 6A, lane 2), the first
JNK reported to do so. This interaction is robust, withstanding high salt (300
mM LiCl) stringency (data not shown). CSN-5, like GLH-1, has a consensus D
site (Table 1), perhaps
facilitating the binding of KGB-1 to CSN-5.
Since CSN-5 and KGB-1 bind one another and bind GLH-1 in vitro and in vivo
(this report) (Smith et al.,
2002), we investigated whether loss of CSN-5 would affect KGB-1
function. After csn-5(RNAi) into N2, only 25% of the injected worms
produced any fertile F1 progeny; by contrast, csn-5(RNAi)
into kgb-1 resulted in 63% of the kgb-1 worms producing a
few fertile F1 progeny (Table
3), a highly significant difference
(P<1x105). Therefore, fertility improves in
kgb-1 worms when csn-5 is also missing (or reduced),
implying a functional interaction between KGB-1 and CSN-5 in C.
elegans.
To test whether the rescue seen for the fertile kgb-1; csn-5(RNAi) worms (Table 3) is due to an equilibration of GLH-1 levels, in effect balancing the roles of two proteins with opposing actions, we compared GLH-1 levels in kgb-1; csn-5(RNAi) fertile animals with uninjected N2s and uninjected kgb-1s (Fig. 6B). We found that the fertile kgb-1; csn-5(RNAi) worms had GLH-1 levels that were, on average, 2.8-fold lower than their uninjected kgb-1 siblings (lane 3 versus lane 2) and these levels were closer to those of aged-matched wild-type animals (lane 1). The results presented in Fig. 6B suggest that KGB-1 and CSN-5 work in opposition to control GLH-1 levels. Perhaps, when KGB-1 is not present to target GLH proteins for degradation, the protective role of CSN-5 might be less necessary.
Vasa interacts with KGB-1 and CSN-5
In searching for D sites and phosphodegrons, we discovered that Vasa, the
well-studied Drosophila polar granule component, also contains
consensus sites for each (Table
1). In fact, GLH-1/Vasa orthologs in many species contain docking
sites and phosphodegrons (not shown). Therefore, we tested whether Vasa might
interact with KGB-1 or CSN-5 when co-expressed in insect cells. We cloned
vas into the pFastBac vector, adding either a 6-His tag or a GST tag
to the full-length vas cDNA (Hay
et al., 1988a). The His-tagged Vasa construct was co-expressed
with KGB-1 or with CSN-5, both tagged with GST. Whereas the Vasa-GST did not
pull down eGFP (Fig. 6C, lane
1), GST-tagged KGB-1 and CSN-5 pulled down Vasa 6-His
(Fig. 6C, lanes 2 and 3). Thus,
Caenorhabditis proteins KGB-1 and CSN-5 recognize and bind to the
Drosophila Vasa germ granule protein.
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Why GLH-1 levels might be important for germline homeostasis
Homeostasis in the germline has been very convincingly shown to balance
numbers of primordial germ cells (PGCs) with those of intermingled somatic
cells in the Drosophila ovary
(Gilboa and Lehmann, 2006). In
C. elegans, we report a homeostatic regulation of GLH-1 that appears
necessary for reproductive success. Regulation of GLH-1 by KGB-1 could ensure
continued fertility by removing excess GLH-1 protein as C. elegans
become post-reproductive; in addition, KGB-1 may remove excess GLH-1 from
those germ cells undergoing apoptosis during pachytene
(Gumienny et al., 1999).
Phosphorylation of GLH-1 by KGB-1 could be the signaling event that removes
GLH-1 (and the P granules) from their peri-nuclear location. There are
precedents for a protein both changing the location of its target and being
involved in its degradation; for example, several of us have recently reported
that C. elegans RLE-1, an E3 ligase, has such an effect on DAF-16
(Li et al., 2007).
