Biomedical subjects

Qingyuan Ge

Publications and source records attributed to Qingyuan Ge.

11 recordsLinked to original sources

PubMed2006

A calcium-regulated MEF2 sumoylation switch controls postsynaptic differentiation.

Postsynaptic differentiation of dendrites is an essential step in synapse formation. We report here a requirement for the transcription factor myocyte enhancer factor 2A (MEF2A) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. A transcriptional repressor form of MEF2A that is sumoylated at lysine-403 promoted dendritic claw differentiation. Activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of MEF2A at serine-408 and, thereby, promoted a switch from sumoylation to acetylation at lysine-403, which led to inhibition of dendritic claw differentiation. Our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain.

Acetylation↗

PubMed2005

Distribution of ERK1/2 and ERK3 during normal rat fetal lung development.

The extracellular regulated kinases-1 and -2 (ERK1/2) are well-characterized mitogen-activated protein kinases (MAPK) that play critical roles in proliferation and differentiation, whereas the function(s) of MAPK ERK3 are currently unknown. To understand better the roles of these kinases in development, the temporal distribution of ERK1, -2, and -3 proteins were investigated in multiple tissues. The ERK3 protein, in contrast to ERK1/2 varied both between and within individual organs over time. To characterize this variability in greater detail, the temporal and spatial distributions of activated ERK1/2 and ERK3 during rat fetal lung development were investigated. The diphosphorylated (activated) forms of ERK1/2 (dp-ERK1/2), ERK3, and its phosphorylated form (P-ERK3) decreased from embryonic day 17 (E17) through E21 while both ERK1 and ERK2 total proteins remained unchanged, indicating that ERK1/2 and ERK3 proteins are expressed independently during fetal lung development. In addition, characterization of the distribution of these proteins by fluorescent immunohistochemistry indicated that phosphorylated ERK1/2 and total ERK1/2 were distributed throughout multiple cell types, with the phosphorylated ERK1/2 colocalizing with prophase mitotic cells. In contrast, ERK3 was restricted to the distal lung epithelium during the pseudoglandular phase (E17) but shifted to the proximal airways, particularly Clara cells during the saccular stage (E21). The P-ERK3 colocalized with the mitotic marker P-histone H3 in fetal lung and in NIH3T3 and HeLa cells, implicating a potential role for P-ERK3 in mitosis. Thus, expression of ERK1/2 and ERK3 and their phosphorylated forms are expressed independently and are temporally and spatially localized during fetal lung morphogenesis. These observations will facilitate detailed functional analysis of these kinases to assess their roles in pulmonary development and diseases.

Animals↗

PubMed2005

Hsp90-Akt phosphorylates ASK1 and inhibits ASK1-mediated apoptosis.

Hsp90 client protein Akt has been shown to inhibit cell apoptosis in part by inhibiting proapoptotic kinase ASK1 (apoptosis signal-regulating kinase 1) activity. In the present study, we show that Hsp90 inhibits hydrogen peroxide (H(2)O(2))-induced ASK1-p38 activation in endothelial cells (EC). The inhibitory effect of Hsp90 on ASK1-p38 activities is diminished when the Akt phosphorylation site on ASK1 (pSer83) is absent or when Akt is genetically deleted in cells, suggesting that Hsp90 and Akt function together to inhibit ASK1-p38 signaling. Thus, inhibition of Hsp90 by 17-allyamino-17-demethoxygeldanamycin (17-AAG) or phosphatidylinositol 3-kinase (PI3K) LY294002 induced and synergized ASK1 activation and ASK1-mediated EC apoptosis. Furthermore, we show that in resting EC Hsp90, Akt and ASK1 form a ternary complex in which both Akt and ASK1 bind to the middle domain of Hsp90, suggesting that Hsp90 may hold Akt and ASK1 in close proximity. The N-terminal domain of ASK1 containing the Akt phosphorylation site (pSer83) associates with Akt in resting state. However, Akt is released from the N-terminal domain concomitant with binding to the C-terminal domain of ASK1 in response to ASK1 activator H(2)O(2), inhibitor of Hsp90 17-AAG and Akt inhibitor LY294002, leading to a more stable Hsp90-Akt-ASK1 complex. We conclude that Hsp90-Akt forms a complex with ASK1 and protect EC from stress-induced apoptosis.

