14-3-3 acts as an intramolecular bridge to regulate cdc25B localization and activity.
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.
Giles
Nichole
N
Cancer Biology Program, Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland 4102, Australia.
Forrest
Alistair
A
Gabrielli
Brian
B
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*
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D010446:Peptide Fragments; D014446:Tyrosine 3-Monooxygenase; C517008:CDC25B protein, human; D020687:cdc25 Phosphatases