Functional genomics approach to identify novel targets of MPK-1 ERK signaling in germline development. Swathi Arur, Mitsue Ohmachi, Sudhir Nayak, Tim Schedl. Dept Genetics, Washington Univ Sch Med, St Louis, MO.

   MPK-1 ERK is critical for spatial co-ordination of meiotic prophase progression and oogenesis in C. elegans and regulates nine different germline processes. The cellular response to signaling occurs as a consequence of ERK activating or inactivating target proteins by phosphorylation. However, targets of MPK-1 that mediate germline development are unknown. To identify downstream targets of MPK-1 signaling, we used an integrated computational, reverse genetic and biochemical approach.
   Computational methods helped identify >2000 proteins from the C. elegans proteome that contained putative ERK docking (e.g., D-domain and FXFP motif) and phospho-acceptor (S/TP) sites. 128 of these were chosen for RNAi screening based on (a) multiple docking sites, (b) conservation of docking sites and (c) germline expression. MPK-1 regulates a number of germline processes and for execution of each process, it likely phosphorylates multiple targets, rather than a single one. Therefore RNAi based gene inactivation of each target is performed in sensitized genetic backgrounds to permit detection of small or partial changes in the pathway that controls a given cellular process. In addition using the sensitized backgrounds allows assessment of whether the downstream target is activated or inactivated by MPK-1 phosphorylation.
   To date, we have identified 21 genes that behave genetically as mpk-1 targets for germline development. RNAi of 15 genes enhances specific phenotypes of an mpk-1 temperature sensitive (ts) loss-of-function mutant at the permissive temperature suggesting that the corresponding gene products are positively regulated by MPK-1 ERK phosphorylation. Conversely, RNAi of 6 gene products enhances specific phenotypes of let-60 RAS ts gain-of-function at the permissive temperature suggesting that the gene products are inactivated by MPK-1 ERK phosphorylation. Ongoing biochemical analysis has thus far shown that 6 of the gene products identified by RNAi as functioning in mpk-1 mediated germline development are direct in vitro substrates of activated mammalian ERK2. Together, these studies have identified at least 6 gene products that are likely direct targets that execute MPK-1 ERK regulated aspects of germline development. Given the conservation of the targets and their docking sites it is likely that the orthologous proteins are previously unknown targets of ERK phosphorylation in other organisms.

Program #: 1198C Presentation Date/Time: TUE, June 28, 2005 1:30-3:00PM Authors: Momoyo Hanazawa, Alessandro Puoti, Tim Schedl Session Type: Poster Location: Pauley Pavilion - POSTER BOARD# 1198C
mog genes and the proliferation versus meiotic development regulatory network. Momoyo Hanazawa1, Alessandro Puoti2, Tim Schedl1. 1) Dept. of Genetics, Washington University School of Medicine, St. Louis, MO, USA; 2) Dept. of Biology, University of Fribourg, Fribourg, Switzerland.

   GLP-1/Notch signaling promotes germ cell proliferation by inhibiting the activity of two redundant pathways that promote meiotic development, the GLD-1/NOS-3 pathway and the GLD-2/GLD-3 pathway. The GLD-1 pathway likely acts, at least in part, to promote meiotic development by translationally repressing proliferation promoting gene products while the GLD-2 pathway likely acts, at least in part, to promote the translation of meiotic gene products. Genetic data indicates that a third redundant pathway exists that promotes meiotic development.
    The mog genes (mog-1, -4, -5 & -6) were originally identified in the Kimble lab based on their masculinized hermaphrodite germline phenotype. Genetic experiments indicate that the mog genes function in the proliferation vs. meiotic development decision; for example mog-6 is a tumorous enhancer of weak glp-1 gf mutations and is synthetic tumorous with gld-3. Our studies of the MOGs have lead to three conclusions. First, the MOGs appear to function downstream of GLP-1 signaling to promote meiotic development. This based on the finding that, for example, the gld-3 mog-6; glp-1 null triple mutant is tumorous. Surprisingly, the gld-2 mog-6; glp-1 null triple mutant has a Glp premature meiotic entry phenotype. (These results indicate that GLD-3 must have a function in addition to acting with GLD-2 to promote meiotic development.) Second, the MOGs may function, at least in part, in the third meiotic entry pathway. This is based on (a) the MOGs functioning outside of the GLD-2/GLD-3 pathways (e.g. gld-2; mog-6 is synthetic tumorous); (b) the MOGs acting, at least in part, outside of the GLD-1 pathway (e.g. gld-1; mog-6 males display overproliferation phenotypes) and (c) the MOGs promoting meiotic development in the absence of the GLD-1 and GLD-2 pathways (e.g. gld-1 gld-2 displays some meiotic development while gld-1 gld-2; mog-6 is completely tumorous). Third, the MOGs appear to function redundantly with GLD-2 to promote GLD-1 accumulation (e.g. gld-2 fog-3; mog-6 fails to accumulate GLD-1). Thus, the MOGs appear to act by at least two mechanisms to promote meiotic development downstream of GLP-1 signaling; functioning in the third pathway and promoting GLD-1 accumulation.

