Person: Jo, Hakryul
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Jo, Hakryul
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Publication Small-Molecule Screen Identifies Reactive Oxygen Species as Key Regulators of Neutrophil Chemotaxis(Proceedings of the National Academy of Sciences, 2010) Hattori, Hidenori; Subramanian, Kulandayan K.; Sakai, Jiro; Jia, Yonghui; Li, Yitang; Porter, Timothy F.; Loison, Fabien; Sarraj, Bara; Kasorn, Anongnard; Jo, Hakryul; Blanchard, Catlyn; Zirkle, Dorothy; McDonald, Douglas; Pai, Sung-Yun; Serhan, Charles; Luo, HongboNeutrophil chemotaxis plays an essential role in innate immunity, but the underlying cellular mechanism is still not fully characterized. Here, using a small-molecule functional screening, we identified NADPH oxidase–dependent reactive oxygen species as key regulators of neutrophil chemotactic migration. Neutrophils with pharmacologically inhibited oxidase, or isolated from chronic granulomatous disease (CGD) patients and mice, formed more frequent multiple pseudopodia and lost their directionality as they migrated up a chemoattractant concentration gradient. Knocking down NADPH oxidase in differentiated neutrophil-like HL60 cells also led to defective chemotaxis. Consistent with the in vitro results, adoptively transferred CGD murine neutrophils showed impaired in vivo recruitment to sites of inflammation. Together, these results present a physiological role for reactive oxygen species in regulating neutrophil functions and shed light on the pathogenesis of CGD.Publication Deactivation of Akt by a Small Molecule Inhibitor Targeting Pleckstrin Homology Domain and Facilitating Akt Ubiquitination(Proceedings of the National Academy of Sciences, 2011) Jo, Hakryul; Lo, Pang-Kuo; Li, Yitang; Loison, Fabien; Green, Sarah; Wang, Jake; Silberstein, Leslie; Ye, Keqiang; Chen, Hexin; Luo, HongboThe phosphatidylinositol-3,4,5-triphosphate (PIP3) binding function of pleckstrin homology (PH) domain is essential for the activation of oncogenic Akt/PKB kinase. Following the PIP3-mediated activation at the membrane, the activated Akt is subjected to other regulatory events, including ubiquitination-mediated deactivation. Here, by identifying and characterizing an allosteric inhibitor, SC66, we show that the facilitated ubiquitination effectively terminates Akt signaling. Mechanistically, SC66 manifests a dual inhibitory activity that directly interferes with the PH domain binding to PIP3 and facilitates Akt ubiquitination. A known PH domain-dependent allosteric inhibitor, which stabilizes Akt, prevents the SC66-induced Akt ubiquitination. A cancer-relevant Akt1 (e17k) mutant is unstable, making it intrinsically sensitive to functional inhibition by SC66 in cellular contexts in which the PI3K inhibition has little inhibitory effect. As a result of its dual inhibitory activity, SC66 manifests a more effective growth suppression of transformed cells that contain a high level of Akt signaling, compared with other inhibitors of PIP3/Akt pathway. Finally, we show the anticancer activity of SC66 by using a soft agar assay as well as a mouse xenograft tumor model. In conclusion, in this study, we not only identify a dual-function Akt inhibitor, but also demonstrate that Akt ubiquitination could be chemically exploited to effectively facilitate its deactivation, thus identifying an avenue for pharmacological intervention in Akt signaling.Publication Small Molecule-Induced Cytosolic Activation of Protein Kinase Akt Rescues Ischemia-Elicited Neuronal Death(Proceedings of the National Academy of Sciences, 2012) Jo, Hakryul; Mondal, Subhanjan; Tan, Dewar; Nagata, Eiichiro; Takizawa, Shunya; Sharma, Alok Kumar; Hou, Qingming; Shanmugasundaram, Kumaran; Prasad, Amit; Tung, Joe K.; Tejeda, Alexander O.; Man, Hengye; Rigby, Alan C.; Luo, HongboElevating Akt activation is an obvious clinical strategy to prevent progressive neuronal death in neurological diseases. However, this endeavor has been hindered because of the lack of specific Akt activators. Here, from a cell-based high-throughput chemical genetic screening, we identified a small molecule SC79 that inhibits Akt membrane translocation, but paradoxically activates Akt in the cytosol. SC79 specifically binds to the PH domain of Akt. SC79-bound Akt adopts a conformation favorable for phosphorylation by upstream protein kinases. In a hippocampal neuronal culture system and a mouse model for ischemic stroke, the cytosolic activation of Akt by SC79 is sufficient to recapitulate the primary cellular function of Akt signaling, resulting in augmented neuronal survival. Thus, SC79 is a unique specific Akt activator that may be used to enhance Akt activity in various physiological and pathological conditions.Publication Exploiting Effectors of Rac GTPase(Elsevier BV, 2012) Jo, Hakryul; Luo, HongboPublication Microtubule Dynamics Regulates Akt Signaling via Dynactin p150(Elsevier BV, 2014) Jo, Hakryul; Loison, Fabien; Luo, HongboFollowing activation at the plasma membrane, Akt