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PTPN12/PTP-PEST Regulates Phosphorylation-Dependent Ubiquitination and Stability Of

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PTPN12/PTP-PEST Regulates Phosphorylation-Dependent Ubiquitination and Stability Of Author Manuscript Published OnlineFirst on May 9, 2018; DOI: 10.1158/0008-5472.CAN-18-0085 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. PTPN12/PTP-PEST Regulates Phosphorylation-Dependent Ubiquitination and Stability of Focal Adhesion Substrates in Invasive Glioblastoma Cells Zhihua Chen*, John E. Morales*, Paola A. Guerrero*, Huandong Sun∑, and Joseph H. McCarty*† *Department of Neurosurgery and ∑Institute for Applied Cancer Sciences, University of Texas M. D. Anderson Cancer Center †Corresponding author Joseph H. McCarty Department of Neurosurgery, Unit 1004 M.D. Anderson Cancer Center 1515 Holcombe Boulevard Houston, TX 77030 Email: [email protected] COI Statement: The authors declare no potential conflicts of interest Running Title: PTP-PEST regulates focal adhesion dynamics in glioma cells Keywords: extracellular matrix, vascular basement membrane, p130Cas, microenvironment, ubiquitin proteasome system, itgb8, glioma 1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 9, 2018; DOI: 10.1158/0008-5472.CAN-18-0085 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract: Glioblastoma (GBM) is an invasive brain cancer with tumor cells that disperse from the primary mass, escaping surgical resection and invariably giving rise to lethal recurrent lesions. Here we report that PTP-PEST, a cytoplasmic protein tyrosine phosphatase, controls GBM cell invasion by physically bridging the focal adhesion protein Crk-associated substrate (Cas) to valosin containing protein (Vcp), an ATP-dependent protein segregase that selectively extracts ubiquitinated proteins from multiprotein complexes and targets them for degradation via the ubiquitin proteasome system. Both Cas and Vcp are substrates for PTP-PEST, with the phosphorylation status of tyrosine 805 (Y805) in Vcp impacting affinity for Cas in focal adhesions and controlling ubiquitination levels and protein stability. Perturbing PTP-PEST- mediated phosphorylation of Cas and Vcp led to alterations in GBM cell invasive growth in vitro and in pre-clinical mouse models. Collectively, these data reveal a novel regulatory mechanism involving PTP-PEST, Vcp, and Cas that dynamically balances phosphorylation-dependent ubiquitination of key focal proteins involved in GBM cell invasion. Statement of significance: PTP-PEST balances GBM cell growth and invasion by interacting with the ATP-dependent ubiquitin segregase Vcp/p97 and regulating phosphorylation and stability of the focal adhesion protein p130Cas. 2 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 9, 2018; DOI: 10.1158/0008-5472.CAN-18-0085 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Patients diagnosed with the malignant cancer GBM have a median survival time of less than two years after diagnosis (1). This poor prognosis is largely due to invasive GBM cells that escape surgical resection and give rise to recurrent lesions that are resistant to chemotherapy such as temozolomide. Targeted therapies such as the anti-vascular endothelial growth factor (VEGF) blocking antibody bevacizumab have yielded disappointing results in GBM clinical trials, with no improvements in overall patient survival. Many patients treated with bevacizumab develop acquired resistance leading to lethal recurrent lesions associated with robust tumor cell invasion (2). While a great deal is known about genes and pathways that promote GBM growth and neovascularization, relatively little is understood about mechanisms that drive GBM cell invasion during progression who following anti-angiogenic therapy. PTP-PEST is a 110 kilo-Dalton (kDa) cytosolic phosphatase that contains a 30 kDa N- terminal catalytic domain and a C-terminus with several proline, glutamate, serine and threonine-rich (PEST) sequences. PTP-PEST plays important roles in promoting tissue morphogenesis, with deletion of the murine PTP-PEST gene (Ptpn12) in all cells leading to embryonic lethality (3). Structural studies of the PTP-PEST catalytic domain reveal that it recognizes phosphotyrosine (pY) motifs in diverse substrates (4), including Rho GEFs, GAPs and focal adhesion proteins such as paxillin and focal adhesion kinase (FAK). Cultured PTP- PEST-/- cells show defective polarity and migration due, in part, to