About

John Sondek is a Professor of Pharmacology at the University of North Carolina at Chapel Hill, with a joint appointment in Biochemistry and Biophysics. His research focuses on understanding signal transduction cascades controlled by heterotrimeric G proteins (Gabg) and small GTPases related to Ras. He primarily employs biophysical techniques to dissect these cascades at atomic and molecular resolution, aiming to understand and manipulate cellular processes. His work includes guiding rational drug design, developing high throughput screens to identify modulators of key signaling components, and producing biosensors to monitor these cascades in real-time within cells. Sondek has made significant contributions to the understanding of the regulation and activation of phospholipase C-γ isozymes, GTPase and GEF network connectivity, and the structural basis of signaling mechanisms, with implications for cancer and other diseases.

Research topics

• Biology
• Computer Science
• Computational biology
• Materials science
• Neuroscience
• Biochemistry
• Immunology
• Genetics
• Nanotechnology
• Pathology
• Telecommunications
• Medicine
• Cell biology
• Chemistry

Selected publications

• A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes

  UNC Libraries · 2025-09-27

  articleOpen accessSenior author
  PublisherDOI

• Optogenetic control of PLC-γ1 activity directs cell motility

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-10-11

  preprintOpen access

  ABSTRACT Phospholipase C-γ1 (PLC-γ1) signaling is required for mesenchymal chemotaxis, but is it sufficient to bias motility? PLC-γ1 enzyme activity is basally autoinhibited, and light-controlled membrane recruitment of wild-type PLC-γ1 (OptoPLC-γ1) in Plcg1- null fibroblasts does not trigger lipid hydrolysis, complicating efforts to isolate its contribution. Utilizing cancer-associated mutations to investigate the regulatory logic of PLC-γ1, we demonstrate that a hallmark of enzyme activity, phosphorylated Tyr783 (pTyr783), is not a proxy for activity level, but is rather a marker of dysregulated autoinhibition. Accordingly, OptoPLC-γ1 with a deregulating mutation (P867R, S345F, or D1165H) exhibits elevated phosphorylation, and membrane localization of such is sufficient to activate substrate hydrolysis and concomitant motility responses. In particular, local recruitment of OptoPLC-γ1 S345F polarizes cell motility and migration on demand. This response is spatially dose-sensitive and only partially reduced by blocking canonical PLC-γ1 signaling yet is lipase-dependent. Our findings reframe the interpretation of PLC-γ1 regulation and demonstrate that local activation of PLC-γ1 is sufficient to direct cell motility.

  PublisherOA PDFDOI

• Hyperactive PLCG1 induces cell-autonomous and bystander T cell activation and drug resistance

  EMBO Reports · 2025-08-12 · 5 citations

  articleOpen access

  Phospholipase C gamma 1 (PLCG1) has been identified as the most frequently mutated gene in adult T-cell leukemia/lymphoma, suggesting a critical function of PLCG1 in driving T cell activation. However, it remains unclear how these mutations regulate T cell physiology and pathology. Here, we investigate three common leukemia/lymphoma-associated mutations (R48W, S345F, and D1165H). We discover that these mutations induce hyperactive T cell signaling and cause pro-survival phenotypes. PLCG1 mutants enhance LAT condensation, calcium influx, and ERK activation. They also promote T cell proliferation, upregulate cell adhesion molecules, induce cell aggregation, and confer resistance to Vorinostat, an FDA-approved drug for cutaneous T-cell lymphoma. The resistance depends on ERK signaling and can be reversed with an ERK inhibitor. Interestingly, PLCG1 mutants also induce bystander drug resistance in nearby cells expressing wild-type PLCG1. Mechanistically, alpha smooth muscle actin, which is specifically induced by PLCG1 mutants, directly binds PLCG1 to promote its activation. These results demonstrate that hyperactive PLCG1 promotes T cell survival and drug resistance by inducing non-canonical signaling.

  PublisherOA PDFDOI

• A high-throughput assay platform to discover small molecule activators of the phospholipase PLC-γ2 to treat Alzheimer's disease

  Journal of Biological Chemistry · 2025-02-25 · 4 citations

  articleOpen access

  A naturally occurring missense variant of the phospholipase C isozyme, PLC-γ2, harboring a single substitution (P522R) protects against several neurodegenerative diseases, including Alzheimer's disease. The phospholipase activity of PLC-γ2 (P522R) is slightly elevated relative to its WT counterpart, and the general consensus is that this increased activity in microglia confers protection against neurodegeneration. In order to phenocopy this protection, we have developed a high-throughput assay to identify small molecule activators of PLC-γ2. The assay takes advantage of the fluorescent reporter, XY-69, embedded in lipid vesicles to readout the allosteric activation of PLC-γ2. The assay is highly reproducible and capable of identifying compounds with a large range of efficacies. A series of secondary assays have been established to define the selectivity of compounds for PLC-γ2, establish relevant activation of PLC-γ2 by compounds in a microglia cell line, and measure affinities between PLC-γ2 and hit compounds. The established workflow was prototyped using approximately 6000 compounds to produce several promising hits, but more importantly, enables screens of much larger chemical libraries to identify selective activators of PLC-γ2 to be used as chemical probes and drug leads.

