12 Feb 2016

Regulation of Cdc25B during G2/M Progression

Puji Astuti, February 2010
The University of Queensland

Abstract
Cdc25B is a key regulator for entry into mitosis, and stringent control of cdc25B expression and activation is essential for normal G2/M progression and exit from G2 phase checkpoint arrest. Cdc25B activity is highly regulated by multiple mechanisms including phosphorylations, changes in localisation and binding of 14-3-3 dimer. The phosphorylation of cdc25B at the N-terminal regulatory domain by a number of kinases has been shown to positively regulate the activity of the phosphatase  as well as influencing its stability. In contrast, phosphorylation by checkpoint kinases such as Chk1 and MK-2 act as inhibitory signals, blocking cdc25B activity during normal G2/M progression and in response to G2 phase checkpoint signaling. These inhibitory  phosphorylations occur within the conserved 14-3-3 binding motifs and create 14-3-3 binding sites.  There are three 14-3-3 binding sites in cdc25B, Ser323 which is a high affinity binding site, Ser151 and Ser230 in the N-terminal domain which appear to act in concert and are relatively lower affinity binding sites. The binding of 14-3-3 to these sites has been shown to regulate both the localisation and activity of cdc25B. The phosphorylation of the inhibitory 14-3-3 binding sites appears to be relatively stable through G2 phase and into mitosis,  when cdc25B is known to be active. This observation raises the question of how cdc25B is activated prior to entry into mitosis, and how the inhibitory signals are relieved to allow cdc25B activation?  The fact that some of the other phosphorylation sites on cdc25B also appear to regulate cdc25B activity and localisation suggest that these phosphorylation sites may also  in some way influence 14-3-3 binding. Here I have examined five of these phosphorylation sites, Ser169, Ser186/7, Ser249, Ser321 and Ser353, chosen due to their proximity to the three 14-3-3 binding sites.

Data presented in this thesis have identified several mechanisms regulating cdc25B during G2/M transition. Cdc25B is shown to be destabilised in response to MAPK activation during TPA-induced G2 phase, and this is  shown to be mediated through Ser249 phosphorylation, independently of 14-3-3 interaction.  Phosphorylation of this site may be targeted by cdk1 in mitosis and may be involved  in the destablisation of cdc25B in mitosis. Thus this single site may be regulated by different stimuli to result in the same endpoint.

Phosphorylation on Ser353 is responsible for cytoplasmic retention of cdc25B, an effect which is also independent of 14-3-3 interaction. The other three proximal sites Ser321, Ser169 and Ser186/7, modulate cdc25B activity by disrupting 14-3-3 binding. In this study I have demonstrated that the inhibitory Ser323 phosphorylation is maintained through mitosis, but phosphorylation of Ser321 disrupts 14-3-3 binding to Ser323, mimicking the effect of inhibiting Ser323 phosphorylation on both  cdc25B activity and localisation. Unphosphorylated Ser321 appears to have a role in stabilization of 14-3-3 binding to Ser323, and the introduction of a highly charged phosphoryl group at this site disrupts 14-3-3 binding. The mitotic Ser321 phosphorylation seems to not interfere with the Ser323 phosphorylation but rather exposes Ser323 to phosphatases which dephosphorylate Ser323. The Ser323 phosphorylation was increased in mitosis and dependent on cdk1 activity. This ensures the maximal activation of cdc25B in mitosis by blocking the rebinding of 14-3-3 to this site.

Phosphorylation of Ser169 or Ser186/7  activates cdc25B  through a distinct mechanism, disrupting 14-3-3 binding to Ser151 and/or Ser230 with the consequences of the disruption being cdc25B relocation to the cytoplasm and increased access of substrates to the catalytic site. Introducing a phosphoryl group to either Ser169 or Ser186/7 did not affect the phosphorylation status of Ser151 or Ser230 but rather appeared to reduce the affinity of 14-3-3 binding to its N-terminal sites, thereby activating cdc25B. This study provides a mechanism by which the inhibitory 14-3-3 binding can be disrupted to allow the activation of cdc25B without dephosphorylation of the N-terminal 14-3-3 binding sites.

In conclusion, the study conducted here demonstrates the mechanisms through which cdc25B is regulated during G2/M progression. Phosphorylation of cdc25B by a number of kinases affects cdc25B stability and activity through 14-3-3 binding-dependent and independent mechanisms. The kinases which are responsible for phosphorylation, in 
particular the N-terminal sites, are likely to be critically important for G2/M progression, and particularly for exit from the G2 phase checkpoint arrest which is specifically dependent on cdc25B.

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