The KGB-1 signaling pathway might be complex
Previous work has shown that KGB-1 is activated by the MAPKK, MEK-1
(Mizuno et al., 2004), yet we
found that loss of MEK-1 leads to a much less robust accumulation of GLH-1
protein than does the loss of KGB-1 (W.L. and K.L.B., unpublished). This
suggests that alternate MAPK pathways, perhaps including the SEK-1/PMK-1
p38-type MAPK pathway (Mizuno et al.,
2004), might influence the activity of KGB-1, broadening the
signals to which KGB-1 is receptive and adding to its potential
responsiveness. KGB-2, a protein very similar to KGB-1, only appears to have a
small role in regulating the GLH proteins, as kgb-1; kgb-2 double
mutants show only slightly higher GLH-1 levels than kgb-1 alone (W.L.
and K.L.B., unpublished). Therefore, in regulating GLH-1, KGB-1 and KGB-2
might function redundantly or in parallel pathways and KGB-2 might respond to
as yet unrecognized stimuli. Although we expect that GLH-1 is a major target
of KGB-1, as the rescue of kgb-1 by glh-1(RNAi) is only a
partial return to fertility, it is possible that KGB-1 has additional germline
targets that remain to be discovered. It is not surprising that KGB-1, a MAP
kinase, may be regulated by multiple MAPKKs and/or may recognize multiple
target substrates, as most MAP kinases participate in complex cross-talk with
various signaling pathways. In addition to KGB-1 and CSN-5, the GLHs are also
likely to respond to multiple regulators, including transcription factors.
Both N2 and kgb-1 worms show a decrease in GLH-1 protein as they age
(Fig. 1B,D); perhaps
glh-1 mRNA levels decline in post-reproductive worms, contributing to
the regulation of GLH-1. However, this report identifies GLH-1 as a likely
target of the KGB-1 MAP kinase and demonstrates dramatic consequences for the
C. elegans germline when the KGB-1/GLH-1 relationship is
disrupted.
CSN-5 as a protective partner
In C. elegans, CSN-5 has been shown to target MEI-1 for
degradation (Pintard et al.,
2003); our report is the first to implicate CSN-5 in a protective
role in C. elegans. We report that CSN-5 contains a conserved MAPK D
site and that CSN-5 interacts both physically and genetically with KGB-1. The
suggestion of a protective role for CSN-5 regarding GLH-1 is also based on
other researchers reporting a protective role for the mammalian CSN-5 homolog,
which protects c-Jun, Id1 and Id3, as well as our finding that loss of C.
elegans CSN-5 closely mimics loss of GLH-1 and GLH-4
(Berse et al., 2004;
Naumann et al., 1999;
Smith et al., 2002).
A working model
A parsimonious explanation for the relationship between GLH-1, KGB-1 and
CSN-5 that takes into account the results presented here would be that CSN-5
antagonizes KGB-1 function, as shown in the model
(Fig. 7), with KGB-1 ultimately
targeting GLH-1 for proteasomal degradation by binding at the D site and using
the phosphodegron motif as a phosphorylation site.
Are GLH and Vasa homologs regulated by similar homeostatic mechanisms?
Homologs of Vasa or GLH-like (DEAD-box proteins that have CCHC zinc
fingers) proteins are present in most if not all animals in which germ
granules have been studied, making this protein family the most highly
conserved germline determinant. We have shown here the MAPK D site is
necessary for the interaction of GLH-1 with KGB-1. In the myriad of Vasa and
GLH homologs, both the D site and the phosphodegron motif are often present
(Table 1 and data not shown).
We also find that Drosophila Vasa binds to both CSN-5 and KGB-1 in
pull-down assays (Fig. 6C).
These results suggest that Drosophila CSN5 and perhaps the closely
related Drosophila BSK protein might have a relationship with Vasa
similar to that reported here for GLH-1 with CSN-5 and KGB-1. Thus, GLH and
Vasa-like proteins might be regulated by degradation in many species, ensuring
homeostasis by monitoring germ granule integrity and maintaining proper levels
of crucial germline components.
|
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Mayo Clinic Jacksonville, Jacksonville, FL 32224, USA
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Adachi-Yamada, T., Fujimura-Kamada, K., Nishida, Y. and
Matsumoto, K. (1999). Distortion of proximodistal information
causes JNK-dependent apoptosis in Drosophila wing.