Program #: 1203B Presentation Date/Time: MON, June 27, 2005 3:00-4:30PM Authors: Jessica Amrozowicz Kerins, Dave Hansen, Laura Berry, Daimon Simmons, Tim Schedl Session Type: Poster Location: Pauley Pavilion - POSTER BOARD# 1203B
The C. elegans Ortholog of Yeast CDC40/PRP17 Functions in the Proliferation versus Meiotic Development Decision. Jessica Amrozowicz Kerins1, Dave Hansen2, Laura Berry1, Daimon Simmons1, Tim Schedl1. 1) Genetics, Washington University, Saint Louis, MO; 2) Biological Sciences, University of Calgary, Alberta.

   GLP-1 is a member of the Notch family of receptors and promotes germ cell proliferation in C. elegans. Activation of the germline GLP-1 receptor is spatially regulated by the distal tip cell transmembrane ligand, LAG-2. Binding of LAG-2 to GLP-1 induces receptor cleavage generating GLP-1(INTRA), which is then presumed to translocate to the nucleus, bind LAG-1, and alter the pattern of transcription. Disruption of this pathway causes all proliferating germ cells to prematurely enter meiosis. Conversely, constitutive activation by the glp-1(oz112gf) mutation results in the formation of a germline tumor where proliferative cells are found throughout the gonad. Two pathways, one containing GLD-1, the other containing GLD-2, function redundantly downstream of GLP-1 to inhibit proliferation and/or promote meiotic development. GLP-1 signaling, in effect, inhibits both pathways to promote germ cell proliferation.
   We have taken a forward genetic approach to identify genes that function downstream of GLP-1 to promote entry into meiosis, or that function to negatively regulate GLP-1. This approach uses sensitized genetic backgrounds to screen for tumorous mutants. One screen utilized the weak glp-1(oz264gf) mutant and identified the allele oz273 as a strong tumorous enhancer. oz273 also displays a Mog (masculinization of the germline) phenotype in the glp-1(+) background, similar to teg-1, another mutant that enhances a weak glp-1(gf) background (see Hansen et al, this meeting). Also similar to teg-1, the gld-2oz273 mutant is tumorous, suggesting that teg-1 and oz273 could be functioning in the same pathway to regulate entry into meiosis. The gene disrupted by the oz273 lesion is F49D11.1, which codes a 567 amino acid protein that is orthologous to yeast CDC40/PRP17 and human PRP17. These proteins are required for the second step of pre-mRNA splicing (Jones et al, 1995; Umen et al, 1995). The oz273 mutation is an 873 bp deletion that removes the first 173 amino acids encoded by exon 2, including four of six WD-40 repeats. Also within the deleted region is a motif that is critical for splicing in yeast (Lindsey-Boltz et al, 2000), suggesting that the oz273 deletion is a strong loss-of-function or null allele. Experiments are currently underway to further characterize the gene and determine if other splicing factors regulate entry into meiosis (also see Hanazawa et al, this meeting).

