title: 14-3-3 acts as an intramolecular bridge to regulate cdc25B localization and activity.
abstract: One of the major regulators of mitosis in somatic cells is cdc25B. cdc25B is tightly regulated at multiple levels. The final activation step involves the regulated binding of 14-3-3 proteins. Previous studies have demonstrated that Ser-323 is a primary 14-3-3 binding site in cdc25B, which influences its activity and cellular localization. 14-3-3 binding to this site appeared to interact with the N-terminal domain of cdc25B to regulate its activity. The presence of consensus 14-3-3 binding sites in the N-terminal domain suggested that the interaction is through direct binding of the 14-3-3 dimer to sites in the N-terminal domain. We have identified Ser-151 and Ser-230 in the N-terminal domain as functional 14-3-3 binding sites utilized by cdc25B in vivo. These low affinity sites cooperate to bind the 14-3-3 dimer bound to the high affinity Ser-323 site, thus forming an intramolecular bridge that constrains cdc25B structure to prevent access of the catalytic site. Loss of 14-3-3 binding to either N-terminal site relaxes cdc25B structure sufficiently to permit access to the catalytic site, and the nuclear export sequence located in the N-terminal domain. Mutation of the Ser-323 site was functionally equivalent to the mutation of all three sites, resulting in the complete loss of 14-3-3 binding, increased access of the catalytic site, and access to nuclear localization sequence.
authors.affiliation: Cancer Biology Program, Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland 4102, Australia.
authors.forename: Nichole
authors.initials: N
authors.lastname: Giles
authors.forename: Alistair
authors.initials: A
authors.lastname: Forrest
authors.forename: Brian
authors.initials: B
authors.lastname: Gabrielli
mesh_terms: D048948:14-3-3 Proteins; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D019281:Dimerization; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D013329:Structure-Activity Relationship; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*; D020687:cdc25 Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D010446:Peptide Fragments; D014446:Tyrosine 3-Monooxygenase; C517008:CDC25B protein, human; D020687:cdc25 Phosphatases