is subsequently deactivated in the cytoplasm. Although activation and deactivation of Akt must sometimes be separated in order to elicit and control cellular responses, the exact details of the spatiotemporal organization of Akt signaling are incompletely understood. Here we show that microtubule dynamics specifically modulate the deactivation phase of Akt signaling. Localization of Akt to microtubules sustains its activity, while disruption of microtubules attenuates Akt signaling independent of its initial activation. Conversely, stabilization of microtubules elevates Akt signaling both in vitro and in muscle tissues in vivo. Localization of Akt to microtubules is mediated by the microtubule binding protein dynactin p150, which is shown to be a direct target of Akt. Finally, microtubule disruption-induced Akt deactivation contributes to delayed cell cycle progression and accelerated cell death. Taken together, we revealed that, after initiation, the overall intensity and duration of oncogenic Akt signaling are determined by microtubule dynamics, a mechanism that could be exploited for therapeutic purposes.Publication Cancer Cell-Derived Clusterin Modulates the Phosphatidylinositol 3'-Kinase-Akt Pathway through Attenuation of Insulin-Like Growth Factor 1 during Serum Deprivation(American Society for Microbiology, 2008) Jo, Hakryul; Jia, Yonghui; Subramanian, Kulandayan K.; Hattori, Hidenori; Luo, HongboCancer cells in their respective microenvironments must endure various growth-constraining stresses. Under these conditions, the cancer cell-derived factors are thought to modulate the signaling pathways between cell growth and dormancy. Here, we describe a cancer cell-derived regulatory system that modulates the phosphatidylinositol 3'-kinase (PI3K)-Akt pathway under serum deprivation stress. Through the use of biochemical purification, we reveal that cancer cell-secreted insulin-like growth factor 1 (IGF-1) and clusterin, an extracellular stress protein, constitute this regulatory system. We show that secreted clusterin associates with IGF-1 and inhibits its binding to the IGF-1 receptor and hence negatively regulates the PI3K-Akt pathway during serum deprivation. This inhibitory function of clusterin appears to prefer IGF-1, as it fails to exert any effects on epidermal growth factor signaling. We demonstrate furthermore that the constitutive activation of oncogenic signaling downstream of IGF-1 confers insensitivity to the inhibitory effects of clusterin. Thus, the interplay between cancer cell-derived clusterin and IGF-1 may dictate the outcome of cell growth and dormancy during tumorigenic progression.Publication Natural Product Celastrol Destabilizes Tubulin Heterodimer and Facilitates Mitotic Cell Death Triggered by Microtubule-Targeting Anti-Cancer Drugs(Public Library of Science, 2010) Jo, Hakryul; Loison, Fabien; Hattori, Hidenori; Silberstein, Leslie; Yu, Hongtao; Luo, HongboBackground: Microtubule drugs are effective anti-cancer agents, primarily due to their ability to induce mitotic arrest and subsequent cell death. However, some cancer cells are intrinsically resistant or acquire a resistance. Lack of apoptosis following mitotic arrest is thought to contribute to drug resistance that limits the efficacy of the microtubule-targeting anti-cancer drugs. Genetic or pharmacological agents that selectively facilitate the apoptosis of mitotic arrested cells present opportunities to strengthen the therapeutic efficacy. Methodology and Principal Findings: We report a natural product Celastrol targets tubulin and facilitates mitotic cell death caused by microtubule drugs. First, in a small molecule screening effort, we identify Celastrol as an inhibitor of neutrophil chemotaxis. Subsequent time-lapse imaging analyses reveal that inhibition of microtubule-mediated cellular processes, including cell migration and mitotic chromosome alignment, is the earliest events affected by Celastrol. Disorganization, not depolymerization, of mitotic spindles appears responsible for mitotic defects. Celastrol directly affects the biochemical properties of tubulin heterodimer in vitro and reduces its protein level in vivo. At the cellular level, Celastrol induces a synergistic apoptosis when combined with conventional microtubule-targeting drugs and manifests an efficacy toward Taxol-resistant cancer cells. Finally, by time-lapse imaging and tracking of microtubule drug-treated cells, we show that Celastrol preferentially induces apoptosis of mitotic arrested cells in a caspase-dependent manner. This selective effect is not due to inhibition of general cell survival pathways or mitotic kinases that have been shown to enhance microtubule drug-induced cell death. Conclusions and Significance: We provide evidence for new cellular pathways that, when perturbed, selectively induce the apoptosis of mitotic arrested cancer cells, identifying a potential new strategy to enhance the therapeutic efficacy of conventional microtubule-targeting anti-cancer drugs.