Fengsong Wang

Septin 7 Interacts with Centromere-associated Protein E and Is Required for Its Kinetochore Localization

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore. Septin (SEPT) belongs to a conserved family of polymerizing GTPases localized to the metaphase spindle during mitosis. Previous study showed that SEPT2 depletion results in chromosome mis-segregation correlated with a loss of centromere-associated protein E (CENP-E) from the kinetochores of congressing chromosomes (1). However, it has remained elusive as to whether CENP-E physically interacts with SEPT and how this interaction orchestrates chromosome segregation in mitosis. Here we show that SEPT7 is required for a stable kinetochore localization of CENP-E in HeLa and MDCK cells. SEPT7 stabilizes the kinetochore association of CENP-E by directly interacting with its C-terminal domain. The region of SEPT7 binding to CENP-E was mapped to its C-terminal domain by glutathione S-transferase pull-down and yeast two-hybrid assays. Immunofluorescence study shows that SEPT7 filaments distribute along the mitotic spindle and terminate at the kinetochore marked by CENP-E. Remarkably, suppression of synthesis of SEPT7 by small interfering RNA abrogated the localization of CENP-E to the kinetochore and caused aberrant chromosome segregation. These mitotic defects and kinetochore localization of CENP-E can be successfully rescued by introducing exogenous GFP-SEPT7 into the SEPT7-depleted cells. These SEPT7-suppressed cells display reduced tension at kinetochores of bi-orientated chromosomes and activated mitotic spindle checkpoint marked by Mad2 and BubR1 labelings on these misaligned chromosomes. These findings reveal a key role for the SEPT7-CENP-E interaction in the distribution of CENP-E to the kinetochore and achieving chromosome alignment. We propose that SEPT7 forms a link between kinetochore distribution of CENP-E and the mitotic spindle checkpoint.

TIP150 interacts with and targets MCAK at the microtubule plus ends

The microtubule (MT) cytoskeleton orchestrates the cellular plasticity and dynamics that underlie morphogenesis and cell division. Growing MT plus ends have emerged as dynamic regulatory machineries in which specialized proteins—called plus‐end tracking proteins (+TIPs)—bind to and control the plus‐end dynamics that are essential for cell division and migration. However, the molecular mechanisms underlying the plus‐end regulation by +TIPs at spindle and astral MTs have remained elusive. Here, we show that TIP150 is a new +TIP that binds to end‐binding protein 1 (EB1) in vitro and co‐localizes with EB1 at the MT plus ends in vivo. Suppression of EB1 eliminates the plus‐end localization of TIP150. Interestingly, TIP150 also binds to mitotic centromere‐associated kinesin (MCAK), an MT depolymerase that localizes to the plus end of MTs. Suppression of TIP150 diminishes the plus‐end localization of MCAK. Importantly, aurora B‐mediated phosphorylation disrupts the TIP150–MCAK association in vitro. We reason that TIP150 facilitates the EB1‐dependent loading of MCAK onto MT plus ends and orchestrates the dynamics at the plus end of MTs.

Helicobacter pylori VacA Disrupts Apical Membrane-Cytoskeletal Interactions in Gastric Parietal Cells

Helicobacter pylori persistently colonize the human stomach and have been linked to atrophic gastritis and gastric carcinoma. Although it is well known that H. pylori infection can result in hypochlorhydria, the molecular mechanisms underlying this phenomenon remain poorly understood. Here we show that VacA permeabilizes the apical membrane of gastric parietal cells and induces hypochlorhydria. The functional consequences of VacA infection on parietal cell physiology were studied using freshly isolated rabbit gastric glands and cultured parietal cells. Secretory activity of parietal cells was judged by an aminopyrine uptake assay and confocal microscopic examination. VacA permeabilization induces an influx of extracellular calcium, followed by activation of calpain and subsequent proteolysis of ezrin at Met469-Thr470, which results in the liberation of ezrin from the apical membrane of the parietal cells. VacA treatment inhibits acid secretion by preventing the recruitment of H,K-ATPase-containing tubulovesicles to the apical membrane of gastric parietal cells. Electron microscopic examination revealed that VacA treatment disrupts the radial arrangement of actin filaments in apical microvilli due to the loss of ezrin integrity in parietal cells. Significantly, expression of calpain-resistant ezrin restored the functional activity of parietal cells in the presence of VacA. Proteolysis of ezrin in VacA-infected parietal cells is a novel mechanism underlying H. pylori-induced inhibition of acid secretion. Our results indicate that VacA disrupts the apical membrane-cytoskeletal interactions in gastric parietal cells and thereby causes hypochlorhydria.