Program #: 271 Presentation Date/Time: TUE, June 28, 2005 at 11:30AM Authors: Min-Ho Lee, Valerie Reinke, Ting Wang, Gary Stormo, Tim Schedl Session Type: Parallel Location: Grand Horizon Ballroom

GLD-1 mRNA Targets and Binding Specificity. Min-Ho Lee1, Valerie Reinke2, Ting Wang1, Gary Stormo1, Tim Schedl1. 1) Genetics, Washington Univ, St Louis, MO; 2) Genetics, Yale Univ, New Haven, CT.

   The germline is an excellent system for investigating translational control as it is a major mechanism for regulating gene expression. GLD-1 is a germline specific, maxi-KH motif containing RNA binding protein that controls multiple aspects of C. elegans germ cell development, suggesting that it regulates multiple mRNAs. To understand how GLD-1 regulates mRNAs to control germ cell development, we have previously identified multiple in vivo mRNA targets of GLD-1 that were co-immunoprecipitated (IP) with GLD-1 from cytosol extracts and recovered following subtractive hybridizationa, b. These target mRNAs are preferentially expressed in the germline and several have essential functions in oocyte differentiation and early embryogenesis. GLD-1 is a translational repressor, based on the analysis of mRNA and protein levels of five mRNA targets (rme-2, puf-5, cep-1, oma-1, and oma-2) in wild-type and gld-1 null germlines. However, it has been clear that we do not have the complete list of GLD-1 mRNA targets. Thus, we used microarray analysis to identify additional targets that are co-IPed with GLD-1. We identified 129 genes that are enriched more than two fold (p<0.05) in the GLD-1 IP over the control IP. Essentially all of our previously identified targets are recovered in the new screen. To determine the in vivo binding specificity of GLD-1 or GLD-1 containing complexes, we have used GLD-1 from wild-type cytosol extracts to identify 12 GLD-1 binding regions in six targets thus far. GLD-1 can bind the 5’UTR, 3’UTR or in the coding region depend on the target. Using the sequences of 13 GLD-1 binding regions (12 in the six targets and the tra-2 3’UTR) and the corresponding, orthologous C. briggsae regions, we found 3 potential GLD-1 binding motifs with PhyloCon, a program that identifies regulatory motifs among co-regulated orthologous gene setsc. One of the motifs is essentially identical to the hexanucleotide consensus that was identified in the tra-2 3’UTR by Ryder et al, 2004d. Interestingly, the tra-2 3’UTR has only this motif four times. Most other binding regions have two different motifs and three regions do not contain the hexanucleotide consensus. Biochemical analysis of the motifs indicates that two motifs, including the hexanucleotide consensus, are important for GLD-1 binding and synergistic interactions between motifs likely occur. Therefore, the data from the IP/microarray analysis and the binding studies suggest that, in addition to the hexanucleotide consensus, GLD-1/GLD-1 containing complexes likely utilize other sequences to achieve specificity. a, b Lee and Schedl, Genes Dev, 2001 & 2004; c, Wang and Stormo Bioinformatics, 2003; d, Ryder et al. Nat Struct Mol Biol, 2004.

Program #: 1215B Presentation Date/Time: MON, June 27, 2005 3:00-4:30PM Authors: Pallavi R. Mantina, Laura Wilson-Berry, Lindsay MacDonald, Tim Schedl, Dave Hansen Session Type: Poster Location: Pauley Pavilion - POSTER BOARD# 1215B
Characterization of the teg genes in regulating proliferation in the germ line. Pallavi R Mantina1, Laura Wilson-Berry2, Lindsay MacDonald1, Tim Schedl2, Dave Hansen1. 1) Biological Sciences, University of Calgary, Calgary, Alberta, Canada; 2) Dept Genetics, Washington University School of Med., St. Louis, MO USA.