Nature 400,166
-169.[CrossRef][Medline]
Bech-Otschir, D., Kraft, R., Huang, X., Henklein, P., Kapelari,
B., Pollmann, C. and Dubiel, W. (2001). COP9
signalosome-specific phosphorylation targets p53 to degradation by the
ubiquitin system. EMBO J.
20,1630
-1639.[CrossRef][Medline]
Berse, M., Bounpheng, M., Huang, X., Christy, B., Pollmann, C.
and Dubiel, W. (2004). Ubiquitin-dependent degradation of Id1
and Id3 is mediated by the COP9 signalosome. J. Mol.
Biol. 343,361
-370.[CrossRef][Medline]
Bosch, M., Serras, F., Martin-Blanco, E. and Baguna, J.
(2005). JNK signaling pathway required for wound healing in
regenerating Drosophila wing imaginal discs. Dev.
Biol. 280,73
-86.[CrossRef][Medline]
Chamovitz, D. A. and Glickman, M. (2002). The
COP9 signalosome. Curr. Biol.
12, R232.[CrossRef][Medline]
Church, D. L., Guan, K.-L. and Lambie, E. J.
(1995). Three genes of the MAP kinase cascade, mek-2,
mpk-1/sur-1 and let-60 ras, are required for meiotic cell cycle
progression in Caenorhabditis elegans.
Development 121,2525
-2535.[Abstract]
Cope, G. A. and Deshaies, R. J. (2003). COP9
signalosome: a multifunctional regulator of SCF and other cullin-based
ubiquitin ligases. Cell
114,663
-671.[CrossRef][Medline]
DeRenzo, C., Reese, K. J. and Seydoux, G.
(2003). Exclusion of germ plasm proteins from somatic lineages by
cullin-dependent degradation. Nature
424,685
-689.[CrossRef][Medline]
Epstein, H. F. and Shakes, D. C. (1995).
Caenorhabditis elegans: Modern Biological Analysis of an Organism
(Methods in Cell Biology). San Diego: Academic Press.
Gao, G., Raikar, S., Davenport, B., Mutapcic, L., Montgomery,
R., Kuzmin, E. and Bennett, K. L. (2006). Cross-species RNAi:
selected Ascaris suum dsRNAs can sterilize Caenorhabditis
elegans. Mol. Biochem. Parasitol.
146,124
-128.[CrossRef][Medline]
Gilboa, L. and Lehmann, R. (2006).
Soma-germline interactions coordinate homeostasis and growth in the
Drosophila gonad. Nature
443,97
-100.[CrossRef][Medline]
Gruidl, M. E., Smith, P. A., Kuznicki, K. A., McCrone, J. S.,
Kirchner, J., Strome, S. and Bennett, K. L. (1996). Multiple
potential germline helicases are components of the germline-specific P
granules of Caenorhabditis elegans. Proc. Natl. Acad. Sci.
USA 93,13837
-13842.
Gumienny, T. L., Lambie, E., Hartwieg, E., Horvitz, H. R. and
Hengartner, M. O. (1999). Genetic control of programmed cell
death in the Caenorhabditis elegans hermaphrodite germline.
Development 126,1011
-1022.[Abstract]
Hay, B., Ackerman, L., Barbel, S., Jan, L. Y. and Jan, Y. N.
(1988a). Identification of a component of Drosophila
polar granules. Development
103,625
-640.
Hay, B., Jan, L. Y. and Jan, Y. N. (1988b). A
protein component of Drosophila polar granules is encoded by
vasa and has extensive sequence similarity to ATP-dependent
helicases. Cell 55,577
-587.[CrossRef][Medline]
Huffman, D. L., Abrami, L., Sasik, R., Corbeil, J., van der
Goot, F. G. and Aroian, R. V. (2004). Mitogen-activated
protein kinase pathways defend against bacterial pore-forming toxins.
Proc. Natl. Acad. Sci. USA
101,10995
-20000.
