Molecular Mechanisms by Which Adapter Protein SH2B1(beta) Facilitates NGF-Dependent Neuronal Differentiation.

Nerve Growth Factor (NGF) has long been recognized as a critical factor in the survival and maintenance of sympathetic neurons. Recent findings have shown that NGF is also required for the sympathetic neuron’s axonal growth and appropriate target organ innervation during development. However, the molecular mechanisms by which NGF elicits these effects are largely unknown. The ubiquitously expressed adapter protein SH2B1 binds to active NGF receptor TrkA and has been implicated in NGF-mediated differentiation and survival of sympathetic neurons. This work provides evidence that SH2B1β facilitates the NGF-dependent nuclear export of FoxO1, a pro-apoptotic transcription factor. While SH2B1 was originally thought to localize and function only at the cell membrane, more recent studies indicated that SH2B1β undergoes nucleocytoplasmic shuttling. The work described in this thesis identifies a functional nuclear localization sequence and provides evidence that nuclear cycling of SH2B1β is critical to promote NGF-mediated differentiation of the preneuronal PC12 cell line. SH2B1β was found to specifically enhance the NGF-induced transcription of a primary response gene required for neuronal differentiation, urokinase plasminogen activator receptor (uPAR). Preventing translocation either into or out of the nucleus abolished the ability of SH2B1β to enhance the transcription of uPAR in response to NGF. Similarly, NGF-dependent neurite outgrowth was inhibited in PC12 cells stably expressing a nuclear import defective SH2B1β. Knocking down endogenous levels of SH2B1 inhibited the NGF-induced transcription of uPAR as well as NGF-dependent neurite outgrowth, suggesting that endogenous SH2B1 is required for both NGF-dependent gene expression and neurite outgrowth. TAP tagged-SH2B1β was used to identify nuclear binding partners of SH2B1, including a putative transcription factor that inhibits NGF-dependent neurite outgrowth. Taken together, these results suggest a nuclear role for SH2B1β during NGF-dependent differentiation and survival. The ability of SH2B1β to influence the subcellular localization of FoxO1 and bind to and counteract the function of a novel transcription factor raises the possibility SH2B1β cycling between the nucleus and cytoplasm is required to shuttle transcription factors into or out of the nucleus.Ph.D.Cellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/60645/1/maurest_1.pd

Nucleocytoplasmic Shuttling of the Adapter Protein SH2B1β (SH2-Bβ) Is Required for Nerve Growth Factor (NGF)-Dependent Neurite Outgrowth and Enhancement of Expression of a Subset of NGF-Responsive Genes

[[abstract]]The adapter protein SH2B1 (SH2-B, PSM) is recruited to multiple ligand-activated receptor tyrosine kinases, including the receptors for nerve growth factor (NGF), insulin, and IGF-I as well as the cytokine receptor-associated Janus kinase family kinases. In this study, we examine SH2B1’s function in NGF signaling. We show that depleting endogenous SH2B1 using short hairpin RNA against SH2B1 inhibits NGF-dependent neurite outgrowth, but not NGF-mediated phosphorylation of Akt or ERKs 1/2. SH2B1 has been hypothesized to localize and function at the plasma membrane. We identify a nuclear localization signal within SH2B1 and show that it is required for nuclear translocation of SH2B1β. Mutation of the nuclear localization signal has no effect on NGF-induced activation of TrkA and ERKs 1/2 but prevents SH2B1β from enhancing NGF-induced neurite outgrowth. Disruption of SH2B1β nuclear import also prevents SH2B1β from enhancing NGF-induced transcription of genes important for neuronal differentiation, including those encoding urokinase plasminogen activator receptor, and matrix metalloproteinases 3 and 10. Disruption of SH2B1β nuclear export by mutation of its nuclear export sequence similarly prevents SH2B1β enhancement of NGF-induced transcription of those genes. Nuclear translocation of the highly homologous family member SH2B2(APS) was not observed. Together, these data suggest that rather than simply acting as an adapter protein linking signaling proteins to the activated TrkA receptor at the plasma membrane, SH2B1β must shuttle between the plasma membrane and nucleus to function as a critical component of NGF-induced gene expression and neuronal differentiation.[[fileno]]2050132010011[[department]]生科

Identification of AMPK Phosphorylation Sites Reveals a Network of Proteins Involved in Cell Invasion and Facilitates Large-Scale Substrate Prediction

AMP-activated protein kinase (AMPK) is a central energy gauge that regulates metabolism and has been increasingly involved in non-metabolic processes and diseases. However, AMPK's direct substrates in non-metabolic contexts are largely unknown. To better understand the AMPK network, we use a chemical genetics screen coupled to a peptide capture approach in whole cells, resulting in identification of direct AMPK phosphorylation sites. Interestingly, the high-confidence AMPK substrates contain many proteins involved in cell motility, adhesion, and invasion. AMPK phosphorylation of the RHOA guanine nucleotide exchange factor NET1A inhibits extracellular matrix degradation, an early step in cell invasion. The identification of direct AMPK phosphorylation sites also facilitates large-scale prediction of AMPK substrates. We provide an AMPK motif matrix and a pipeline to predict additional AMPK substrates from quantitative phosphoproteomics datasets. As AMPK is emerging as a critical node in aging and pathological processes, our study identifies potential targets for therapeutic strategies

BRD2 inhibition blocks SARS-CoV-2 infection by reducing transcription of the host cell receptor ACE2

International audienceSARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. Here we show that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a therapeutic target for COVID-19

BRD2 inhibition blocks SARS-CoV-2 infection by reducing transcription of the host cell receptor ACE2.

SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. Here we show that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a therapeutic target for COVID-19