Structural and biochemical analysis of atypically low dephosphorylating activity of human dual-specificity phosphatase 28

Abstract

Dual-specificity phosphatases (DUSPs) constitute a subfamily of protein tyrosine phosphatases, and are intimately involved in the regulation of diverse parameters of cellular signaling and essential biological processes. DUSP28 is one of the DUSP subfamily members that is known to be implicated in the progression of hepatocellular and pancreatic cancers, and its biological functions and enzymatic characteristics are mostly unknown. Herein, we present the crystal structure of human DUSP28 determined to 2.1 Å resolution. DUSP28 adopts a typical DUSP fold, which is composed of a central β-sheet covered by α-helices on both sides and contains a well-ordered activation loop, as do other enzymatically active DUSP proteins. The catalytic pocket of DUSP28, however, appears hardly accessible to a substrate because of the presence of nonconserved bulky residues in the protein tyrosine phosphatase signature motif. Accordingly, DUSP28 showed an atypically low phosphatase activity in the biochemical assay, which was remarkably improved by mutations of two nonconserved residues in the activation loop. Overall, this work reports the structural and biochemical basis for understanding a putative oncological therapeutic target, DUSP28, and also provides a unique mechanism for the regulation of enzymatic activity in the DUSP subfamily proteins.

Fig 1. Crystal structure of human DUSP28.

(A) DUSP28 is presented as secondary structure-labeled ribbon models, where α-helices are violet, β-strands are green, and the remaining structures are white. The serine-substituted catalytic residue is shown as a stick model and is labeled as “C103S”. (B) Stereo views of three superimposed DUSP proteins shown as C_α trace representation. Dashed circles indicate the DUSP28-specific N-terminal extension region. (C) The N-terminal region of DUSP proteins. (Left) N-terminal residues ahead of β1 from three DUSP proteins are presented as ribbon models and are labeled. (Right) Sequence alignment of N-terminal residues from DUSP28 and 10 other DUSP proteins ordered by decreasing Z-scores (noted in parenthesis). Four nonconserved proline residues in DUSP28 are labeled in red in both panels.

Fig 2. Structural analysis of the active site of DUSP28.

(A) DUSP28 is shown as electrostatic surface representation together with a phosphate ion presented as sticks in a dashed circle. (B) Stereo views of the active site of DUSP28. Residues constituting the P-loop are shown violet, and a general acid/base residue Asp72 is green. Electrostatic interaction and hydrogen bonds mediated by a phosphate ion are presented as dotted lines. A 2Fo-Fc electron density omit map of the phosphate ion contoured at 5.0 σ is shown also. The temperature factor of the phosphate ion is 34.7. (C) ASC types. Four DUSP proteins are structurally aligned, representing their catalytic residues and general acid/base residues as sticks with labels. PDB codes are 1ZZW for DUSP10, 1MKP for DUSP6, and 2IMG for DUSP23a. (D) Structural comparison with the sulfate ion-bound active site of DUSP10. Active sites of two DUSP proteins are shown as sticks (top) or as electrostatic surface representation (bottom). Anions bound to the active sites are also presented as sticks. Rectangles indicate positions of two nonconserved residues in the PTP signature motif of DUSP28 and those of the corresponding residues of DUSP10.

Fig 3. Effects of nonconserved residues in the P-loop of DUSP28.

(A) Sequence alignment. The sequences of the P-loop of DUSP members and three non-DUSP PTPs are aligned with the PTP signature motif shown at the top. Conserved residues are shaded in cyan. Three nonconserved residues in the P-loop of DUSP28 are marked in red. (B) The active site pocket of twelve DUSP proteins are shown as electrostatic surface representation. Anions bound to the active sites are presented as sticks. (C and D) Structural alignment. The P-loop region of DUSP 28 is superimposed onto that of phosphotyrosine mimetic-bound DUSP22 (C, left; PDB code 1WRM) or DUSP18 (C, right; PDB code 2ESB), or onto that of DUSP3 bound to HEPES (D, left; PDB code 1VHR) or a peptide (D, right; PDB code 1J4X). The fourth and sixth residues in the PTP signature motif of the indicated DUSP proteins are labeled, together with DUSP28’s Pro14 residue, which is located in the N-terminal extension region of the protein (see Fig 1C).

Fig 4. Characterization of phosphatase activity of DUSP28.

Enzymatic assays were performed as described in the Materials and Methods section. RFU stands for relative fluorescence units. pH was 6.0 in A and C−F, and as indicated in panel B. Protein concentrations were 2 μM in B, and as indicated in A and C−F. Substrates were DuFMUP in A−D and indicated phosphoamino acids in E and F. (A and B) Phosphatase activities of wild-type and C103S mutant DUSP28 proteins were assayed at various protein concentrations (A) or at various pH levels (B) and were compared. (C and D) Enzymatic activities of four DUSP proteins (DUSP1, DUSP3, DUSP15, and DUSP28) and three kinds of DUSP28 mutants were assayed at various protein concentrations and were compared. CPD indicates the cysteine protease domain of the Vibrio cholera MARTX toxin protein. It was fused with DUSP28 proteins to improve protein solubility and stability of DUSP28(N105A•R107V) and DUSP28(Y102H) that severely precipitated during purification without tagging with the CPD protein. DUSP28 and DUSP28(C103S) were also tagged with the identical tag for comparing their activity in the same condition. (E and F) Dephosphorylating activities of three DUSP28 constructs toward phosphoamino acids. Phosphotyrosine and phosphothreonine are denoted as p-Tyr and p-Thr, respectively. Phosphatase activities were measured using the malachite green phosphate assay kit as described in Material and Methods section in detail.

Fig 5. Structural analysis of the atypical Tyr102 residue in DUSP28.

(A and B) Structural roles of His123 in DUSP3 (A) and of Tyr102 in DUSP28 (B) are aligned and compared. Dashed lines indicate hydrogen bonds associated with those residues. A water molecule involved in the DUSP3 His123-mediated hydrogen bond network is shown as a red sphere (A, top). Directions of the backbone amide of the catalytic site residue are highlighted by arrows in both structures (A and B, bottom). (C) ϕ and ψ dihedral angles of the catalytic residue of five DUSP proteins and two non-DUSP proteins are presented on the Ramachandran plot. Extended and detailed information about the other members of the DUSP subfamily is shown in S3 Fig.