Igaki, T., Kanda, H., Yamamoto-Goto, Y., Kanuka, H., Kuranaga,
E., Aigaki, T. and Miura, M. (2002). Eiger, a TNF superfamily
ligand that triggers the Drosophila JNK pathway. EMBO
J. 21,3009
-3018.[CrossRef][Medline]
Ip, Y. T. and Davis, R. J. (1998). Signal
transduction by the c-Jun N-terminal kinase (JNK) - from inflammation to
development. Curr. Opin. Cell Biol.
10,205
-219.[CrossRef][Medline]
Jacinto, A., Woolner, S. and Martin, P. (2002).
Dynamic analysis of dorsal closure in Drosophila: from genetics to
cell biology. Dev. Cell
3, 9-19.[CrossRef][Medline]
Jacobs, D., Glossip, D., Xing, H., Muslin, A. J. and Kornfeld,
K. (1999). Multiple docking sites on substrate proteins form
a modular system that mediates recognition by ERK MAP kinase. Genes
Dev. 13,163
-175.
Jones, A. R., Francis, R. and Schedl, T.
(1996). GLD-1, a cytoplasmic protein essential for oocyte
differentiation, shows stage- and sex-specific expression during
Caenorhabditis elegans germline development. Dev.
Biol. 180,165
-183.[CrossRef][Medline]
Kawasaki, I., Shim, Y. H., Kirchner, J., Kaminker, J., Wood, W.
B. and Strome, S. (1998). PGL-1, a predicted RNA-binding
component of germ granules, is essential for fertility in C. elegans.
Cell 94,635
-645.[CrossRef][Medline]
Koga, M., Zwaal, R., Guan, K. L., Avery, L. and Ohshima, Y.
(2000). A Caenorhabditis elegans MAP kinase kinase,
MEK-1, is involved in stress responses. EMBO J.
19,5148
-5156.[CrossRef][Medline]
Kuznicki, K. A., Smith, P. A., Leung-Chiu, W. M., Estevez, A.
O., Scott, H. C. and Bennett, K. L. (2000). Combinatorial RNA
interference indicates GLH-4 can compensate for GLH-1; these two P granule
components are critical for fertility in C. elegans.
Development 127,2907
-2916.[Abstract]
Lasko, P. F. and Ashburner, M. (1988). The
product of the Drosophila gene vasa is very similar to
eukaryotic initiation factor-4A. Nature
335,611
-617.[CrossRef][Medline]
Li, S., Liu, X. and Ascoli, M. (2000). p38JAB1
binds to the intracellular precursor of the lutropin/choriogonadotropin
receptor and promotes its degradation. J. Biol. Chem.
275,13386
-13393.
Li, S., Armstrong, C. M., Bertin, N., Ge, H., Milstein, S.,
Boxem, M., Vidalain, P. O., Han, J. D., Chesneau, A., Hao, T. et al.
(2004). A map of the interactome network of the metazoan C.
elegans. Science
303,540
-543.
Li, W., Gao, B., Lee, S.-M., Bennett, K. and Fang, D.
(2007). RLE-1, an E3 ubiquitin ligase, regulates C.
elegans aging by catalyzing DAF-16 polyubiquitination. Dev.
Cell 12,1
-12.[CrossRef][Medline]
Mattila, J., Omelyanchuk, L., Kyttälä, S., Turunen, H.
and Nokkala, S. (2005). Role of Jun N-terminal kinase (JNK)
signaling in the wound healing and regeneration of a Drosophila
melanogaster wing imaginal disc. Int. J. Dev.
Biol. 49,391
-399.[CrossRef][Medline]
McCarter, J., Bartlett, B., Dang, T. and Schedl, T.
(1999). On the control of oocyte meiotic maturation and ovulation
in Caenorhabditis elegans. Dev. Biol.
205,111
-128.[CrossRef][Medline]
Miller, M. A., Nguyen, V. Q., Lee, M.-H., Kosinski, M., Schedl,
T., Caprioli, R. M. and Greenstein, D. (2001). A sperm
cytoskeletal protein that signals ooctye meiotic maturation and ovulation.
Science 291,2144
-2147.
Mizuno, T., Hisamoto, N., Terada, T., Kondo, T., Adachi, M.,
Nishida, E., Kim, D. H., Ausubel, F. M. and Matsumoto, K.