PALS1 specifies the localization of ezrin to the apical membrane of gastric parietal cells

The ERM (ezrin/radixin/moesin) proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to regulated HCl secretion in gastric parietal cells. Here, we show that the integrity of ezrin is essential for parietal cell activation and provide the first evidence that ezrin interacts with PALS1, an evolutionarily conserved PDZ and SH3 domain-containing protein. Our biochemical study verifies that ezrin binds to PALS1 via its N terminus and is co-localized with PALS1 to the apical membrane of gastric parietal cells. Furthermore, our study shows that PALS1 is essential for the apical localization of ezrin, as either suppression of PALS1 protein accumulation or deletion of the PALS1-binding domain of ezrin eliminated the apical localization of ezrin. Finally, our study demonstrates the essential role of ezrin-PALS1 interaction in the apical membrane remodeling associated with parietal cell secretion. Taken together, these results define a novel molecular mechanism linking ezrin to the conserved apical polarity complexes and their roles in polarized epithelial secretion of gastric parietal cells.

Proteomic Identification and Functional Characterization of a Novel ARF6 GTPase-activating Protein, ACAP4

ARF6 GTPase is a conserved regulator of membrane trafficking and actin-based cytoskeleton dynamics at the leading edge of migrating cells. A key determinant of ARF6 function is the lifetime of the GTP-bound active state, which is orchestrated by GTPase-activating protein (GAP) and GTP-GDP exchanging factor. However, very little is known about the molecular mechanisms underlying ARF6-mediated cell migration. To systematically analyze proteins that regulate ARF6 activity during cell migration, we performed a proteomic analysis of proteins selectively bound to active ARF6 using mass spectrometry and identified a novel ARF6-specific GAP, ACAP4. ACAP4 encodes 903 amino acids and contains two coiled coils, one pleckstrin homology domain, one GAP motif, and two ankyrin repeats. Our biochemical characterization demonstrated that ACAP4 has a phosphatidylinositol 4,5-bisphosphate-dependent GAP activity specific for ARF6. The co-localization of ACAP4 with ARF6 occurred in ruffling membranes formed upon AIF4 and epidermal growth factor stimulation. ACAP4 overexpression limited the recruitment of ARF6 to the membrane ruffles in the absence of epidermal growth factor stimulation. Expression of GTP hydrolysis-resistant ARF6Q67L resulted in accumulations of ACAP4 and ARF6 in the cytoplasmic membrane, suggesting that GTP hydrolysis is required for the ARF6-dependent membrane remodeling. Significantly the depletion of ACAP4 by small interfering RNA or inhibition of ARF6 GTP hydrolysis by overexpressing GAP-deficient ACAP4 suppressed ARF6-dependent cell migration in wound healing, demonstrating the importance of ACAP4 in cell migration. Thus, our study sheds new light on the biological function of ARF6-mediated cell migration.

Phospho-regulated ACAP4-Ezrin Interaction Is Essential for Histamine-stimulated Parietal Cell Secretion

The ezrin-radixin-moesin proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to the regulated HCl secretion. Our recent proteomic study revealed a protein complex of ezrin-ACAP4-ARF6 essential for volatile membrane remodeling (Fang, Z., Miao, Y., Ding, X., Deng, H., Liu, S., Wang, F., Zhou, R., Watson, C., Fu, C., Hu, Q., Lillard, J. W., Jr., Powell, M., Chen, Y., Forte, J. G., and Yao, X. (2006) Mol. Cell Proteomics 5, 1437–1449). However, knowledge of whether ACAP4 physically interacts with ezrin and how their interaction is integrated into membrane-cytoskeletal remodeling has remained elusive. Here we provide the first evidence that ezrin interacts with ACAP4 in a protein kinase A-mediated phosphorylation-dependent manner through the N-terminal 400 amino acids of ACAP4. ACAP4 locates in the cytoplasmic membrane in resting parietal cells but translocates to the apical plasma membrane upon histamine stimulation. ACAP4 was precipitated with ezrin from secreting but not resting parietal cell lysates, suggesting a phospho-regulated interaction. Indeed, this interaction is abolished by phosphatase treatment and validated by an in vitro reconstitution assay using phospho-mimicking ezrinS66D. Importantly, ezrin specifies the apical distribution of ACAP4 in secreting parietal cells because either suppression of ezrin or overexpression of non-phosphorylatable ezrin prevents the apical localization of ACAP4. In addition, overexpressing GTPase-activating protein-deficient ACAP4 results in an inhibition of apical membrane-cytoskeletal remodeling and gastric acid secretion. Taken together, these results define a novel molecular mechanism linking ACAP4-ezrin interaction to polarized epithelial secretion.

Single-molecule detection of phosphorylation-induced plasticity changes during ezrin activation.

Ezrin–radixin–moesin protein family provides a regulated link between the cortical actin cytoskeleton and the plasma membrane. Phosphorylation of ezrin has been functionally linked to membrane dynamics and plasticity. Our recent study demonstrated that phosphorylation of the conserved T567 residue of ezrin alters the physiology of gastric parietal cells. However, the molecular mechanism of phosphorylation‐induced ezrin activation has remained elusive. Here we use atomic force microscopy (AFM) to probe phosphorylation‐mediated activation of ezrin in single molecules. The phospho‐mimicking and non‐phosphorylatable mutant ezrin proteins were generated and purified to homogeneity. Comparative analyses of two ezrin mutants by AFM demonstrate the unfolding of the N‐ and C‐terminal domains upon the phospho‐activation. To measure the physical force underlying the inter‐domain contact during mechanical unfolding, we probed the defined region of ezrin using the N‐terminal ezrin coated onto the AFM tip. Comparative force measurements indicate that T567 phosphorylation‐induced unfolding of ezrin favors the inter‐molecular association. Taken together, these results provide molecular illustration of phosphorylation elicited functional activation of ERM proteins and indicate that stimulus‐induced protein conformational change can be used as a signaling mechanism orchestrating cellular dynamics.

