Specific interaction between 14-3-3 isoforms and the human CDC25B phosphatase

CDC25 dual-specificity phosphatases are essential regulators that activate cyclin-dependent kinases (CDKs) at critical stages of the cell cycle. In hu man cells, CDC25A and C are involved in the control of G1/S and G2/M respec tively, whereas CDC25B is proposed to act both in S phase and G2/M. Evidenc e for an interaction between CDC25 phosphatases and members of the 14-3-3 p rotein family has been obtained in vitro and in vivo in several organisms. On the basis of the work performed with CDC25C, it has been proposed that p hosphorylation is required to mediate the interaction with 14-3-3. Here we have examined the molecular basis of the interaction between CDC25B phospha tases and 14-3-3 proteins. We show that in the two-hybrid assay all three s plice variants of CDC25B interact similarly and strongly with 14-3-3 eta, b eta and zeta proteins, but poorly with epsilon and theta. In vitro, CDC25B interacts at a low level with 14-3-3 beta, epsilon, zeta, eta, and theta is oforms. This interaction is not increased upon phosphorylation of CDC25B by CHK1 and is not abolished by dephosphorylation. In contrast, a specific, s trong interaction between CDC25B and 14-3-3 zeta and eta isoforms is reveal ed by a deletion of 288 residues in the amino-terminal region of CDC25B. Th is interaction requires the integrity of Ser 323, although it is independen t of phosphorylation. Thus, interaction between 14-3-3 proteins and CDC25B is regulated in a manner that is different from that with CDC25C. We propos e that, in addition to a low affinity binding site that is available for al l 14-3-3 isoforms, post-translational modification of CDC25B in vivo expose s a high-affinity binding site that is specific for the zeta and eta 14-3-3 isoforms.