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The Caenorhabditis elegans CDT-2 ubiquitin ligase is required for attenuation of EGFR signalling in vulva precursor cells.

Poulin GB, Ahringer J - BMC Dev. Biol. (2010)

Bottom Line: We have characterised CDT-2's role during vulva development and found that it is a novel attenuator of LET-23 signalling.In this study, we have shown that CDT-2 and CUL-4, members of the CUL-4/DDB-1/CDT-2 E3 ubiquitin ligase complex attenuate LET-23 signalling in vulval precursor cells.This work has uncovered a novel function for the CUL-4/DDB-1/CDT-2 E3 ligase that may be relevant for its mammalian oncogenic activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK. Gino.Poulin@manchester.ac.uk

ABSTRACT

Background: Attenuation of the EGFR (Epidermal Growth Factor Receptor) signalling cascade is crucial to control cell fate during development. A candidate-based RNAi approach in C. elegans identified CDT-2 as an attenuator of LET-23 (EGFR) signalling. Human CDT2 is a component of the conserved CDT2/CUL4/DDB1 ubiquitin ligase complex that plays a critical role in DNA replication and G2/M checkpoint. Within this complex, CDT2 is responsible for substrate recognition. This ubiquitin ligase complex has been shown in various organisms, including C. elegans, to target the replication-licensing factor CDT1, and the CDK inhibitor p21. However, no previous link to EGFR signalling has been identified.

Results: We have characterised CDT-2's role during vulva development and found that it is a novel attenuator of LET-23 signalling. CDT-2 acts redundantly with negative modulators of LET-23 signalling and CDT-2 or CUL-4 downregulation causes persistent expression of the egl-17::cfp transgene, a marker of LET-23 signalling during vulva development. In addition, we show that CDT-2 physically interacts with SEM-5 (GRB2), a known negative modulator of LET-23 signalling that directly binds LET-23, and provide genetic evidence consistent with CDT-2 functioning at or downstream of LET-23. Interestingly, both SEM-5 and CDT-2 were identified independently in a screen for genes involved in receptor-mediated endocytosis in oocytes, suggesting that attenuation of LET-23 by CDT-2 might be through regulation of endocytosis.

Conclusions: In this study, we have shown that CDT-2 and CUL-4, members of the CUL-4/DDB-1/CDT-2 E3 ubiquitin ligase complex attenuate LET-23 signalling in vulval precursor cells. In future, it will be interesting to investigate the potential link to endocytosis and to determine whether other signalling pathways dependent on endocytosis, e.g. LIN-12 (Notch) could be regulated by this ubiquitin ligase complex. This work has uncovered a novel function for the CUL-4/DDB-1/CDT-2 E3 ligase that may be relevant for its mammalian oncogenic activity.

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CDT-2 can interact physically with SEM-5, the C. elegans GRB2 homologue. Pull down using in vitro labelled CDT-2 and purified GST, GST::SLI-1 or GST::SEM-5 shows that SEM-5 but not SLI-1 or GST can interact with CDT-2. As an additional negative control, we used labelled luciferase and did not detect interactions with either SEM-5 or SLI-1. Input is set at 5%.
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Figure 4: CDT-2 can interact physically with SEM-5, the C. elegans GRB2 homologue. Pull down using in vitro labelled CDT-2 and purified GST, GST::SLI-1 or GST::SEM-5 shows that SEM-5 but not SLI-1 or GST can interact with CDT-2. As an additional negative control, we used labelled luciferase and did not detect interactions with either SEM-5 or SLI-1. Input is set at 5%.

Mentions: Since an absence of genetic interaction can sometimes suggest a physical interaction [35,36], we tested whether CDT-2 could physically interact with either SLI-1 or SEM-5. We produced in vitro labelled CDT-2 and purified SLI-1 and SEM-5 from bacteria. We found that CDT-2 could physically associate with SEM-5, but not with SLI-1 (Figure 4). Together, the genetic and physical interaction data suggest that CDT-2 may prevent excessive signalling regulating LET-23 through SEM-5.


