Showing papers by "Shao En Ong published in 2022"

Proteomic Identification of Phosphorylation-Dependent Septin 7 Interactors that Drive Dendritic Spine Formation

Abstract: Septins are a family of cytoskeletal proteins that regulate several important aspects of neuronal development. Septin 7 (Sept7) is enriched at the base of dendritic spines in excitatory neurons and mediates both spine formation and spine and synapse maturation. Phosphorylation at a conserved C-terminal tail residue of Sept7 mediates its translocation into the dendritic spine head to allow spine and synapse maturation. The mechanistic basis for postsynaptic stability and compartmentalization conferred by phosphorylated Sept7, however, is unclear. We report herein the proteomic identification of Sept7 phosphorylation-dependent neuronal interactors. Using Sept7 C-terminal phosphopeptide pulldown and biochemical assays, we show that the 14-3-3 family of proteins specifically interacts with Sept7 when phosphorylated at the T426 residue. Biochemically, we validate the interaction between Sept7 and 14-3-3 isoform gamma and show that 14-3-3 gamma is also enriched in the mature dendritic spine head. Furthermore, we demonstrate that interaction of phosphorylated Sept7 with 14-3-3 protects it from dephosphorylation, as expression of a 14-3-3 antagonist significantly decreases phosphorylated Sept7 in neurons. This study identifies 14-3-3 proteins as an important physiological regulator of Sept7 function in neuronal development.

Neurodevelopmental disorder associated mutations in TAOK1 reveal its function as a plasma membrane remodeling kinase

Abstract: Mutations in TAOK1, a serine-threonine kinase encoding gene are strongly associated with both autism spectrum disorder (ASD) and neurodevelopmental delay (NDD). However, molecular function of this evolutionarily conserved kinase and the mechanisms through which TAOK1 mutations lead to neuropathology are unknown. Here, we showed that TAOK1 is highly expressed in neurons within the brain, and has a functional role in remodeling the plasma membrane through direct association with phosphoinositides. We characterized four NDD-associated TAOK1 mutations, and demonstrated that these mutations render TAOK1 catalytically dead. Kinase dead TAOK1 mutants were aberrantly trapped in membrane-bound state, which induced exuberant membrane protrusions. Expression of TAOK1 disease mutants in hippocampal neurons led to abnormal growth of the dendritic arbor. The coiled-coil region C-terminal to the kinase domain are predicted to fold into a triple helix. We showed that this triple helix directly bound phospholipids, and was required for both TAOK1 membrane association and induction of aberrant protrusions. Further, TAOK1 mutants were rescued from their membrane-trapped state by exogenous expression of the isolated kinase domain. Utilizing mass-spectrometry, we identified critical residues in the triple helix phosphorylated by TAOK1 that autoregulated its plasma membrane association. These findings define a previously unknown function of TAOK1 as a unique plasma membrane remodeling kinase, and reveal the underlying mechanisms through which TAOK1 dysfunction leads to neurodevelopmental disorders.

Proteomic identification of phosphorylation dependent Septin 7 interactors that drive dendritic spine formation

Abstract: Septins are a family of cytoskeletal proteins that regulate several important aspects of neuronal development. Septin 7 (Sept7) is enriched at the base of dendritic spines in excitatory neurons and mediates both spine formation and spine-synapse maturation. Phosphorylation at a conserved C-terminal tail residue of Sept7 mediates its translocation into the dendritic spine head to allow spine-synapse maturation. The mechanistic basis for postsynaptic stability and compartmentalization conferred by phosphorylated Sept7, however, is not known. We report herein the proteomic identification of Sept7 phosphorylation dependent neuronal interactors. Using Sept7 C-terminal phosphopeptide pulldown and biochemical assays, we show that the 14-3-3 family of proteins specifically interact with Sept7 when phosphorylated at the T426 residue. Biochemically, we validate the interaction between Sept7 and 14-3-3 isoform gamma, and show that 14-3-3 gamma is also enriched in mature dendritic spine head. Further, we demonstrate that interaction of phosphorylated Sept7 with 14-3-3 protects it from dephosphorylation, as expression of a 14-3-3 antagonist significantly decreases phosphorylated Sept7 in neurons. This study identifies 14-3-3 proteins as an important physiological regulator of Sept7 function in neuronal development.

Designed sensors reveal normal and oncogenic Ras signaling in endomembranes and condensates

Abstract: While the ability of Ras to dynamically shuttle around the cell is well characterized, the activity, mechanism of activation, and function of non-plasma membrane-localized Ras is less well understood due to lack of suitable tools. Here, we describe the use of the Latching Orthogonal Cage-Key pRotein (LOCKR) switch platform to generate first-in-class intracellular sensors of endogenous Ras activity (Ras-LOCKR-S) and signaling environment (Ras-LOCKR-PL). By targeting these tools to endomembranes and oncogenic condensates, we defined subcellular Ras activity and identified upstream Ras effectors (guanine exchange factors and SAM68) responsible for signaling in these locations. We also found that Major Vault Protein drives RasG12C inhibitor resistance by enhancing wild type Ras-mediated signaling at the golgi and mutant Ras signaling at mitochondria. Together, these results highlight the importance of non-plasma membrane Ras signaling (endomembranes and condensates), and our new sensors should accelerate the discovery of new therapeutic targets.