Structural basis for recognition of the tumor suppressor protein PTPN14 by the oncoprotein E7 of human papillomavirus

Abstract

Human papillomaviruses (HPVs) are causative agents of various diseases associated with cellular hyperproliferation, including cervical cancer, one of the most prevalent tumors in women. E7 is one of the two HPV-encoded oncoproteins and directs recruitment and subsequent degradation of tumor-suppressive proteins such as retinoblastoma protein (pRb) via its LxCxE motif. E7 also triggers tumorigenesis in a pRb-independent pathway through its C-terminal domain, which has yet been largely undetermined, with a lack of structural information in a complex form with a host protein. Herein, we present the crystal structure of the E7 C-terminal domain of HPV18 belonging to the high-risk HPV genotypes bound to the catalytic domain of human nonreceptor-type protein tyrosine phosphatase 14 (PTPN14). They interact directly and potently with each other, with a dissociation constant of 18.2 nM. Ensuing structural analysis revealed the molecular basis of the PTPN14-binding specificity of E7 over other protein tyrosine phosphatases and also led to the identification of PTPN21 as a direct interacting partner of E7. Disruption of HPV18 E7 binding to PTPN14 by structure-based mutagenesis impaired E7’s ability to promote keratinocyte proliferation and migration. Likewise, E7 binding-defective PTPN14 was resistant for degradation via proteasome, and it was much more effective than wild-type PTPN14 in attenuating the activity of downstream effectors of Hippo signaling and negatively regulating cell proliferation, migration, and invasion when examined in HPV18-positive HeLa cells. These results therefore demonstrated the significance and therapeutic potential of the intermolecular interaction between HPV E7 and host PTPN14 in HPV-mediated cell transformation and tumorigenesis.

Molecular Analysis of the Interaction between Human PTPN21 and the Oncoprotein E7 from Human Papillomavirus Genotype 18

“…this protein was sensitive to proteasome inhibitor MG132 treatment (Yun et al, 2019). Contrastively, the WT and binding-defective PTPN21 protein levels were comparable to each other and hardly affected by the treatment of the proteasome inhibitor MG132 when expressed in HeLa cells (Fig.…”

“…We carried out vacuum degasification for 20 min prior to all experiments. The measurements were performed on a VP-ITC microcalorimetry system (MicroCal, UK) at 25°C and analyzed using Origin software (OriginLab, USA) as described previously ( Yun et al, 2019 ).…”

“…CR3, which covers the C-terminal half of the E7 protein, adopts a homodimeric domain structure ( Liu et al, 2006 ; Ohlenschlager et al, 2006 ) and regulates multiple viral processes involved in high-risk HPV-mediated tumorigenesis by interacting with several host factors ( Avvakumov et al, 2003 ; Berezutskaya and Bagchi, 1997 ; Bodily et al, 2011 ; Brehm et al, 1999 ; Poirson et al, 2017 ; Todorovic et al, 2012 ). We recently determined the crystal structure of HPV18 E7 CR3 bound to the protein tyrosine phosphatase (PTP) domain of PTPN14, and this was the first report of the E7 CR3-mediated complex structure ( Yun et al, 2019 ).…”

“…PTPN14 or PTPD2 is a cytosolic protein that contains an N-terminal four-point-one, ezrin-radixin-moesin (FERM) domain, central proline-rich motifs, and a C-terminal catalytic PTP domain. Given that PTPN14 is an important antitumor protein that cooperates with the p53 transcription factor and negatively regulates Hippo signaling-involved cell proliferation ( Huang et al, 2013 ; Liu et al, 2013 ; Mello et al, 2017 ; Michaloglou et al, 2013 ; Wilson et al, 2014 ), it is not surprising that this protein is targeted by HPV E7 via direct binding, which leads to its proteasomal degradation ( Hatterschide et al, 2019 ; Szalmas et al, 2017 ; White et al, 2016 ; Yun et al, 2019 ). Our previous study also revealed that PTPN21 or PTPD1 can directly interact with CR3 of HPV18 E7 ( Yun et al, 2019 ), consistent with previous systematic proteomic analyses ( Rozenblatt-Rosen et al, 2012 ; White et al, 2012 ).…”

“…Given that PTPN14 is an important antitumor protein that cooperates with the p53 transcription factor and negatively regulates Hippo signaling-involved cell proliferation ( Huang et al, 2013 ; Liu et al, 2013 ; Mello et al, 2017 ; Michaloglou et al, 2013 ; Wilson et al, 2014 ), it is not surprising that this protein is targeted by HPV E7 via direct binding, which leads to its proteasomal degradation ( Hatterschide et al, 2019 ; Szalmas et al, 2017 ; White et al, 2016 ; Yun et al, 2019 ). Our previous study also revealed that PTPN21 or PTPD1 can directly interact with CR3 of HPV18 E7 ( Yun et al, 2019 ), consistent with previous systematic proteomic analyses ( Rozenblatt-Rosen et al, 2012 ; White et al, 2012 ). However, unlike PTPN14, which is defined as a tumor suppressor and particularly controls the Hippo signaling pathway ( Huang et al, 2013 ; Liu et al, 2013 ; Mello et al, 2017 ; Michaloglou et al, 2013 ; Wilson et al, 2014 ), PTPN21 is known to be upregulated in several types of cancer cells and is associated with EGFR activation and intracellular trafficking, implying that this protein might be potentially oncogenic rather than tumor-suppressive ( Carlucci et al, 2010 ; Plani-Lam et al, 2016 ; Roda-Navarro and Bastiaens, 2014 ; Wu et al, 2010 ).…”