However, RNAi of ddb 1 did not cause obvious LET 23 signalling defects. This might be due to incomplete knockdown, or alternatively, CDT 2 and CUL 4 could act independently selleck compound of DDB 1 in this context. We also provided in vitro evidence that CDT 2 can associate with SEM 5 directly. CDT 2 and SEM 5 share two functions, they attenuate LET 23 signalling during vulva development and are required for receptor mediated endocytosis during oogenesis. Linking these two functions together, we hypothesise that the CUL 4 DDB 1 CDT 2 E3 ubiquitin ligase might interact with SEM 5 to affect LET 23 endocytosis and attenuation of the LET 23 signalling cascade. However, our studies do not rule out an effect through other signalling pathways involved in vulva development such as Wnt or Notch.

The rereplication defect and LET 23 signalling The rereplication defect caused by depletion of CDT 2 or CUL 4 has been previously characterised as well as the cell cycle arrest phenotype. AV-951 However, it is difficult to explain that these defects could cause exces sive LET 23 signalling during vulva development. Indeed, experiments using hydroxyurea to arrest the VPC cell cycle have shown that egl 17 expression remains restricted to P6. p. Therefore, a replication block after first division as in the case of cul 4 deletion mutants is unlikely to cause persistent expression of egl 17,cfp. Furthermore, we observed increased LET 23 sig nalling in cdt 2 RNAi animals, and an increase in vulval fate adoption in gap 1, cdt 2 animals, under con ditions where the cell cycle proceeds normally.

There fore, the role of CDT 2 in preventing rereplication is likely to be independent of its function in preventing excess LET 23 signalling. CDT 2 may attenuate LET 23 signalling as a component of the CUL 4 DDB 1 E3 ligase complex RNAi by feeding in C. elegans has significant false nega tive rate, but false positives are rare. Hence, the finding that a deletion of cul 4 can cause the same vul val phenotype as cdt 2 suggests that both CUL 4 and CDT 2 are novel attenua tors of LET 23 signalling. Since purification of the human CUL 4 DDB 1 E3 ligase complex by different groups has identified CDT 2 as the substrate recognition unit, it is likely that CUL 4 and CDT 2 function together in the process of LET 23 attenuation. Even though, this study cannot rule out that CUL 4 could act in parallel to attenuate LET 23 signalling.

SEM 5 and attenuation of signalling SEM 5, the GRB2 homologue, has two activities linked to Receptor Tyrosine Kinase signalling. It can act as a positive regulator of signalling by http://www.selleckchem.com/products/baricitinib-ly3009104.html recruiting SOS 1, or act as a negative modulator by recruiting SLI 1, the CBL homologue. SLI 1 is an E3 ubiqui tin ligase that can associate with SEM 5 to target RTKs and promote lysosomal degradation.