(2004). The Caenorhabditis elegans MAPK phosphatase
VHP-1 mediates a novel JNK-like signaling pathway in stress response.
EMBO J. 23,2226
-2234.[CrossRef][Medline]
Naumann, M., Bech-Otschir, D., Huang, X., Ferrell, K. and
Dubiel, W. (1999). COP9 signalosome-directed c-Jun
activation/stabilization is independent of JNK. J. Biol.
Chem. 274,35297
-35300.
Navarro, R. E., Shim, E. Y., Kohara, Y., Singson, A. and
Blackwell, T. K. (2001). cgh-1, a conserved predicted RNA
helicase required for gametogenesis and protection from physiological germline
apoptosis in C. elegans. Development
128,3221
-3232.
Nelson, G. A., Lew, K. K. and Ward, S. (1978).
Intersex, a temperature-sensitive mutant of the nematode Caenorhabditis
elegans. Dev. Biol.
66,386
-409.[CrossRef][Medline]
Nishi, Y. and Lin, R. (2005). DYRK2 and GSK-3
phosphorylate and promote the timely degradation of OMA-1, a key regulator of
the oocyte-to-embryo transition in C. elegans. Dev.
Biol. 288,139
-149.[CrossRef][Medline]
Olmstead, J. B. (1986). Analysis of
cytoskeletal structures using blot purified monospecific antibodies.
Methods Enzymol. 134,467
-472.[Medline]
Orlicky, S., Tang, X., Willems, A., Tyers, M. and Sicheri,
F. (2003). Structural basis for phosphodependent substrate
selection and orientation by the SCF Cdc4 ubiquitin ligase.
Cell 112,243
-256.[CrossRef][Medline]
Page, B. D., Guedes, S., Waring, D. and Priess, J. R.
(2001). The C. elegans E2F- and DP-related proteins are
required for embryonic asymmetry and negatively regulate Ras/MAPK signaling.
Mol. Cell 7,451
-460.[Medline]
Pellettieri, J., Reinke, V., Kim, S. K. and Seydoux, G.
(2003). Coordinate activation of maternal protein degradation
during the egg-to-embryo transition in C. elegans. Dev.
Cell 5,451
-462.[CrossRef][Medline]
Pintard, L., Kurz, T., Glaser, S., Willis, J. H., Peter, M. and
Bowerman, B. (2003). Neddylation and deneddylation of CUL-3
is required to target MEI-1/katanin for degradation at the meiosis-to-mitosis
transition in C. elegans. Curr. Biol.
13,911
-921.[CrossRef][Medline]
Pitt, J. N., Schisa, J. A. and Priess, J. R.
(2000). P granules in the germ cells of Caenorhabditis
elegans adults are associates with clusters of nuclear pores and contain
RNA. Dev. Biol. 219,315
-333.[CrossRef][Medline]
Reese, K. J., Dunn, M. A., Waddle, J. A. and Seydoux, G.
(2000). Asymmetric segregation of PIE-1 in C. elegans is
mediated by two complementary mechanisms that act through separate PIE-1
protein domains. Mol. Cell
6, 445-455.[CrossRef][Medline]
Reinke, V., Smith, H. E., Nance, J., Wang, J., Van Doren, C.,
Begley, R., Jones, S. J., Davis, E. B., Scherer, S., Ward, S. et al.
(2000). A global profile of germline gene expression in C.
elegans. Mol. Cell
6, 605-616.[CrossRef][Medline]
Robinson, M. J. and Cobb, M. H. (1997).
Mitogen-activated protein kinase pathways. Curr. Opin. Cell
Biol. 9,180
-186.[CrossRef][Medline]
Roussell, D. L. and Bennett, K. L. (1993).
glh-1: a germline putative RNA helicase from Caenorhabditis
has four zinc fingers. Proc. Natl. Acad. Sci. USA
90,9300
-9304.
Schwechheimer, C., Serino, G., Callis, J., Crosby, W. L.,
Lyapina, S., Deshaies, R. J., Gray, W. M., Estelle, M. and Deng, X. W.
