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.
Highlights
The final activation step involves the regulated binding of 14-3-3 proteins
Its protein levels are tightly controlled, accumulating in early G2 phase and destroyed by proteasome-mediated destruction during early mitosis [12]. It accumulates in the nucleus in early G2 phase, but at prophase it translocates to cytoplasm, and its appearance in this compartment is tightly associated with the activation of cdc2/cyclin B1 in the cytoplasm and the appearance of cytoplasmic mitotic features such as increased microtubule nucleation at the centrosome and changes in microtubule dynamics [3, 6]. cdc25B is responsible for the activation of not
To identify which of these sites were capable of supporting 14-3-3 binding in vitro, bacterially expressed GST-cdc25B3 N-terminal protein fragments were produced with point mutations of the Ser residues Ser-151 and Ser-230 changed to Ala, either individually or in tandem, and a truncation of the N terminus that included only the Ser-49 site deleting the Ser-151 and
Summary
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. 14-3-3 binding to this site appeared to interact with the N-terminal domain of cdc25B to regulate its activity. Whereas cdc25B dominant-negative overexpression blocked all evidence of mitosis, cells overexpressing the cdc25C dominant-negative mutant often displayed evidence of mitotic events in the cytoplasm, with a complete block in nuclear mitotic events [3]. Its protein levels are tightly controlled, accumulating in early G2 phase and destroyed by proteasome-mediated destruction during early mitosis [12] It accumulates in the nucleus in early G2 phase, but at prophase it translocates to cytoplasm, and its appearance in this compartment is tightly associated with the activation of cdc2/cyclin B1 in the cytoplasm and the appearance of cytoplasmic mitotic features such as increased microtubule nucleation at the centrosome and changes in microtubule dynamics [3, 6]. The factors that control 14-3-3 binding are likely to be important regulators of both normal G2/M progression and in the G2 checkpoint control
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