The C-terminal tail of the dual-specificity Cdc25B phosphatase mediates modular substrate recognition.
Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases (Cdk/cyclins), thus triggering initiation and progression of successive phases of the cell cycle. In our efforts to elucidate the interaction between Cdc25B and the natural substrate, bis-phosphorylated Cdk2/CycA (Cdk2-pTpY/CycA), we have previously found that the 17 residues of the C-terminal tail mediate a factor of 10 in substrate recognition. In the studies reported here, we localize the majority of this interaction using site-directed mutagenesis to two arginine residues (Arg556 and Arg562) located within this C-terminal region. We also show that the catalytic domain of Cdc25C, which differs most significantly from Cdc25B in this tail region, has a 100-fold lower activity toward Cdk2-pTpY/CycA. We further demonstrate that the proper presentation of the C-terminal tail of Cdc25B can be achieved in a "gain-of-function" chimeric protein consisting of the C-terminal tail of Cdc25B fused onto the catalytic core of Cdc25C. The >10-fold increase in activity seen only in the chimeric protein containing the two critical arginine residues demonstrates that the modular C-terminal tail of Cdc25B is the basis for most of the catalytic advantage of Cdc25B versus Cdc25C toward the Cdk2-pTpY/CycA substrate.
Wilborn
M
M
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Free
S
S
Ban
A
A
Rudolph
J
J
D000595:Amino Acid Sequence; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019925:Cyclin A / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D020687:cdc25 Phosphatases / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D019925:Cyclin A; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D020687:cdc25 Phosphatases