Session WOC. There are 4 abstracts in this session.



Session: CELL BIOLOGY: REGULATION AND FUNCTION OF PROTEIN PHOSPHORYLATION, time: 09:50 AM - 10:15 AM

Substrate specificity and kinase opposition of PP2A holoenzymes

Arminja Kettenbach

Protein Phosphatase 2A (PP2A) is an essential protein phosphatase that regulates most cellular processes. PP2A forms heterotrimers composed of one catalytic, one scaffolding, and one of several regulatory subunits. In mitosis, specific B regulatory subunit-containing PP2A holoenzymes are inhibited to allow entry into mitosis, while others are active to ensure faithful chromosome segregation and progression through mitosis. While the role of PP2A in many aspects of cellular physiology is well established, only a small number of PP2A dephosphorylation sites are known. This is in part due to the lack of specific and selective inhibitors. Recently it was shown that substrate identification by PP2A occurs through short linear motifs (SLiMs). The PP2A regulatory subunits recognize SLiMs in the substrates resulting in PP2A and substrate interaction. However, it remains unclear how, upon substrate binding, specific phosphorylation sites are selected for dephosphorylation. To address this gap in knowledge, we have developed phosphoproteomic strategies to identify PP2A regulatory subunit-specific dephosphorylation sites. Using our approach, we have identified thousands of dephosphorylation sites of PP2A holoenzymes containing the B55 or B56 regulatory subunit in different cell cycle phases. We discovered that the B-subunits regulate the phosphorylation site preference and thereby kinase opposition of the PP2A catalytic subunit. Furthermore, we discovered that in addition to direct SLiM-mediated interactions, SLiM-mediated scaffolding is a major mechanism of PP2A-B56 substrate recruitment. These differences in site-specific dephosphorylation contribute to our understanding of the differential regulation of PP2A holoenzymes in mitosis, which is essential for successful chromosome segregation.
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Session: CELL BIOLOGY: REGULATION AND FUNCTION OF PROTEIN PHOSPHORYLATION, time: 10:15 AM - 10:40 AM

Pending

Judit Villen

Pending
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Session: CELL BIOLOGY: REGULATION AND FUNCTION OF PROTEIN PHOSPHORYLATION, time: 10:40 AM - 10:55 AM

Dynamic Lysosome Interactomes In vitro and In vivo in Aging and Neurodegeneration

Saadia Hasan2; Ashley Frankenfield1; Michael Ward2; Ling Hao1
1George Washington University, Washington, DC; 2NIH/NINDS, Bethesda, MD

Autophagy-lysosomal pathways are critical to cell survival by recycling misfolded proteins and damaged organelles. This is especially true for neurons, which cannot dilute theses insults by cell division. Lysosome dysfunctions have been associated with aging and neurodegeneration, but the molecular mechanisms remain unclear. Here, we developed lysosomal proximity-labeling proteomics strategies both in vitro (human neuron culture) and in vivo (fixed mouse brain tissues) targeting on the bait protein, lysosomal-associated membrane protein1 (LAMP1), to evaluate lysosome interacting proteins in neurodegeneration. 

In human iPSC-neuron culture, we stably expressed an ascorbate peroxidase (APEX2) enzyme on the LAMP1 protein, which biotinylates proteins within ~20 nm of lysosomes in living neurons. In fixed mouse brain tissues, we stained the tissue slices with LAMP1 antibody and then developed a LAMP1 antibody-guided proximity labeling method which biotinylates lysosomal adjacent proteins. Biotinylated proteins were enriched by streptavidin magnetic beads for subsequent untargeted proteomic analysis. Comprehensive method optimization and validation were conducted for the optimal sensitivity, specificity and confidence of lysosome interacting proteomics. The proximity-labeling results demonstrated specific and complementary identification of lysosome interacting proteins between neurons in culture and mouse brain tissues, in comparison to a cytosolic bait as a spatial control to exclude nonspecific bindings. Using LAMP1-APEX proximity labeling combined with live cell imaging, we have discovered that Annexin A11 (an ALS-associated protein) is used as a molecular tether that links RNA granules to lysosomes during the long-distance transport of RNA binding proteins in neuronal axons (Cell, 2019). Annexin A11 also showed reduced recruitment to the lysosome when we knocked out GRN gene, whose loss-of-function mutations lead to frontotemporal dementia. In summary, the established lysosome proximity labeling methods provide unique insights of dynamic lysosome interactome both in vitro and in vivo with great specificity and sensitivity for the study of lysosome dysfunctions in neurodegeneration. 

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Session: CELL BIOLOGY: REGULATION AND FUNCTION OF PROTEIN PHOSPHORYLATION, time: 10:55 AM - 11:10 AM

Phosphoproteome analysis of metastatic breast cancer cells following treatment with a tumor-selective NQO1 Bioactivatable Drug

Naveen Singh1; Gitanjali Roy1; Emma Doud1; Edward Motea1; Xiumei Huang1; Paul Hergenrother2; David Boothman1; Amber Mosley1
1Indiana University School of Medicine, Indianapolis, IN; 2University of Illinois at Urbana-Champaign,, Urbana, IL

Proteins that are constitutively overexpressed as a consequence of gene amplification or other cellular oncogenic transformation mechanisms are potential targets for precision cancer therapy. One such protein, NAD(P)H:quinone oxidoreductase-1 (NQO1), has been found to be over-expressed in multiple solid cancers including triple negative breast cancer. The NQO1 enzyme overproduction can be targeted using specific quinones which are bioactivated by NQO1 including β-lapachone, deoxynyboquinone, and isobutyl-DNQ (IB-DNQ). In this work, we have explored the phosphoproteome of triple negative breast cancer cells treated with IB-DNQ, the poly ADP ribose polymerase (PARP) inhibitor Rucaparib, or a combination of IB-DNQ and Rucaparib.  Numerous significant changes were observed in the phosphoproteome following combination therapy, including large decreases in phosphorylation associated with active RNA Polymerase II transcription. 

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