  PublisherOA PDFDOI

• 783 Oncogenic PLCG1 drives aberrant intra- and intercellular T cell activation

  Regular and Young Investigator Award Abstracts · 2025-11-01

  articleOpen access
  PublisherOA PDFDOI

• Hyperactive PLCG1 drives non-canonical signaling to promote cell survival

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-12-18 · 2 citations

  preprintOpen access

  ) was identified as the most frequently mutated gene in adult T-cell leukemia/lymphoma, suggesting a critical function of PLCG1 in driving T cell activation. However, it remains unclear how these mutations regulate T cell physiology and pathology. Here we investigated three common leukemia/lymphoma associated mutations (R48W, S345F, and D1165H). We discovered that these mutations induced hyperactive T cell signaling and caused pro-survival phenotypes. PLCG1 mutants enhanced LAT condensation, calcium influx, and ERK activation. They promoted T cell proliferation, induced cell aggregation, and rendered resistance to vorinostat, an FDA-approved drug for cutaneous T-cell lymphoma. The resistance to vorinostat depended on ERK signaling and can be reversed with an ERK inhibitor. Mechanistically, alpha smooth muscle actin, which was specifically induced by PLCG1 mutants, directly bound PLCG1 to promote its activation. Together, these results demonstrated that hyperactive PLCG1 promoted T cell survival and drug resistance through inducing non-canonical signaling.

  PublisherOA PDFDOI

• De novo design of stable proteins that efficaciously inhibit oncogenic G proteins

  UNC Libraries · 2024-07-27

  articleOpen access

  Many protein therapeutics are competitive inhibitors that function by binding to endogenous proteins and preventing them from interacting with native partners. One effective strategy for engineering competitive inhibitors is to graft structural motifs from a native partner into a host protein. Here, we develop and experimentally test a computational protocol for embedding binding motifs in de novo designed proteins. The protocol uses an "inside-out" approach: Starting with a structural model of the binding motif docked against the target protein, the de novo protein is built by growing new structural elements off the termini of the binding motif. During backbone assembly, a score function favors backbones that introduce new tertiary contacts within the designed protein and do not introduce clashes with the target binding partner. Final sequences are designed and optimized using the molecular modeling program Rosetta. To test our protocol, we designed small helical proteins to inhibit the interaction between Gαq and its effector PLC-β isozymes. Several of the designed proteins remain folded above 90°C and bind to Gαq with equilibrium dissociation constants tighter than 80nM. In cellular assays with oncogenic variants of Gαq , the designed proteins inhibit activation of PLC-β isozymes and Dbl-family RhoGEFs. Our results demonstrate that computational protein design, in combination with motif grafting, can be used to directly generate potent inhibitors without further optimization via high throughput screening or selection.

  PublisherDOI

• Designer proteins that competitively inhibit Gαq by targeting its effector site