(2001). Interactions of the COP9 signalosome with the E3
ubiquitin ligase SCFTIRI in mediating auxin response.
Science 292,1379
-1382.
Sharrocks, A. D., Yang, S. H. and Galanis, A.
(2000). Docking domains and substrate-specificity determination
for MAP kinases. Trends Biochem. Sci.
25,448
-453.[CrossRef][Medline]
Shirayama, M., Soto, M. C., Ishidate, T., Kim, S., Nakamura, K.,
Bei, Y., van den Heuvel, S. and Mello, C. C. (2006). The
conserved kinases CDK-1, GSK-3, KIN-19, and MBK-2 promote OMA-1 destruction to
regulate the oocyte-to-embryo transition in C. elegans.
Curr. Biol. 16,47
-55.[CrossRef][Medline]
Smith, P. A., Leung-Chiu, W. M., Montgomery, R., Orsborn, A.,
Kuznicki, K. A., Gressman-Coberly, E., Mutapcic, L. and Bennett, K. L.
(2002). The GLH proteins, Caenorhabditis elegans P
granule components, associate with CSN-5 and KGB-1, proteins necessary for
fertility, and with ZYX-1, a predicted cytoskeletal protein. Dev.
Biol. 251,333
-347.[CrossRef][Medline]
Stitzel, M. L., Pellettieri, J. and Seydoux, G.
(2006). The C. elegans DYRK kinase MBK-2 marks oocyte
proteins for degradation in response to meiotic maturation. Curr.
Biol. 16,56
-62.[CrossRef][Medline]
Strome, S. and Wood, W. B. (1982).
Immunofluorescence visualization of germ-line-specific cytoplasmic granules in
embryos, larvae, and adults of Caenorhabditis elegans.
Proc. Natl. Acad. Sci. USA
79,1558
-1562.
Strome, S. and Wood, W. B. (1983). Generation
of asymmetry and segregation of germ-line granules in early C.
elegans embryos. Cell
35, 15-25.[CrossRef][Medline]
Sun, Y., Wilson, M. P. and Majerus, P. W.
(2002). Inositol 1,3,4-trisphosphate 5/6-kinase associates with
the COP9 signalosome by binding to CSN1. J. Biol.
Chem. 277,45759
-45764.
Uhle, S., Medalia, O., Waldron, R., Dumdey, R., Henklein, P.,
Bech-Otschir, D., Huang, X., Berse, M., Sperling, J., Schade, R. et al.
(2003). Protein kinase CK2 and protein kinase D are associated
with the COP9 signalosome. EMBO J.
22,1302
-1312.[CrossRef][Medline]
Ye, X., Nalepa, G., Welcker, M., Kessler, B. M., Spooner, E.,
Qin, J., Elledge, S. J., Clurman, B. E. and Harper, J. W.
(2004). Recognition of phosphodegron motifs in human cyclin E by
the SCF Fbw7 ubiquitin ligase. J. Biol. Chem.
279,50110
-50119.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  R. K. Miller, H. Qadota, T. J. Stark, K. B. Mercer, T. S. Wortham, A. Anyanful, and G. M. Benian CSN-5, a Component of the COP9 Signalosome Complex, Regulates the Levels of UNC-96 and UNC-98, Two Components of M-lines in Caenorhabditis elegans Muscle Mol. Biol. Cell, August 1, 2009; 20(15): 3608 - 3616. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. P. Choe and K. Strange Genome-wide RNAi screen and in vivo protein aggregation reporters identify degradation of damaged proteins as an essential hypertonic stress response Am J Physiol Cell Physiol, December 1, 2008; 295(6): C1488 - C1498. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Spike, N. Meyer, E. Racen, A. Orsborn, J. Kirchner, K. Kuznicki, C. Yee, K. Bennett, and S. Strome Genetic Analysis of the Caenorhabditis elegans GLH Family of P-Granule Proteins Genetics, April 1, 2008; 178(4): 1973 - 1987. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Spike, J. Bader, V. Reinke, and S. Strome DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells Development, March 1, 2008; 135(5): 983 - 993. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