   In the C. elegans germ line, the proliferative cells reside at the distal ends of each of the gonad arms. As the cells move proximally, towards the uterus, they cease proliferating and enter into meiotic prophase. The balance between proliferation and meiotic entry is controlled by the GLP-1/Notch signalling pathway. If the activity of this signalling pathway is rendered inactive, germ cells enter into meiosis prematurely and the population of proliferative cells is depleted. Conversely, constitutive activation of the pathway (through a strong glp-1(gf) mutation) causes cells to remain proliferative, never entering into meiotic prophase. In order to identify additional factors that are involved in controlling the balance between proliferation and meiotic entry, we conducted a genetic screen in which mutations were isolated that enhance the tumorous phenotype of a weak glp-1(gf) mutant. Genes identified in this screen are likely negative regulators of the activity of the GLP-1/Notch signalling pathway, or positive regulators of meiotic entry functioning downstream of GLP-1/Notch signalling. We are currently characterizing three genes identified in this screen; teg-1, teg-2 and teg-4 (tumorous enhancer of glp-1(gf)).
   We have cloned teg-1 and found that it encodes a protein conserved in many species, from yeast to humans; however, the functions these homologous proteins are not yet known. TEG-1 contains a GYF motif, which is thought to be involved in protein binding. Therefore, we are performing a yeast two-hybrid screen in order to identify proteins that interact with TEG-1. Antibodies against TEG-1 show that it is expressed in the nuclei of all cells in the germ line, the somatic gonad, as well as other somatic cells. We are also performing genetic epistasis and further expression analysis to determine where teg-1 functions in the genetic regulatory network that controls the proliferation vs. meiotic entry decision. We are also performing the same genetic analysis with teg-2 and teg-4. We have mapped teg-2 and teg-4 to relatively small regions on chromosomes II and I, respectively. Finally, we are determining if any of the teg genes interact with the Notch signalling pathway in other cell fate decisions. This will help us determine if they function as general negative regulators of Notch signalling or as downstream regulators of meiotic entry.

Program #: 788A Presentation Date/Time: SUN, June 26, 2005 1:30-3:00PM Authors: Sudhir B. Nayak, Paul Fox, Tim Schedl Session Type: Poster Location: Pauley Pavilion - POSTER BOARD# 788A
Downregulation of GLD-1 in the Proximal Germ Line may be mediated by an SCF-type E3. Sudhir B. Nayak, Paul Fox, Tim Schedl. Dept Genetics, Washington Univ, St Louis, MO.

   The KH domain containing RNA binding protein GLD-1 has functions in promoting spermatogenesis in the hermaphrodite, progression through female meiotic prophase, and oocyte differentiation. The proper accumulation of GLD-1 at the distal region and loss of GLD-1 at the loop are critical for its functions and maintaining germ line polarity. Unlike the requirements for GLD-1 accumulation in the distal region, the mechanisms by which GLD-1 levels fall abruptly and are maintained at low levels even though the gld-1 mRNA is abundant in oocytes have not been defined. GLD-1 functions in the translation repression of a variety of mRNA targets required during oogenesis to spatially restrict their expression. For one target, the rme-2 (yolk receptor) mRNA, GLD-1 acts to spatially restrict RME-2 accumulation to developing oocytes. The rme-2 mRNA is present throughout the germ line, however high GLD-1 levels in the transition zone and pachytene region repress its translation. During late oogenesis, when RME-2 is required, degradation of GLD-1 at the loop region allows for the efficient translation of the rme-2 mRNA. One potential mechanism that could mediate the rapid fall of GLD-1 levels at the loop region is ubiquitin mediated degradation, which is a common means of controlling the levels of important regulators. One type of E3 ubiquitin ligase is the SCF (Skp1(SKR-1/2)/Cullin(CUL-1)/F-box), which selects specific proteins for ubiquitin mediated degradation by using a variable adaptor protein (F-box) to bring protein substrate to a core catalytic complex made up of a Cullin, Skp1, and the Rbx1/Roc1 RING finger proteins. Consistent with this possibility, we find that RNAi of the C. elegans core SCF components CUL-1 and SKR-1/2 result in the proximal extension of GLD-1 expression beyond the loop region into developing oocytes. Over time the ectopic expression of GLD-1 results in the inappropriate repression of GLD-1 mRNA targets, such as the rme-2 mRNA, and abnormal oogenesis. The FOG-2 F-box protein binds GLD-1 but is not essential for its degradation as the expression pattern of GLD-1 and oogenesis are normal in fog-2(null) animals. We will present our data on the potential targeted degradation of GLD-1 and progress toward isolating the F-box component that mediates the recruitment of GLD-1 to the SCF.

