Browsing by Subject "Protein Serine-Threonine Kinases"
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Item Open Access ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1.(Proceedings of the National Academy of Sciences of the United States of America, 2023-04) Mayro, Benjamin; Hoj, Jacob P; Cerda-Smith, Christian G; Hutchinson, Haley M; Caminear, Michael W; Thrash, Hannah L; Winter, Peter S; Wardell, Suzanne E; McDonnell, Donald P; Wu, Colleen; Wood, Kris C; Pendergast, Ann MarieThe hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC.Item Open Access Host protein kinases required for SARS-CoV-2 nucleocapsid phosphorylation and viral replication.(Science signaling, 2022-10) Yaron, Tomer M; Heaton, Brook E; Levy, Tyler M; Johnson, Jared L; Jordan, Tristan X; Cohen, Benjamin M; Kerelsky, Alexander; Lin, Ting-Yu; Liberatore, Katarina M; Bulaon, Danielle K; Van Nest, Samantha J; Koundouros, Nikos; Kastenhuber, Edward R; Mercadante, Marisa N; Shobana-Ganesh, Kripa; He, Long; Schwartz, Robert E; Chen, Shuibing; Weinstein, Harel; Elemento, Olivier; Piskounova, Elena; Nilsson-Payant, Benjamin E; Lee, Gina; Trimarco, Joseph D; Burke, Kaitlyn N; Hamele, Cait E; Chaparian, Ryan R; Harding, Alfred T; Tata, Aleksandra; Zhu, Xinyu; Tata, Purushothama Rao; Smith, Clare M; Possemato, Anthony P; Tkachev, Sasha L; Hornbeck, Peter V; Beausoleil, Sean A; Anand, Shankara K; Aguet, François; Getz, Gad; Davidson, Andrew D; Heesom, Kate; Kavanagh-Williamson, Maia; Matthews, David A; tenOever, Benjamin R; Cantley, Lewis C; Blenis, John; Heaton, Nicholas SMultiple coronaviruses have emerged independently in the past 20 years that cause lethal human diseases. Although vaccine development targeting these viruses has been accelerated substantially, there remain patients requiring treatment who cannot be vaccinated or who experience breakthrough infections. Understanding the common host factors necessary for the life cycles of coronaviruses may reveal conserved therapeutic targets. Here, we used the known substrate specificities of mammalian protein kinases to deconvolute the sequence of phosphorylation events mediated by three host protein kinase families (SRPK, GSK-3, and CK1) that coordinately phosphorylate a cluster of serine and threonine residues in the viral N protein, which is required for viral replication. We also showed that loss or inhibition of SRPK1/2, which we propose initiates the N protein phosphorylation cascade, compromised the viral replication cycle. Because these phosphorylation sites are highly conserved across coronaviruses, inhibitors of these protein kinases not only may have therapeutic potential against COVID-19 but also may be broadly useful against coronavirus-mediated diseases.Item Open Access Sucrose Nonfermenting 1-Related Protein Kinase 1 Phosphorylates a Geminivirus Rep Protein to Impair Viral Replication and Infection.(Plant physiology, 2018-09) Shen, Wei; Bobay, Benjamin G; Greeley, Laura A; Reyes, Maria I; Rajabu, Cyprian A; Blackburn, R Kevin; Dallas, Mary Beth; Goshe, Michael B; Ascencio-Ibáñez, Jose T; Hanley-Bowdoin, LindaGeminiviruses are single-stranded DNA viruses that infect a wide variety of plants and cause severe crop losses worldwide. The geminivirus replication initiator protein (Rep) binds to the viral replication origin and catalyzes DNA cleavage and ligation to initiate rolling circle replication. In this study, we found that the Tomato golden mosaic virus (TGMV) Rep is phosphorylated at serine-97 by sucrose nonfermenting 1-related protein kinase 1 (SnRK1), a master regulator of plant energy homeostasis and metabolism. Phosphorylation of Rep or the phosphomimic S97D mutation impaired Rep binding to viral DNA. A TGMV DNA-A replicon containing the Rep S97D mutation replicated less efficiently in tobacco (Nicotiana tabacum) protoplasts than in wild-type or Rep phosphorylation-deficient replicons. The TGMV Rep-S97D mutant also was less infectious than the wild-type virus in Nicotiana benthamiana and was unable to infect tomato (Solanum lycopersicum). Nearly all geminivirus Rep proteins have a serine residue at the position equivalent to TGMV Rep serine-97. SnRK1 phosphorylated the equivalent serines in the Rep proteins of Tomato mottle virus and Tomato yellow leaf curl virus and reduced DNA binding, suggesting that SnRK1 plays a key role in combating geminivirus infection. These results established that SnRK1 phosphorylates Rep and interferes with geminivirus replication and infection, underscoring the emerging role for SnRK1 in the host defense response against plant pathogens.Item Open Access Targeted genomic CRISPR-Cas9 screen identifies MAP4K4 as essential for glioblastoma invasion.(Scientific reports, 2019-09) Prolo, Laura M; Li, Amy; Owen, Scott F; Parker, Jonathon J; Foshay, Kara; Nitta, Ryan T; Morgens, David W; Bolin, Sara; Wilson, Christy M; Vega L, Johana CM; Luo, Emily J; Nwagbo, Gigi; Waziri, Allen; Li, Gordon; Reimer, Richard J; Bassik, Michael C; Grant, Gerald AAmong high-grade brain tumors, glioblastoma is particularly difficult to treat, in part due to its highly infiltrative nature which contributes to the malignant phenotype and high mortality in patients. In order to better understand the signaling pathways underlying glioblastoma invasion, we performed the first large-scale CRISPR-Cas9 loss of function screen specifically designed to identify genes that facilitate cell invasion. We tested 4,574 genes predicted to be involved in trafficking and motility. Using a transwell invasion assay, we discovered 33 genes essential for invasion. Of the 11 genes we selected for secondary testing using a wound healing assay, 6 demonstrated a significant decrease in migration. The strongest regulator of invasion was mitogen-activated protein kinase 4 (MAP4K4). Targeting of MAP4K4 with single guide RNAs or a MAP4K4 inhibitor reduced migration and invasion in vitro. This effect was consistent across three additional patient derived glioblastoma cell lines. Analysis of epithelial-mesenchymal transition markers in U138 cells with lack or inhibition of MAP4K4 demonstrated protein expression consistent with a non-invasive state. Importantly, MAP4K4 inhibition limited migration in a subset of human glioma organotypic slice cultures. Our results identify MAP4K4 as a novel potential therapeutic target to limit glioblastoma invasion.