Henry T. Phan

Bio: Henry T. Phan is an academic researcher from Cornell University. The author has contributed to research in topics: Palmitoylation & Transmembrane protein. The author has co-authored 2 publications.

A palmitoylation code controls PI4KIIIα complex formation and PI(4,5)P2 homeostasis at the plasma membrane.

Abstract: PI4KIIIα is the major enzyme responsible for generating the phosphoinositide PI(4)P at the plasma membrane. This lipid kinase forms two multicomponent complexes, both including a palmitoylated anchor, EFR3. Whereas both PI4KIIIα complexes support production of PI(4)P, the distinct functions of each complex and mechanisms underlying the interplay between them remain unknown. Here, we present roles for differential palmitoylation patterns within a tri-Cys motif in EFR3B (Cys5/Cys7/Cys8) in controlling the distribution of PI4KIIIα between these two complexes at the plasma membrane and corresponding functions in phosphoinositide homeostasis. Spacing of palmitoyl groups within three doubly palmitoylated EFR3B "lipoforms" affects both its interactions with TMEM150A, a transmembrane protein governing formation of a PI4KIIIα complex functioning in rapid PI(4,5)P2 resynthesis following PLC signaling, and its partitioning within liquid-ordered and -disordered regions of the plasma membrane. This work identifies a palmitoylation code in controlling protein-protein and protein-lipid interactions affecting a plasma membrane-resident lipid biosynthetic pathway.

A palmitoylation code controls PI4KIIIα complex formation and PI(4,5)P2 homeostasis at the plasma membrane

Abstract: PI4KIIIα is the major enzyme responsible for generating the phosphoinositide PI(4)P at the plasma membrane. This lipid kinase forms two multicomponent complexes, both including a palmitoylated anchor, EFR3. Whereas both PI4KIIIα complexes support production of PI(4)P, the distinct functions of each complex and mechanisms underlying the interplay between them remain unknown. Here, we present roles for differential palmitoylation patterns within a tri-Cys motif in EFR3B (Cys5/Cys7/Cys8) in controlling the distribution of PI4KIIIα between these two complexes at the plasma membrane and corresponding functions in phosphoinositide homeostasis. Spacing of palmitoyl groups within three doubly palmitoylated EFR3B “lipoforms” affects both its interactions with TMEM150A, a transmembrane protein governing formation of a PI4KIIIα complex functioning in rapid PI(4,5)P2 resynthesis following PLC signaling, and its partitioning within liquid-ordered and -disordered regions of the plasma membrane. This work identifies a palmitoylation code in controlling protein–protein and protein–lipid interactions affecting a plasma membrane-resident lipid biosynthetic pathway. SUMMARY STATEMENT Different palmitoylation patterns on a lipid kinase adaptor protein control partitioning of the kinase between two spatiotemporally and functionally distinct complexes within the plasma membrane.

Molecular mechanisms of PI4K regulation and their involvement in viral replication

Abstract: Lipid phosphoinositides are master signaling molecules in eukaryotic cells and key markers of organelle identity. Because of these important roles, the kinases and phosphatases that generate phosphoinositides must be tightly regulated. Viruses can manipulate this regulation, with the Type III phosphatidylinositol 4‐kinases (PI4KA and PI4KB) being hijacked by many RNA viruses to mediate their intracellular replication through the formation of phosphatidylinositol 4‐phosphate (PI4P)‐enriched replication organelles (ROs). Different viruses have evolved unique approaches toward activating PI4K enzymes to form ROs, through both direct binding of PI4Ks and modulation of PI4K accessory proteins. This review will focus on PI4KA and PI4KB and discuss their roles in signaling, functions in membrane trafficking and manipulation by viruses. Our focus will be the molecular basis for how PI4KA and PI4KB are activated by both protein‐binding partners and post‐translational modifications, with an emphasis on understanding the different molecular mechanisms viruses have evolved to usurp PI4Ks. We will also discuss the chemical tools available to study the role of PI4Ks in viral infection.

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes

Abstract: Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.

Knockdown of Transmembrane Protein 150A (TMEM150A) Results in Increased Production of Multiple Cytokines.

Abstract: Lipopolysaccharide (LPS)-induced signaling through Toll-like receptor 4 (TLR4) is mediated by the plasma membrane lipid, phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] and its derivatives diacylglycerol and inositol trisphosphate. Levels of PI(4,5)P2 are controlled enzymatically and fluctuate in LPS-stimulated cells. Recently, transmembrane protein 150A (TMEM150A/TM6P1/damage-regulated autophagy modulator 5) has been shown to regulate PI(4,5)P2 production at the plasma membrane by modifying the composition of the phosphatidylinositol 4-kinase enzyme complex. To determine if TMEM150A function impacts TLR4 signaling, TMEM150A was knocked down in TLR4-expressing epithelial cells and cytokine expression quantified after LPS stimulation. In general, decreased expression of TMEM150A led to increased levels of LPS-induced cytokine secretion and transcript levels. Unexpectedly, knockdown of TMEM150A in a lung epithelial cell line (H292) also led to increased cytokine levels in the unstimulated conditions suggesting TMEM150A plays an important role in cellular homeostasis. Future studies will investigate if TMEM150A plays a similar role for other TLR agonists and in other cell lineages.

Periodicity, Mixed-Mode Oscillations, and Multiple Timescale in a Phosphoinositide-Rho GTPase Network

Abstract: While rhythmic contractile behavior is prevalent on the cortex of living cells, current experimental observation and mechanistic understanding primarily tackle a small subset of dynamical behavior including excitable or periodic events that can be described by simple activator-delayed inhibitor mechanisms. In this work we found that the oscillatory activation of Rho GTPase in nocodazole-treated mitotic rat basophilic leukemia (RBL) cells exhibited both simple and complex mixed-mode oscillations, with periodicity ranging from 30 sec to 5 min. Complex mixed-mode oscillations require at least two instability-generating mechanisms. We show that Rho oscillations at the fast timescale (20-30 sec) is regulated by phosphatidylinositol (3,4,5)-trisphosphate (PIP3) via an activator-delayed inhibitor mechanism, while the period of the slow reaction (minutes) is regulated by phosphatidylinositol 4-phosphate (PI(4)P) via an activator-substrate depletion mechanism where replenishment of phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is rate-limiting. Conversion from simple to complex oscillations could be induced by modulating PIP3 metabolism or membrane contact site dynamics. In particular, a period-doubling intermediate can be captured by PTEN depletion. Both period doubling and mixed-mode oscillations are intermediate states towards chaos. Collectively, these results suggest that phosphoinositide-Rho GTPase signaling network is poised at the edge of chaos and small parameter changes in the phosphoinositide metabolism network could confer cells the flexibility to rapidly transit into a number of ordered states with different periodicities.