Program #: 789B Presentation Date/Time: MON, June 27, 2005 3:00-4:30PM Authors: Sudhir B. Nayak, Daimon Simmons, Omar Arnaout, Tim Schedl Session Type: Poster Location: Pauley Pavilion - POSTER BOARD# 789B
Attempts to Improve Recovery of Transgene Expression in the Germ Line Using a Dual Selection System. Sudhir B. Nayak, Daimon Simmons, Omar Arnaout, Tim Schedl. Dept Genetics, Washington Univ, St Louis, MO.

   A variety of methods such as injection, micromechanical piercing, and ballistic transofmation have been used to stably introduce transgenic DNA into C. elegans. While all the techniques have been successful for genes expressed in the soma, high-copy suppression and transgene-mediated co-suppression have made the expression of transgenes in germ line difficult. Of these methods the ballistic transformation rescue of unc-119 has the potential to generate low-copy integrants, and even homologous recombinants, that express transgenes under endogenous promoters. While technically less demanding than injection the high material cost of maintaining large populations of C. elegans and the low frequency of animals with stable germ line expression, have been significant barriers. To resolve these issues, we have attempted to develop methods for generating large populations of unc-119 animals and enrich for germ line expression by requiring it for viability or fertility.
   We have been successful in generating multiple integrated lines of C. elegans that express transgenes stably in the germ line using both the standard and modified versions of the ballistic transformation rescue of unc-119 protocols. However, we found that the vast majority of lines rescued for unc-119 did not have germ line expression of the transgene and when germ line expression was observed the majority of animals did not maintain transgene expression even if a potential integration event had occurred. Thus, the large number of lines rescued for the somatic unc-119 marker but negative for germ line expression makes testing each positive line a cumbersome task. The expression of GFP fusion proteins facilitates the screening for stable germ line expressing lines, however, two issues make this approach problematic. First, lines with appropriate levels of GFP fusion protein expression below our detection limit will be lost. Second, constructs where GFP interferes with protein function cannot reliability used for downstream analysis. To reduce the number of “false positives” and eliminate the requirement of using a GFP fusion protein for screening we would like to develop a highly sensitive system for selecting only those animals with germ line expression. We reasoned that if germ line expression was required for fertility or viability the number of false positives should be greatly reduced. To test this hypothesis we have generated constructs carrying unc-119(+) for primary selection and a secondary selectable marker either (fem-3(+) or emb-27(+)) that is required in the germ line. Our progress in the assessment of this dual selection system will be presented.

Program #: 1250A Presentation Date/Time: SUN, June 26, 2005 1:30-3:00PM Authors: Mitsue Ohmachi, Swathi Arur, Sudhir Nayak, Tim Schedl Session Type: Poster Location: Pauley Pavilion - POSTER BOARD# 1250A
rskn-1, a C. elegans homolog of p90 ribosomal S6 kinases, acts downstream of MPK-1 ERK signaling in germline development. Mitsue Ohmachi, Swathi Arur, Sudhir Nayak, Tim Schedl. Dept Genetics, Washington Univ, St Louis, MO.