abnormal activation of Rho GTPase signaling and imbalances in cell-ECM adhesion (5,6). Focal adhesions are multiprotein complexes that connect the cytoskeleton to the extracellular matrix (ECM) via integrins (7). Integrin-ECM adhesions continually develop and disassemble as a cell moves, with intermediate structures (nascent adhesions) forming and growing into larger focal adhesions at the leading edge, and subsequently disassembling under the cell body (8). A key regulatory event in the formation and disassembly of focal adhesions is 3 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 9, 2018; DOI: 10.1158/0008-5472.CAN-18-0085 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. post-translational tyrosine phosphorylation, related to activities of tyrosine kinases such as Src and FAK (9). Crk-associated substrate (Cas) is a 130 k-Da protein that was originally identified as a substrate of Src (10). There are five members of the Cas protein family: Cas, also known as breast cancer anti-estrogen resistance (Bcar1), Nedd9, Cass4, and embryonal Fyn substrate (Efs) (11). Cas is a core component of focal adhesions where it bridges multiple signaling proteins to modulate adhesion and motility (12). Cas-deficient cells show normal focal adhesion assembly, but dramatically impaired disassembly, leading to defective migration and invasion (13). Phosphorylation and ubiquitination are tightly coupled processes, with ‘phosphodegron’ sequences in target proteins recruiting E3 ubiquitin ligases and other proteins involved in degradation by the ubiquitin proteasome system (14). Proteins are covalently tagged with ubiquitin via the activities of three enzymes, termed E1, E2 and E3 (15). Ubiquitinated proteins within multicellular complexes are selectively removed via chaperone activities associated with Vcp, a 97 kDa evolutionarily conserved protein (16). Vcp catalyzes the segregation of ubiquitinated proteins from organelles, chromatin, and multiprotein complexes, and promotes destruction by the proteasome (17). Vcp protein contains two AAA+ adenosine triphosphatase (ATPase) domains and an N-domain, which interacts with lipids in the plasma membrane and other proteins, including E2 and E3 enzymes (18). A Peptide:N-glycanase/UBA or UBX (PUB) domain-interaction sequence (PBS) in the Vcp C-terminus mediates associations with PUB domain-containing proteins and other factors (19). Src phosphorylation of Vcp tyrosine 805 (Y805) in the PBS blocks interactions with ubiquitinated PUB domain-containing proteins (20). Here, we report that PTP-PEST dynamically regulates GBM cell invasive growth via the formation of a complex with Cas and Vcp. PTP-PEST enzymatic activities create phosphodegrons in Cas that target it for extraction by Vcp, thus stabilizing focal adhesions and balancing GBM cell invasive growth. Cells expressing PTP-PEST are less invasive due to lower levels of phosphorylated Cas and the presence of more stable focal adhesions. GBM cells that 4 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 9, 2018; DOI: 10.1158/0008-5472.CAN-18-0085 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. express low levels of PTP-PEST display enhanced invasion due to increased Cas phosphorylation and more dynamic focal adhesion disassembly. Collectively, these results not only elucidate novel signaling pathways that link the phosphorylation and ubiquitination pathways. 5 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 9, 2018; DOI: 10.1158/0008-5472.CAN-18-0085 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and Methods Ethics statement Approval for the use of human specimens was obtained from the Institutional Review Board (IRB) at the University of Texas MD Anderson Cancer Center. The IRB waived the requirement for informed consent for previously collected residual tissues from surgical procedures stripped of unique patient identifiers according to the Declaration of Helsinki guidelines. All animal procedures and experiments conducted in this study were reviewed and approved by the University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee (IACUC). GBM cell culturing and analysis Primary human GBM cells from patient samples were cultured in the following growth media: DMEM-F12 (Mediatech), 20 ng/ml EGF and bFGF (Gibco), B27 supplement (Life Technologies) and one unit per ml penicillin-streptomycin (Gibco). After 7 to 10 days spheroids were passaged by accutase (Sigma #A 6964) treatment
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