Remote hot spots mediate protein substrate recognition for the Cdc25 phosphatase

Abstract

Cdc25B is a phosphatase that catalyzes the dephosphorylation and activation of the cyclin-dependent kinases, thus driving cell cycle progression. We have identified two residues, R488 and Y497, located >20 Å from the active site, that mediate protein substrate recognition without affecting activity toward small-molecule substrates. Injection of Cdc25B wild-type but not the R488L or Y497A variants induces germinal vesicle breakdown and cyclin-dependent kinase activation in Xenopus oocytes. The conditional knockout of the cdc25 homolog (mih1) in Saccharomyces cerevisiae can be complemented by the wild type but not by the hot spot variants, indicating that protein substrate recognition by the Cdc25 phosphatases is an essential and evolutionarily conserved feature. protein-protein interactions The hot spot theory of protein-protein interactions suggests that the binding energy between two proteins is governed in large part by just a few critical residues at the binding interface. In typical interfaces of 1,200-2,000 Å 2 , only Ϸ5% of the residues from each protein contribute Ͼ2 kcal͞mol to the binding interaction (1). Although the establishment of the hot spot theory in pioneering studies by the Wells and Fersht laboratories using the high-affinity systems of human growth hormone binding protein (2) and barnase-barstar (3) has been subsequently validated in other similar systems, it is less clear to what extent hot spot theory applies to the transient protein-protein interactions involved in enzyme catalysis. Given that many such transient interactions govern crucial intracellular signaling processes (for example, protein kinases and phosphatases), it is of great interest to identify and characterize such potential hot spots. Identification of these hot spots would allow for experiments that probe the details of specific transient protein-protein interactions in vivo. Additionally, such sites could serve as targets for the development of small-molecule compounds that interfere specifically with these protein-protein interactions and serve as leads for drug development.Cdc25 phosphatases are regulators of the eukaryotic cell cycle that dephosphorylate and thus activate the main gatekeepers of this process, the cyclin-dependent kinases (Cdks͞cyclins) (4). Confirming their importance in cell cycle regulation, Cdc25A and Cdc25B contribute to oncogenic transformation and are overexpressed in many diverse types of cancer (5). Crystal structures of the catalytic domains of Cdc25A and Cdc25B have revealed a shallow active site with no obvious features for mediating substrate recognition, suggesting a broad protein interface rather than a lock-and-key interaction (6, 7). This finding is confirmed by the activity of the Cdc25 phosphatases toward Cdk͞cyclin protein substrates, which is six orders of magnitude greater than that of peptidic substrates that contain the same primary sequence (8). The shallow active site also correlates with the lack of potent and specific inhibitors of the Cdc25 phosphatases, despite extensive eff...

Mitotic Phosphorylation of Cdc25B Ser321 Disrupts 14-3-3 Binding to the High Affinity Ser323 Site

“…The mutant Cdc25B had severely reduced substrate binding. This was clearly demonstrated with the substrate trapping mutant of Cdc25B3, where the substrate binding Y511A mutation was introduced, Cdk1 phosphorylated at Tyr 15 (pTyr 15 ) and its major cyclin partner cyclin B1 was reduced by Ͼ90% (Fig. 1A).…”

“…The antibody complexes were precipitated with 50 l of 50% slurry of protein A-Sepharose (Amersham Biosciences), washed three times with 14-3-3 lysis buffer, and eluted with SDS sample buffer. Samples were resolved on 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antibodies against Cdc25B, 14-3-3␤, -, or -⑀ (Santa Cruz Biotechnology), Myc tag (Cell Signaling), cyclin A (Santa Cruz Biotechnology), cyclin B (1), pTyr 15 Cdk1 and phospho-Ser 216 Cdc25C (Cell Signaling), phospho-Ser 323 Cdc25B (20), and detected using ECL (PerkinElmer Life Sciences). The phospho-Cdc25B and phospho-Cdc25C antibodies both detected the phosphorylation of Ser 323 on Cdc25B and were used interchangeably through this work.…”

“…The Cdc25B isoform is a dual-specificity phosphate, which is proposed to function as an initiator of entry into mitosis (1,2) The phosphatase activates its substrates cyclin/Cdks by dephosphorylating inhibitory residues on the Cdk subunits at Thr 14 and Tyr 15 , which drives entry into mitosis (1,3,4). The phosphatase also activates cyclin A/Cdk2 in the early G 2 phase (5).…”

“…In this study, we have produced an inert form of Cdc25B by mutating the substrate binding sites, to allow us to examine its 14-3-3 binding and localization without perturbing the cell cycle. Tyr 511 (using Cdc25B3 numbering) was identified as a critical substrate binding site residue, and its mutation has been reported to reduce the activity of Cdc25B to Ͻ0.5% compared with wild type Cdc25B, without altering the active site (15,16 (17). As these regions in Cdc25B and Cdc25C are identical, we reasoned that these residues would likely perform a similar role in regulating Cdc25B activity.…”

Kinetic Template‐Guided Tethering of Fragments

“…[70,71] However, a hotspot over 20 Å from the active site pocket was reported for cdc25B as crucial for binding to cdk substrates, and may potentially represent a new pocket to target for cdc25-cdk inhibition. [72][73][74] Disulfide tethering was initially chosen as a means to target this pocket in a site specific manner, but difficulties were encountered. During the synthesis of unsymmetrical disulfides, it was noted that a significant number of compounds were highly labile, and rapidly led to statistical mixtures of products whilst as a solution in organic * Carried out by RHN as part of MRes degree.…”