   MPK-1 ERK signaling is important for several aspects of germline development such as pachytene progression, oocyte growth and differentiation, oocyte meiotic maturation and sex determination. While core components of the RAS/MAP kinase pathway function in these processes, downstream targets as well as upstream signals are not known. One possible downstream target of MPK-1 ERK signaling is RSK (p90 ribosomal S6 kinases), which was among the first substrates of ERK to be discovered in vertebrate systems and has been proposed to be a widespread mediator of ERK signaling. Database analysis identifies two C.elegans paralogous genes, T01H8.1 and C54G4.1, as being RSK homolog. We named these genes rskn-1 and rskn-2 respectively. Here we describe rskn-1, which is the closest homolog of human RSK1. RSK proteins have N-terminal and C-terminal kinase domains and the allele rskn-1(ok159) deletes both and thus is likely a null mutation. The germline of rskn-1(ok159) is morphologically normal at all temperatures, although there is a low frequency Him phenotype (1.3%). mpk-1(ga111) is a temperature sensitive loss-of-function mutant that is essentially normal at 20C. The rskn-1(ok159); mpk-1(ga111) mutant is sterile at 20C, with small oocytes that are abnormally arranged in the proximal germline. RSKN-1 is efficiently phosphorylated by mammalian activated ERK2 in vitro consistent with results in vertebrate systems. Together, these results support the conclusion that RSKN-1 is positively regulated by MPK-1 ERK phosphorylation. In wild-type hermaphrodite germlines activated, di-phosphorylated, MPK-1 ERK is found in the last half of pachytene and in the most proximal 1 to 3 oocytes, with a low level in the intervening diplotene and diakinesis oocytes. In rskn-1(ok159) mutant hermaphrodites, high levels of activated MPK-1 ERK are found throughout diplotene and diakinesis indicating that RSKN-1 functions to down-regulate MPK-1 ERK activation. In wild-type females, activated MPK-1 ERK is not observed in the proximal gonad in the absence of the MSP sperm signal. In rskn-1(ok159) females, activated MPK-1 ERK is to a large extent absent, indicating that RSKN-1 mediated down-regulation of MPK-1 ERK activation is for the most part mediated by sperm signaling. The above results suggest that RSKN-1 functions in a sperm dependent negative feedback mechanism to down-regulate MPK-1 ERK activation in the proximal germline. Interestingly, in rskn-1(ok159) females a single late diplotene stage growing oocyte stains strongly for activated MPK-1 ERK. In this case, RSKN-1 may be acting in a sperm-independent manner to down-regulate MPK-1 ERK for oocyte growth control.

Program #: 1214A Presentation Date/Time: SUN, June 26, 2005 1:30-3:00PM Authors: Valarie Vought, Min-Ho Lee, Momoyo Hanazawa, Larissa Wirlo, Katy Michalak, Deb Springer, Valerie Reinke, Tim Schedl, Eleanor Maine Session Type: Poster Location: Pauley Pavilion - POSTER BOARD# 1214A
DNA replication and the proliferation versus meiosis decision. Valarie Vought1, Min-Ho Lee2, Momoyo Hanazawa2, Larissa Wirlo1, Katy Michalak1, Deb Springer1, Valerie Reinke3, Tim Schedl2, Eleanor Maine1. 1) Dept Biol, Syracuse Univ, Syracuse, NY; 2) Dept Genetics, Washington Univ School of Medicine, St Louis, MO; 3) Dept Genetics, Yale Univ School of Medicine, New Haven, CT.

   Germ cell proliferation in C. elegans is induced by the somatic gonad and mediated by GLP-1/Notch signaling. GLP-1 maintains proliferation by inhibiting GLD-1 and GLD-2 activity in distal germ cells. GLD-1 and GLD-2 are translational regulators whose complementary activities are required for germ cells to enter meiosis. GLD-1 may induce entry into meiotic prophase by (at least in part) inhibiting the translation of mRNAs that promote proliferation while GLD-2 may promote entry into meiotic prophase by (at least in part) activating the translation of meiotic mRNAs. ego [enhancer of glp-1] genes interact genetically with GLP-1 signaling and promote germline proliferation and/or inhibit meiotic entry. We found that the ego-5 gene encodes the B subunit of DNA polymerase (pol) alpha-primase complex, also called DIV-1 in C. elegans. We then investigated whether regulation of DNA replication is an aspect of the proliferation/meiosis decision.
    When GLP-1 signaling is absent, germ cells prematurely stop proliferation and enter meiosis; this phenotype is distinct from a simple mitotic defect, where germ cells stop proliferation but fail to enter meiosis. We found that a weak glp-1(lf) mutation is enhanced by partial RNAi-mediated depletion of other DNA replication proteins besides DIV-1/EGO-5. In contrast, partial depletion of other cell cycle proteins (that do not promote DNA replication) generally does not enhance glp-1.
   We recently identified ~125 new putative GLD-1 mRNA targets based on a co-IP/ amplification/ microarray detection strategy. Among them are mRNAs encoding several DNA replication proteins. We chose the pri-1 mRNA for validation. PRI-1 is a primase subunit and the only GLD-1 target from the DNA pol-alpha primase complex. We found that pri-1(RNAi) enhances glp-1(lf) mutations, and pri-1 mRNA contains two GLD-1 binding sites. In other organisms, meiotic S phase is several-fold longer than mitotic S phase, which may allow factors to associate with the chromosomes that are required for later meiotic events. We propose that GLD-1 promotes meiotic entry, in part, by repressing translation of mRNAs that encode certain DNA replication proteins, including PRI-1. To better understand the mechanism of enhancement, we are now evaluating whether depletion of DNA replication proteins enhances glp-1 in a rad-5 mutant background, where S phase checkpoint control is disabled.