Animals↗

PubMed2005

Xenopus p21-activated kinase 5 regulates blastomeres' adhesive properties during convergent extension movements.

The p21-activated kinase (PAK) proteins regulate many cellular events including cell cycle progression, cell death and survival, and cytoskeleton rearrangements. We previously identified X-PAK5 that binds the actin and microtubule networks, and could potentially regulate their coordinated dynamics during cell motility. In this study, we investigated the functional importance of this kinase during gastrulation in Xenopus. X-PAK5 is mainly expressed in regions of the embryo that undergo extensive cell movements during gastrula such as the animal hemisphere and the marginal zone. Expression of a kinase-dead mutant inhibits convergent extension movements in whole embryos and in activin-treated animal cap by modifying behavior of cells. This phenotype is rescued in embryo by adding back X-PAK5 catalytic activity. The active kinase decreases cell adhesiveness when expressed in animal hemisphere and inhibits the calcium-dependent reassociation of cells, while dead X-PAK5 kinase localizes to cell-cell junctions and increases cell adhesion. In addition, endogenous X-PAK5 colocalizes with adherens junction proteins and its activity is regulated by extracellular calcium. Taken together, our results suggest that X-PAK5 regulates convergent extension movements in vivo by modulating the calcium-mediated cell-cell adhesion.

Adherens Junctions↗

PubMed2004

A stable aspirin-triggered lipoxin A4 analog blocks phosphorylation of leukocyte-specific protein 1 in human neutrophils.

Lipoxins and their aspirin-triggered 15-epimers are endogenous anti-inflammatory agents that block neutrophil chemotaxis in vitro and inhibit neutrophil influx in several models of acute inflammation. In this study, we examined the effects of 15-epi-16-(p-fluoro)-phenoxy-lipoxin A(4) methyl ester, an aspirin-triggered lipoxin A(4)-stable analog (ATLa), on the protein phosphorylation pattern of human neutrophils. Neutrophils stimulated with the chemoattractant fMLP were found to exhibit intense phosphorylation of a 55-kDa protein that was blocked by ATLa (10-50 nM). This 55-kDa protein was identified as leukocyte-specific protein 1, a downstream component of the p38-MAPK cascade in neutrophils, by mass spectrometry, Western blotting, and immunoprecipitation experiments. ATLa (50 nM) also reduced phosphorylation/activation of several components of the p38-MAPK pathway in these cells (MAPK kinase 3/MAPK kinase 6, p38-MAPK, MAPK-activated protein kinase-2). These results indicate that ATLa exerts its anti-inflammatory effects, at least in part, by blocking activation of the p38-MAPK cascade in neutrophils, which is known to promote chemotaxis and other proinflammatory responses by these cells.

Amino Acid Sequence↗

PubMed2004

Regulation of the p300 HAT domain via a novel activation loop.

The transcriptional coactivator p300 is a histone acetyltransferase (HAT) whose function is critical for regulating gene expression in mammalian cells. However, the molecular events that regulate p300 HAT activity are poorly understood. We evaluated autoacetylation of the p300 HAT protein domain to determine its function. Using expressed protein ligation, the p300 HAT protein domain was generated in hypoacetylated form and found to have reduced catalytic activity. This basal catalytic rate was stimulated by autoacetylation of several key lysine sites within an apparent activation loop motif. This post-translational modification and catalytic regulation of p300 HAT activity is conceptually analogous to the activation of most protein kinases by autophosphorylation. We therefore propose that this autoregulatory loop could influence the impact of p300 on a wide variety of signaling and transcriptional events.

Acetyl Coenzyme A↗

PubMed2004

Persistent activation by constitutive Ste7 promotes Kss1-mediated invasive growth but fails to support Fus3-dependent mating in yeast.