ACAP4 Protein Cooperates with Grb2 Protein to Orchestrate Epidermal Growth Factor-stimulated Integrin β1 Recycling in Cell Migration

Background: ARF6 GTPase orchestrates membrane trafficking and actin-based cytoskeleton dynamics in migrating cells. Results: ACAP4 regulates integrin β1 dynamics in EGF-stimulated cell migration by interaction with Grb2 via Tyr-733 phosphorylation. Conclusion: These results revealed the function of the ACAP4-Grb2-integrin β1 axis in EGF-elicited cell migration. Significance: This study sheds light on a better understanding of aberrant tyrosine phosphorylation in tumor metastasis. ARF6 GTPase is an important regulator of membrane trafficking and actin-based cytoskeleton dynamics active at the leading edge of migrating cells. The integrin family heterodimeric transmembrane proteins serve as major receptors for extracellular matrix proteins, which play essential roles in cell adhesion and migration. Our recent proteomic analyses of ARF6 effectors have identified a novel ARF6 GTPase-activating protein, ACAP4, essential for EGF-induced cell migration. However, molecular mechanisms underlying ACAP4-mediated cell migration have remained elusive. Here, we show that ACAP4 regulates integrin β1 dynamics during EGF-stimulated cell migration by interaction with Grb2. Our biochemical study shows that EGF stimulation induces phosphorylation of tyrosine 733, which enables ACAP4 to bind Grb2. This interaction of ACAP4 with Grb2 regulates integrin β1 recycling to the plasma membrane. Importantly, knockdown of ACAP4 by siRNA or overexpression of ACAP4 decreased recycling of integrin β1 to the plasma membrane and reduced integrin-mediated cell migration. Taken together, these results suggest a novel function for ACAP4 in the regulation of cell migration through controlling integrin β1 dynamics.

IRE1–RACK1 axis orchestrates ER stress preconditioning-elicited cytoprotection from ischemia/reperfusion injury in liver

Endoplasmic reticulum (ER) stress is involved in ischemic preconditioning that protects various organs from ischemia/reperfusion (I/R) injury. We established an in vivo ER stress preconditioning model in which tunicamycin was injected into rats before hepatic I/R. The hepatic I/R injury, demonstrated by serum aminotransferase level and the ultra-structure of the liver, was alleviated by administration of tunicamycin, which induced ER stress in rat liver by activating inositol-requiring enzyme 1 (IRE1) and upregulating 78 kDa glucose-regulated protein (GRP78). The proteomic identification for IRE1 binders revealed interaction and cooperation among receptor for activated C kinase 1 (RACK1), phosphorylated AMPK, and IRE1 under ER stress conditions in a spatiotemporal manner. Furthermore, in vitro ER stress preconditioning was induced by thapsigargin and tunicamycin in L02 and HepG2 cells. Surprisingly, BCL2 was found to be phosphorylated by IRE1 under ER stress conditions to prevent apoptotic process by activation of autophagy. In conclusion, ER stress preconditioning protects against hepatic I/R injury, which is orchestrated by IRE1-RACK1 axis through the activation of BCL2. Our findings provide novel insights into the molecular pathways underlying ER stress preconditioning-elicited cytoprotective effect against hepatic I/R injury.

Phosphorylation of the Bin, Amphiphysin, and RSV161/167 (BAR) domain of ACAP4 regulates membrane tubulation

ArfGAP With Coiled-Coil, Ankyrin Repeat And PH Domains 4 (ACAP4) is an ADP-ribosylation factor 6 (ARF6) GTPase-activating protein essential for EGF-elicited cell migration. However, how ACAP4 regulates membrane dynamics and curvature in response to EGF stimulation is unknown. Here, we show that phosphorylation of the N-terminal region of ACAP4, named the Bin, Amphiphysin, and RSV161/167 (BAR) domain, at Tyr34 is necessary for EGF-elicited membrane remodeling. Domain structure analysis demonstrates that the BAR domain regulates membrane curvature. EGF stimulation of cells causes phosphorylation of ACAP4 at Tyr34, which subsequently promotes ACAP4 homodimer curvature. The phospho-mimicking mutant of ACAP4 demonstrates lipid-binding activity and tubulation in vitro, and ARF6 enrichment at the membrane is associated with ruffles of EGF-stimulated cells. Expression of the phospho-mimicking ACAP4 mutant promotes ARF6-dependent cell migration. Thus, the results present a previously undefined mechanism by which EGF-elicited phosphorylation of the BAR domain controls ACAP4 molecular plasticity and plasma membrane dynamics during cell migration.