cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The cell cycle progression is tightly regulated by specific protein kinases and phosphatases.
• The p34cdc2 kinase complex is a key regulator of the G2 to M phase transition.
• The precise mechanism of p34cdc2 activation by the cdc25 gene product was not fully understood.

Purpose of the Study:

• To elucidate the role of the human cdc25 gene product (cdc25Hs) in regulating p34cdc2 activity.
• To identify the functional domain of the cdc25Hs protein responsible for its activity.

Main Methods:

• Overproduction of full-length cdc25Hs and deletion mutants as glutathione-S-transferase (GST) fusion proteins in bacteria.
• Functional assays including induction of meiotic maturation in Xenopus oocytes, activation of histone H1 kinase activity, and activation of p34cdc2/cyclin B complexes.
• In vitro tyrosine dephosphorylation assays and p-nitrophenylphosphate hydrolysis.

Main Results:

• Recombinant p80GST-cdc25 demonstrated functional activity in various assays, including inducing oocyte maturation and activating p34cdc2 kinase.
• p80GST-cdc25 directly stimulated the tyrosine dephosphorylation of p34cdc2/cyclin complexes.
• Deletion analysis localized the functional domain of cdc25Hs to its carboxy-terminus.

Conclusions:

• The human cdc25Hs protein functions as a phosphatase.
• cdc25Hs directly mediates the tyrosine dephosphorylation and activation of p34cdc2.
• This phosphatase activity is critical for the G2 to M transition in the cell cycle.