Fig 2: HPV18 E7 relies on interaction with PTPN14 to promote keratinocyte proliferation and migration.The indicated HPV18 E7 constructs were stably expressed in HaCaT cells. *P < 0.05; **P < 0.01; ***P < 0.001 in the Student t test. The numerical data are included in S1 Data. (A) Protein levels of endogenous PTPN14 upon expression of the indicated E7 constructs were detected by immunoblotting. Relative protein amounts were quantified using Vilber Lourmat. (B) mRNA levels of PTPN14 and three indicated keratinocyte differentiation markers with or without WT or the AA mutant HPV18 E7 were measured by qRT-PCR. (C) Proliferation assay. Growth curves of HaCaT cells stably expressing the indicated constructs were compared from day 0 to day 4. (D) Clonogenic assay using HaCaT cells grown for 6 days. (Left) Representative cell images stained with crystal violet. (Right) Crystal violet-stained colony area per well was quantified and shown as a bar graph. (E) Migration assay. Motility of HaCaT cells with or without WT or the AA mutant HPV18 E7 were analyzed and compared. (Left) Representative cell images stained with crystal violet. The scale bars indicate 50 μm. (Right) The area of migrated cells per field was displayed as bar graphs. AA, R84A and L91A; HA, hemagglutinin; HPV, human papillomavirus; Hsp90, heat shock protein 90; KRT, keratin; n.s., not significant; PTPN14, nonreceptor-type protein tyrosine phosphatase 14; qRT-PCR, quantitative real-time polymerase chain reaction; WT, wild type.

Fig 3: Analysis of the binding specificity of HPV18 E7 to PTP proteins.(A) Sequence alignment. Sequence of the HPV18 E7–binding interface of PTPN14 is aligned with those of the corresponding regions of 16 other nonreceptor-type PTP proteins. The secondary structures of PTPN14 are shown at the top. Conserved residues are shaded in orange. Four hydrophobic residues in PTPN14 that play a key role in the intermolecular interaction with HPV18 E7 are marked by red arrows. (B) Structural alignment. The PTP domains of two representative nonreceptor-type PTP proteins, PTP1B and PTPN3, are superimposed onto that of PTPN14 bound to HPV18 E7. Shown in sticks and labeled are PTPN14 residues interacting with Arg84 or Leu91 of HPV18 E7 and PTP1B and PTPN3 residues corresponding to the represented PTPN14 residues. (C) ITC measurements. The KD values between the C-terminal domain of HPV18 E7 and a series of PTP domains were calculated, as in Fig 1B. Deducible dissociation constants were shown only when PTPN14 or PTPN21 was subjected to the experiment. (D) SEC-MALS analysis. The complex formation was shown between the dimeric C-terminal domain of HPV18 E7 and the monomeric PTP domain of human PTPN21. Solid lines, molar masses in kg/mol; dotted lines, refractive indexes. Constructs are listed in S1 Table. The numerical data are included in S1 Data. a.a., amino acids; HPV, human papillomavirus; ITC, isothermal titration calorimetry; MBP, maltose binding protein; Mn, number-average molar mass; Mw, weight-average molar mass; PTP, protein tyrosine phosphatase; PTPN14, nonreceptor-type PTP 14; SEC-MALS, size-exclusion chromatography–multiangle light scattering.

Fig 4: Crystal structure of the PTPN14–HPV18 E7 complex.(A) Crystal structure of the complex. PTPN14 (violet) and HPV E7 (green) are presented as ribbon drawings with the secondary structure labels according to the order of their appearance in the primary sequence. The catalytic cysteine residue of PTPN14 (Cys1121) and zinc ion (present as a red sphere)–coordinating four cysteine residues of HPV18 E7 (Cys65, Cys68, Cys98, and Cys101) are shown in sticks and labeled. (B) Intermolecular interaction is shown in two views. PTPN14 and HPV18 E7 residues critically involved in the complex formation are shown in sticks and labeled. Residues that are selected to be mutated in c are marked with underlines. Dotted lines represent electrostatic interaction between Glu1095 of PTPN14 and Arg84 of HPV18 E7. (C) ITC measurements. The KD values between wild-type or mutant proteins were calculated as was in Fig 1B. Introduction of R84A and L91A mutations into HPV18 E7 or F1044S, G1055Q, and E1095A mutations into PTPN14 abrogated the binding interaction between the two proteins. HPV, human papillomavirus; ITC, isothermal titration calorimetry; MBP, maltose binding protein; PTPN14, nonreceptor-type protein tyrosine phosphatase 14.

Fig 5: Tumor-suppressive activity of PTPN14 was recovered in HeLa cells by disrupting interaction with HPV18 E7.The indicated PTPN14 constructs were transiently (A and B) or constitutively (C–E) expressed in HeLa cells. *P < 0.05; **P < 0.01; ***P < 0.001 in the Student t test. The numerical data are included in S1 Data. (A) Dual luciferase assay. Transcriptional activity of YAP with or without WT or the SQA mutant PTPN14 was monitored by quantifying luciferase activity. (B) mRNA levels of CYR61 and CTGF with or without WT or the SQA mutant PTPN14 were measured by qRT-PCR and compared. (C) Subcellular localization of YAP/TAZ with or without wild-type or mutant PTPN14 was detected by immunostaining using an anti-YAP/TAZ antibody (green), anti-PTPN14 antibody (red), and DAPI (blue). Nuclear exclusion of YAP/TAZ was indicated by arrows. Percentages of YAP/TAZ located in both nucleus and cytoplasm (“N/C”) or in cytoplasm (“C”) are shown as graphs. Images were captured using 40x oil immersion objectives. The scale bars indicate 10 μm. (D) Short-term cell proliferation assay. Growth curves of HeLa cells constitutively expressing empty-vector control (“Mock”) or wild-type or the SQA mutant PTPN14 are compared from day 0 to days 4. (E) Clonogenic assay using HeLa cells grown for 15 days. (Left) Representative cell images stained with crystal violet. (Right) The number of colonies were quantified as bar graphs. (F) Migration and invasion assay. Motility and invasiveness of HeLa cells with or without wild-type or the SQA mutant PTPN14 were analyzed and compared. (Left) Representative cell images stained with crystal violet. The scale bars indicate 50 μm. (Right) The number of migrated (top) or invasive (bottom) cells were quantified as bar graphs. CTGF, connective tissue growth factor; CYR61, cysteine-rich angiogenic inducer 61; HPV, human papillomavirus; n.s., not significant; PTPN14, nonreceptor-type protein tyrosine phosphatase 14; qRT-PCR, quantitative real-time polymerase chain reaction; SQA, F1044S, G1055Q, and E1095A; TAZ, tafazzin; WT, wild type; YAP, yes-associated protein.