Mitogen-activated protein kinase kinase kinase-Ste11 (MAPKKK-Ste11), MAPKK-Ste7, and MAPK-Kss1 mediate pheromone-induced mating differentiation and nutrient-responsive invasive growth in Saccharomyces cerevisiae. The mating pathway also requires the scaffold-Ste5 and the additional MAPK-Fus3. One contribution to specificity in this system is thought to come from stimulus-dependent recruitment of the MAPK cascade to upstream activators that are unique to one or the other pathway. To test this premise, we asked if stimulus-independent signaling by constitutive Ste7 would lead to a loss of biological specificity. Instead, we found that constitutive Ste7 promotes invasion without supporting mating responses. This specificity occurs because constitutive Ste7 activates Kss1, but not Fus3, in vivo and promotes filamentation gene expression while suppressing mating gene expression. Differences in the ability of constitutive Ste7 variants to bind the MAPKs and Ste5 account for the selective activation of Kss1. These findings support the model that Fus3 activation in vivo requires binding to both Ste7 and the scaffold-Ste5 but that Kss1 activation is independent of Ste5. This scaffold-independent activation of Kss1 by constitutive Ste7 and the existence of mechanisms for pathway-specific promoter discrimination impose a unique developmental fate independently of any distinguishing external stimuli.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Mechanism of p21-activated kinase 6-mediated inhibition of androgen receptor signaling.

PAK6 was first identified as an androgen receptor (AR)-interacting protein able to inhibit AR-mediated transcriptional responses. PAK6 is a serine/threonine kinase belonging to the p21-activated kinase (PAK) family implicated in actin reorganization and cell motility, gene transcription, apoptosis, and cell transformation. We investigated the biochemical basis for inhibition of AR signaling by PAK6. We compared the kinase activity of PAK6 with two other well characterized members of the PAK family, PAK1 and PAK4. Like PAK4, PAK6 possesses a constitutive basal kinase activity that, unlike PAK1, is not modulated by the binding of active Rac or Cdc42 GTPases. In order to test the involvement of PAK6 kinase activity in suppression of AR-mediated transcription, we generated kinase-dead (K436A) and kinase-active (S531N) mutants of PAK6. We show that PAK6 kinase activity is required for effective PAK6-induced repression of AR signaling. Suppression does not depend upon GTPase binding to PAK6 and is not mimicked by the closely related PAK1 and PAK4 isoforms. Kinase-dependent inhibition by PAK6 extended to the enhanced AR-mediated transcription seen in the presence of coactivating molecules and to the action of AR coinhibitors. Active PAK6 inhibited nuclear translocation of the stimulated AR, suggesting a possible mechanism for inhibition of AR responsiveness. Finally, we observe that autophosphorylated, active PAK6 protein is differently expressed among prostate cancer cell lines. Modulation of PAK6 activity may be responsible for regulation of AR signaling in various forms of prostate cancer.

Androgen Receptor Antagonists↗

PubMed2003

Protein phosphorylation in neutrophils monitored with phosphospecific antibodies.

Protein phosphorylation in neutrophils was monitored with two phosphospecific antibodies (pAbs) [termed pPKC(S1) Ab and pPKC(S2) Ab] that recognize products of protein kinase C (PKC) and other Arg/Lys-directed Ser/Thr protein kinases. The pPKC(S1) Ab bound preferentially to p-Ser/p-Thr residues with Arg or Lys in the -3 and -5 positions or the -2 and -3 positions, whereas the pPKC(S2) Ab bound preferentially to p-Ser with Arg or Lys in the -2 and +2 positions and with a hydrophobic residue at the +1 position. Phosphorylated pleckstrin, myristoylated alanine-rich C-kinase substrate (MARCKS), the 47-kDa subunit of the phagocyte oxidase (p47-phox) and numerous unidentified proteins that underwent phosphorylation during neutrophil stimulation were readily detected with these pAbs. Priming effects of tumor necrosis factor alpha (TNF-alpha) and the susceptibility of certain reactions in neutrophils to inhibitors of protein kinases could also be easily investigated with these reagents. Compared to the commonly used 32P-labeling/autoradiographic method, Western blotting with pAbs was found to be a faster, safer, more specific and in many cases more sensitive approach for monitoring protein phosphorylation events in neutrophils. These pAbs may facilitate the identification of several new phosphorylation reactions involved in neutrophil stimulation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2003

p21-activated kinase 2 in neutrophils can be regulated by phosphorylation at multiple sites and by a variety of protein phosphatases.