The Caenorhabditis elegans CDT-2 ubiquitin ligase is required for attenuation of EGFR signalling in vulva precursor cells.

Poulin GB, Ahringer J - BMC Dev. Biol. (2010)

CDT-2 can interact physically with SEM-5, the C. elegans GRB2 homologue. Pull down using in vitro labelled CDT-2 and purified GST, GST::SLI-1 or GST::SEM-5 shows that SEM-5 but not SLI-1 or GST can interact with CDT-2. As an additional negative control, we used labelled luciferase and did not detect interactions with either SEM-5 or SLI-1. Input is set at 5%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2984460&req=5

Figure 4: CDT-2 can interact physically with SEM-5, the C. elegans GRB2 homologue. Pull down using in vitro labelled CDT-2 and purified GST, GST::SLI-1 or GST::SEM-5 shows that SEM-5 but not SLI-1 or GST can interact with CDT-2. As an additional negative control, we used labelled luciferase and did not detect interactions with either SEM-5 or SLI-1. Input is set at 5%.
Mentions: Since an absence of genetic interaction can sometimes suggest a physical interaction [35,36], we tested whether CDT-2 could physically interact with either SLI-1 or SEM-5. We produced in vitro labelled CDT-2 and purified SLI-1 and SEM-5 from bacteria. We found that CDT-2 could physically associate with SEM-5, but not with SLI-1 (Figure 4). Together, the genetic and physical interaction data suggest that CDT-2 may prevent excessive signalling regulating LET-23 through SEM-5.

Bottom Line: We have characterised CDT-2's role during vulva development and found that it is a novel attenuator of LET-23 signalling.In this study, we have shown that CDT-2 and CUL-4, members of the CUL-4/DDB-1/CDT-2 E3 ubiquitin ligase complex attenuate LET-23 signalling in vulval precursor cells.This work has uncovered a novel function for the CUL-4/DDB-1/CDT-2 E3 ligase that may be relevant for its mammalian oncogenic activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK. Gino.Poulin@manchester.ac.uk

ABSTRACT

Background: Attenuation of the EGFR (Epidermal Growth Factor Receptor) signalling cascade is crucial to control cell fate during development. A candidate-based RNAi approach in C. elegans identified CDT-2 as an attenuator of LET-23 (EGFR) signalling. Human CDT2 is a component of the conserved CDT2/CUL4/DDB1 ubiquitin ligase complex that plays a critical role in DNA replication and G2/M checkpoint. Within this complex, CDT2 is responsible for substrate recognition. This ubiquitin ligase complex has been shown in various organisms, including C. elegans, to target the replication-licensing factor CDT1, and the CDK inhibitor p21. However, no previous link to EGFR signalling has been identified.

Results: We have characterised CDT-2's role during vulva development and found that it is a novel attenuator of LET-23 signalling. CDT-2 acts redundantly with negative modulators of LET-23 signalling and CDT-2 or CUL-4 downregulation causes persistent expression of the egl-17::cfp transgene, a marker of LET-23 signalling during vulva development. In addition, we show that CDT-2 physically interacts with SEM-5 (GRB2), a known negative modulator of LET-23 signalling that directly binds LET-23, and provide genetic evidence consistent with CDT-2 functioning at or downstream of LET-23. Interestingly, both SEM-5 and CDT-2 were identified independently in a screen for genes involved in receptor-mediated endocytosis in oocytes, suggesting that attenuation of LET-23 by CDT-2 might be through regulation of endocytosis.

Conclusions: In this study, we have shown that CDT-2 and CUL-4, members of the CUL-4/DDB-1/CDT-2 E3 ubiquitin ligase complex attenuate LET-23 signalling in vulval precursor cells. In future, it will be interesting to investigate the potential link to endocytosis and to determine whether other signalling pathways dependent on endocytosis, e.g. LIN-12 (Notch) could be regulated by this ubiquitin ligase complex. This work has uncovered a novel function for the CUL-4/DDB-1/CDT-2 E3 ligase that may be relevant for its mammalian oncogenic activity.

Show MeSH