Program #: 1242B Presentation Date/Time: MON, June 27, 2005 1:30-3:00PM Authors: Ikuko Yamamoto, Swathi Arur, Tim Schedl, David Greenstein Session Type: Poster Location: Pauley Pavilion - POSTER BOARD# 1242B
The conserved DEAD-box helicase CGH-1 is both a negative regulator of MAP kinase activation and a MAP kinase substrate in C. elegans oocytes. Ikuko Yamamoto1, Swathi Arur2, Tim Schedl2, David Greenstein1. 1) Dept. of Cell & Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA; 2) Dept. of Genetics, Washington University School of Medicine, St. Louis, MO, USA.

   Oocyte meiotic maturation is an essential biological process required for sexual reproduction. In C. elegans, the MSP hormone triggers meiotic maturation and MAP kinase (MAPK) activation in oocytes. In the absence of sperm, VAB-1 Eph/MSP receptor and sheath cell pathways inhibit meiotic maturation. MSP antagonizes these regulatory circuits to promote meiotic maturation and MAPK activation. A genetic screen for meiotic maturation defects defined the CGH-1 DEAD-box helicase as a negative regulator of MAPK activation in oocytes. In the wild type, MAPK is activated when sperm are present, whereas no MAPK activation is observed in proximal oocytes in females. By contrast, both cgh-1 null and antimorphic (gf) mutations result in oocyte MAPK activation in female genetic backgrounds. In the presence of sperm, MAPK activation extends to distal oocytes, suggesting that cgh-1 mutations affect the response threshold to the MSP signal. In addition, cgh-1 has a cell non-autonomous function in inhibiting basal sheath cell contractions. CGH-1 localizes differently in the presence and absence of sperm. In the presence of sperm CGH-1 localizes to a subcortical band that encircles the oocyte; whereas, in the absence of sperm CGH-1 localizes to large aggregates in this subcortical region. In the cgh-1(ok492) null mutant, PGL-1, MEX-3, OMA-2, and AIR-2 localize abnormally to large filamentous aggregates in oocytes. Interestingly, CGH-1 protein itself forms filamentous aggregates in the cgh-1(tn691gf) antimorph. CGH-1 may function in the remodeling of RNP complexes, as proposed for other DExH/D helicases. In a bioinformatic screen for proteins predicted to contain MAPK docking sites (S. Arur et al., this meeting), CGH-1 was found to possess three D-domains and one FXFP-motif and two potential S/TP phosphorylation sites at S67 and T168. In vitro kinetic data suggest that S67 is the primary phosphorylation site for mammalian ERK2. In vivo data indicate that CGH-1 is phosphorylated on serine and that phosphorylation is eliminated in mpk-1(ga117) null and cgh-1(tn691gf) at the non-permissive temperature. Interestingly, the cgh-1(tn691gf) mutation results from a P68L substitution at the phosporylation site in the Q-motif. The Q-motif of DEAD-box helicases regulates ATP binding and hydrolysis, and affinity for RNA substrates. Our current hypothesis is that CGH-1 phosphorylation by MAPK controls a positive feedback loop for MAPK activation.