  UNC Libraries · 2024-07-27

  articleOpen access
  PublisherDOI

• Mechanism of Hyperactive PLCγ1 Signaling in T-Cell Leukemia/Lymphoma

  Blood · 2024-11-05

  article

  The phospholipase PLCγ1 is essential for T cell activation. It is frequently mutated in Adult T-cell leukemia/lymphoma (ATLL), Peripheral T-cell lymphomas (PTCL), and Cutaneous T-cell lymphoma (CTCL). However, the molecular mechanisms of PLCγ1 mutation in pathogenesis of these T cell malignancies remains unclear. Liquid-liquid phase separation (LLPS) is an emerging principle in organizing cellular signaling. The dysregulation of LLPS derived by aberrant protein aggregation is progressively implicated as pathological mechanism in tumorigenesis. We previously reported that PLCγ1 structurally promotes LLPS of linker for activation of T cells (LAT) to form condensates in physiological TCR signaling. These lead us to hypothesize that leukemia associated PLCγ1 mutations enhanced TCR signaling and T cell growth by promoting abnormal LLPS. Three frequent and persistent PLCγ1 mutations (R48W, S345F, and D1165H) were chosen in this study. The mutated PLCγ1 recombinant proteins significantly boosted membrane associated LAT condensation compared with wild type (WT) at physiological concentration in a membrane-based biochemical reconstitution system. Increased condensation of LAT was confirmed in live T cells through total internal reflection fluorescence (TIRF) microcopy. Consequently, the downstream signaling including calcium influx and ERK phosphorylation were enhanced in T cell cancer lines harboring PLCγ1 mutations. PLCγ1 mutations also significantly induced the secretion of cytokines and chemokines such as IL-2 and CXCL10. Ectopically expression of PLCγ1 mutants in human primary T cells promotes T cell proliferation, CD69 expression, and effector memory T cell development. Together, these results suggested that leukemia-associated PLCγ1 mutations drive abnormal LLPS to boost TCR signaling and promote T cell proliferation. We also determined if PLCγ1 mutations render any drug resistance by testing a few T cell lymphoma drugs in clinical use. The T cell lymphoma cell line Hut78 expressing PLCγ1 mutations showed resistance to histone deacetylase (HDAC) inhibitors. Moreover, PLCγ1 mutations reduced apoptosis under HDAC inhibition, with a company of enhanced Bcl-2 expression. The Human protein kinase phosphorylation array assay was performed to reveal the mechanism underlying drug resistance. We found that inhibiting MAPK could reverse the HDAC inhibitor resistance at non-cytotoxicity concentrations. These results demonstrated that PLCγ1-MAPK signaling axis confers the HDAC inhibitor resistance. In summary, leukemia-associated PLCγ1 mutations facilitate abnormal LLPS formation to enhance the T cell receptor signaling. Gain-of-function of PLCγ1 mutations cause drug resistance to HDAC inhibitors, which can be rehabilitated by inhibiting the MAPK pathway. Our work reveals molecular mechanisms underlying T cell lymphomagenesis and provides solutions to patients suffering from drug resistance.

  PublisherDOI

• Structural basis for the activation of PLC-γ isozymes by phosphorylation and cancer-associated mutations

  UNC Libraries · 2024-08-14

  articleOpen access

  Direct activation of the human phospholipase C-γ isozymes (PLC-γ1, -γ2) by tyrosine phosphorylation is fundamental to the control of diverse biological processes, including chemotaxis, platelet aggregation, and adaptive immunity. In turn, aberrant activation of PLC-γ1 and PLC-γ2 is implicated in inflammation, autoimmunity, and cancer. Although structures of isolated domains from PLC-γ isozymes are available, these structures are insufficient to define how release of basal autoinhibition is coupled to phosphorylation-dependent enzyme activation. Here, we describe the first high-resolution structure of a full-length PLC-γ isozyme and use it to underpin a detailed model of their membrane-dependent regulation. Notably, an interlinked set of regulatory domains integrates basal autoinhibition, tyrosine kinase engagement, and additional scaffolding functions with the phosphorylation-dependent, allosteric control of phospholipase activation. The model also explains why mutant forms of the PLC-γ isozymes found in several cancers have a wide spectrum of activities, and highlights how these activities are tuned during disease.

  PublisherDOI

Recent grants

• NIH Grant R01GM062299

  NIH · $2.3M · 2013

• NIH Grant R01GM120291

  NIH · $1.4M · 2021

• NIH Grant R01GM098894

  NIH · $834k · 2015

• Regulation of phospholipase C

  NIH · $7.2M · 1998–2023

• Cancer Genetics Research Program

  NIH · $80.2M · 1997–2027

Frequent coauthors

• T. Kendall Harden

  University of North Carolina at Chapel Hill

  88 shared

• Kent L. Rossman

  59 shared

• Jason T. Snyder

  University of North Carolina at Chapel Hill

  49 shared

• David P. Siderovski

  University of North Texas Health Science Center

  34 shared

• Channing J. Der

  University of North Carolina at Chapel Hill

  24 shared

• Stephanie N. Hicks

  National Institute of Environmental Health Sciences

  24 shared

• David K. Worthylake

  Louisiana State University Health Sciences Center New Orleans

  23 shared

• Marielle E. Yohe

  22 shared

Education

• Postdoc

  Yale University

  1996

• Ph.D., Biochemistry

  Johns Hopkins School of Medicine

  1992

• B.S.

  University of Rochester

  1985

Awards & honors

• Winner, GlaxoSmithKline Discovery Fast Track Competition (20…
• Chair, Gordon Research Conference (2011)
• Keynote Lecture, SER-CAT symposium (2008)
• Pew Scholar in the Biomedical Sciences (1999)