The Cdc14 protein phosphatase family regulates diverse cellular processes, including mitotic exit, cytokinesis, and DNA repair. Cdc14 is a member of the dual-specificity subfamily of protein tyrosine phosphatases (PTPs), but has evolved to selectively recognize specific phosphoserine sites deposited by cyclin-dependent kinases. In humans, it has been linked to hearing loss and male infertility conditions. In several fungal species, Cdc14 has been linked to pathogenesis. The Hall lab identified a novel pseudosubstrate motif in the disordered region of all fungal Cdc14 orthologs. Studies with S. cerevisiae Cdc14 revealed this motif interacts with the Cdc14 active site to stimulate the rate-limiting catalytic step and is functionally important for fungal cell wall integrity. The motif may allow cells to dynamically control Cdc14 activity. Metazoan Cdc14 orthologs do not share this sequence motif. However, we demonstrate here that the human Cdc14A (hCdc14A) C-terminal region also contains a similar catalytic enhancer. AlphaFold structural predictions suggest that a conserved alpha helix in the otherwise disordered C-terminus makes extensive contacts with the active site region. Using site-directed mutagenesis, recombinant protein expression and purification, and steady-state kinetic assays, we show that the hCdc14A pseudosubstrate motif accelerates phosphoenzyme hydrolysis by nearly two orders of magnitude, similar to the fungal motif. Using a mass spectrometry-based kinetics assay, we also demonstrate that hCdc14A substrate specificity is nearly identical to fungal Cdc14s, and that the pseudosubstrate motif in hCdc14A does not influence specificity or substrate binding. Current work is 1) testing the hypothesis that the hCdc14A pseudosubstrate motif also binds directly to the active site to accelerate the rate-limiting catalytic step and 2) identifying the specific amino acids required for active site binding. Future work will focus on characterizing the biological significance of this motif and testing its conservation in human Cdc14B. Our results suggest that fungi and metazoa independently evolved pseudosubstrate motifs to similarly regulate Cdc14 catalytic activity. The direct contribution of sequences outside the core phosphatase domain to catalysis appears to be unique among PTP enzymes.
Support or Funding Information
Purdue University Department of Biochemistry, Purdue University College of Agriculture AgSeed Program, Purdue University Summer Undergraduate Research Fellowship
Purdue University Department of Biochemistry, Purdue University College of Agriculture AgSeed Program,amp;nbsp; Purdue University Summer Undergraduate Research Fellowship