The p21-activated kinase(Pak) 2 undergoes rapid autophosphorylation/activation in neutrophils stimulated with a variety of chemoattractants (e.g., fMLP). Phosphorylation within the activation loop (Thr(402)) and inhibitory domain (Ser(141)) is known to increase the activity of Pak in vitro, whereas phosphorylation within the Nck (Ser(20)) and Pak-interacting guanine nucleotide exchange factor (Ser(192) and Ser(197)) binding sites blocks the interactions of Pak 2 with these proteins. A panel of phosphospecific Abs was used to investigate the phosphorylation of Pak 2 in neutrophils at these sites. Pak 2 underwent rapid (< or =15 s) phosphorylation at Ser(20), Ser(192/197), and Thr(402) in neutrophils stimulated with fMLP. Phosphorylation at Ser(192/197) and Thr(402) were highly transient events, whereas that at Ser(20) was more persistent. In contrast, Pak 2 was constitutively phosphorylated at Ser(141) in unstimulated neutrophils and phosphorylation at this site was less sensitive to cell stimulation than at other residues. Studies with selective inhibitors suggested that a variety of phosphatases might be involved in the rapid dephosphorylation of Pak 2 at Thr(402) in stimulated neutrophils. This was consistent with biochemical studies which showed that the activation loop of GST-Pak 3, which is homologous to that in Pak 2, was a substrate for protein phosphatase 1, 2A, and a Mg(2+)/Mn(2+)-dependent phosphatase(s) which exhibited properties different from those of the conventional isoforms of protein phosphatase 2C. The data indicate that Pak 2 undergoes a complex pattern of phosphorylation in neutrophils and that dephosphorylation at certain sites may involve multiple protein phosphatases that exhibit distinct modes of regulation.

Amino Acid Sequence↗

PubMed2003

Phosphorylation of the Ras-GRF1 exchange factor at Ser916/898 reveals activation of Ras signaling in the cerebral cortex.

The Ras-GRF1 exchange factor, which is regulated by increases in intracellular calcium and the release of G beta gamma subunits from heterotrimeric G proteins, plays a critical role in the activation of neuronal Ras. Activation of G protein-coupled receptors stimulates an increase in the phosphorylation of Ras-GRF1 at certain serine residues. The first of these sites to be identified, Ser(916) in the mouse sequence (equivalent to Ser(898) in the rat sequence), is required for full activation of the Ras exchange factor activity of Ras-GRF1 by muscarinic receptors. We demonstrate here that Ras-GRF1 is highly expressed in rat brain compared with the Sos exchange factor and that there is an increase in incorporation of (32)P into Ser(898) of brain Ras-GRF1 following activation of protein kinase A. Phosphorylation of Ras-GRF1 at Ser(916) is also required for maximal induction of Ras-dependent neurite outgrowth in PC12 cells. A novel antibody (termed 2152) that selectively recognizes Ras-GRF1 when it is phosphorylated at Ser(916/898) confirmed the regulated phosphorylation of Ras-GRF1 by Western blotting in both model systems of transfected COS-7 and PC12 cells and also of the endogenous protein in rat forebrain slices. Indirect confocal immunofluorescence of transfected PC12 cells using antibody 2152 demonstrated reactivity only under conditions in which Ras-GRF1 was phosphorylated at Ser(916/898). Confocal immunofluorescence of cortical slices of rat brain revealed widespread and selective phosphorylation of Ras-GRF1 at Ser(898). In the prefrontal cortex, there was striking phosphorylation of Ras-GRF1 in the dendritic tree, supporting a role for Ras activation and signal transduction in neurotransmission in this area.

Animals↗