Biology subjects

MacMullan, M. A.

Publications and source records attributed to MacMullan, M. A..

3 recordsLinked to original sources

bioRxiv2022

Phospho-proteomic analysis of CAR-T cell signaling following activation by antigen-presenting cancer cells

Chimeric antigen receptors (CARs) are synthetic biomolecules comprised of an extracellular antigen recognition domain and intracellular signaling domains. When expressed in immune cells, CARs direct their host cells to kill diseased cells expressing the antigen recognized by the CAR. Although signaling pathways downstream of CAR activation control the cytotoxic function of CAR-expressing cells, phospho-proteomic studies of CAR signaling have been limited. Most approaches have used antibodies or soluble ligands, rather than cell-displayed antigens, to activate CAR signaling. Here, we demonstrate an efficient and cost-effective label-free phospho-proteomic approach to analyze CAR signaling in immune cells stimulated with antigen-presenting cancer cells. Following co-culture of CAR-T cells with cancer cells, we first preserve phospho-signaling by cross-linking proteins with formalin. Then, we use magnet-activated cell sorting (MACS) to isolate CAR-T cells from the co-culture. Validation experiments demonstrated that formalin fixation did not alter the phospho-proteome and that MACS achieved >90% CAR-T cell purity. Next, we compared the phospho-proteome in CAR-T cells stimulated with either CD19-expressing or non-CD19-expressing SKOV3 ovarian cancer cells. This analysis revealed that CAR signaling activated known pathways including the mitogen- activated protein kinases (MAPKs) ERK1/2. Bioinformatic approaches further showed that CAR activation induced other signaling pathways including the MAPK p38, protein kinase A, and checkpoint kinase 1 (CHK1). Taken together, this work presents an easy and inexpensive method to better understand CAR immunotherapy by label-free phospho-proteomic analysis of CAR signaling in immune cells stimulated by antigen- presenting cancer cells.

immunology↗

bioRxiv2021

Phospho-proteomics reveals that RSK signaling is required for proliferation of natural killer cells stimulated with IL-2 or IL-15

Natural killer (NK) cells are cytotoxic lymphocytes that play a critical role in the innate immune system. Although cytokine signaling is crucial for the development, expansion, and cytotoxicity of NK cells, the signaling pathways stimulated by cytokines are not well understood. Here, we sought to compare the early signaling dynamics induced by the cytokines interleukin (IL)-2 and IL-15 using liquid chromatography-mass spectrometry (LC-MS)-based phospho-proteomics. Following stimulation of the immortalized NK cell line NK-92 with IL-2 or IL-15 for 5, 10, 15, or 30 minutes, we identified 8,692 phospho-peptides from 3,023 proteins. Comparing the kinetic profiles of 3,619 fully quantified phospho-peptides, we found that IL-2 and IL-15 induced highly similar signaling in NK-92 cells. Among the IL-2/IL-15-regulated phospho-sites were both well-known signaling events like the JAK/STAT pathway and novel signaling events with potential functional significance including LCP1 Ser5, PAK2 Ser141, and STK17B Ser12. Using bioinformatic approaches, we sought to identify kinases regulated by IL-2/IL-15 stimulation and found that the p90 ribosomal S6 kinase (p90RSK) family was activated by both cytokines. Using pharmacological inhibitors, we then discovered that RSK signaling is required for IL-2 and IL-15-induced proliferation in NK-92 cells. Taken together, our analysis represents the first phospho-proteomic characterization of cytokine signaling in NK cells and increases our understanding of how cytokine signaling regulates NK cell function.

immunology↗

bioRxiv2021

A Bidirectional Switch in the Shank3 Phosphorylation State Biases Synapses toward Up or Down Scaling

Homeostatic synaptic plasticity requires widespread remodeling of synaptic signaling and scaffolding networks, but the role of posttranslational modifications in this process has not been systematically studied. Using deepscale, quantitative analysis of the phosphoproteome in mouse neocortical neurons, we found wide-spread and temporally complex changes during synaptic scaling up and down. We observed 424 bidirectionally modulated phosphosites that were strongly enriched for synapse-associated proteins, including S1539 in the ASD-associated synaptic scaffold protein Shank3. Using a parallel proteomic analysis performed on Shank3 isolated from rat neocortical neurons by immunoaffinity, we identified two sites that were hypo-phosphorylated during scaling up and hyper-phosphorylated during scaling down: one (rat S1615) that corresponded to S1539 in mouse, and a second highly conserved site, rat S1586. The phosphorylation status of these sites modified the synaptic localization of Shank3 during scaling protocols, and dephosphorylation of these sites via PP2A activity was essential for the maintenance of synaptic scaling up. Finally, phosphomimetic mutations at these sites prevented scaling up but not down, while phosphodeficient mutations prevented scaling down but not up. Thus, an activity-dependent switch between hypo- and hyperphosphorylation at S1586/ S1615 of Shank3 enables scaling up or down, respectively. Collectively our data show that activity-dependent phosphoproteome dynamics are important for the functional reconfiguration of synaptic scaffolds, and can bias synapses toward upward or downward homeostatic plasticity.

neuroscience↗