Maintenance Of Phosphorylation Research Articles

Bioenergetic Requirements and Spatiotemporal Profile of Nerve Growth Factor Induced PI3K-Akt Signaling Along Sensory Axons.

Nerve Growth Factor (NGF) promotes the elaboration of axonal filopodia and branches through PI3K-Akt. NGF activates the TrkA receptor resulting in an initial transient high amplitude burst of PI3K-Akt signaling followed by a maintained lower steady state, hereafter referred to as initiation and steady state phases. Akt initially undergoes phosphorylation at T308 followed by phosphorylation at S473, resulting in maximal kinase activation. We report that during the initiation phase the localization of PI3K signaling, reported by visualizing sites of PIP3 formation, and Akt signaling, reflected by Akt phosphorylation at T308, correlates with the positioning of axonal mitochondria. Mitochondrial oxidative phosphorylation but not glycolysis is required for Akt phosphorylation at T308. In contrast, the phosphorylation of Akt at S473 is not spatially associated with mitochondria and is dependent on both oxidative phosphorylation and glycolysis. Under NGF steady state conditions, maintenance of phosphorylation at T308 shows dual dependence on oxidative phosphorylation and glycolysis. Phosphorylation at S473 is more dependent on glycolysis but also requires oxidative phosphorylation for maintenance over longer time periods. The data indicate that NGF induced PI3K-Akt signaling along axons is preferentially initiated at sites containing mitochondria, in a manner dependent on oxidative phosphorylation. Steady state signaling is discussed in the context of combined contributions by mitochondria and the possibility of glycolysis occurring in association with endocytosed signalosomes.

Skeletal muscle IGF-1 is lower at rest and after resistance exercise in humans with obesity.

Obesity is associated with numerous changes in skeletal muscle including greater muscle mass and muscle fiber cross sectional area (FCSA), yet fasted muscle protein synthesis is lower. Activation of the IGF-1/Akt/mTOR pathway is critical for muscle mass maintenance, muscle hypertrophy, and muscle protein regulation.Resistance exercise (RE) increases muscle mass, FCSA, and IGF-1. Persons with obesity have greater skeletal muscle mass and larger skeletal muscle fiber cross sectional area. The IGF-1/Akt/mTOR pathway is critical for the regulation of skeletal muscle mass. Our study found men and women with obesity have lower skeletal muscle IGF-1 mRNA and protein and higher expression of miR-206 an epigenetic regulator of IGF-1, at rest and following an acute bout of resistance exercise. Despite this, Akt mediated signaling was maintained and maintenance of phosphorylation does not appear to be accounted for by compensatory pathways. Our findings suggest a possible negative feedback mechanism via increased miR-206 and in turn decreased IGF-1 to limit further skeletal muscle hypertrophy in persons with obesity. The current work investigated if: (1) obesity dysregulates basal skeletal muscle IGF-1 pathways; and (2) obesity augments the muscle IGF-1 pathway responses to acute RE. Eight sedentary (no self-reported physical activity), lean (LN) and eight sedentary subjects with obesity (OB) had vastus lateralis biopsies taken at rest, and 15min and 3h post-acute RE for the measurement of the IGF-1 pathway and muscle FCSA. Type II FCSA was larger in OB vs. LN. Skeletal muscle IGF-1 mRNA and IGF-1 protein were lower in OB vs. LN at rest and post-exercise. Acute RE increased IGF-1 protein similarly in both groups. The expression of miR-206, a post-transcriptional inhibitor of IGF-1 expression, was higher in OB vs. LN and linked with lower IGF-1 mRNA (r = - 0.54). In spite of greater muscle FCSA, muscle IGF-1 expression was lower in obesity suggesting negative feedback may be limiting muscle mass expansion in obesity.

Abstract 29: Mechanistic link between phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) activity and PDL1 expression in head and neck squamous cell carcinoma (HNSCC)

Abstract Introduction: HNSCC is an immunosuppressive disease, with multiple functional and quantitative defects contributing to immune evasion and tumor escape. One of the identified areas for therapy development is the Programmed Death-1 (PD-1)/Programmed Death Ligand 1 (PD-L1) pathway, as this pathway has been hypothesized to allow cancer cell evasion of the immune system by promoting T cell anergy and apoptosis. Pembrolizumab, a PD-L1 inhibitor, has recently been approved for treatment in recurrent/metastatic head and neck squamous cell carcinoma. As regulation of PD-L1 expression could play an important role in the effectiveness of therapy, we further explored the regulation of PD-L1 expression by HNSCC cells. Specifically, we targeted our investigation by evaluating the effects on expression of one of the most frequently mutated genes in HNSCC, PiK3CA. We evaluated this with and without interferon-γ which has previously been shown to affect PD-L1 expression, possibly through activation of the STAT1 pathway. Methods: HPV+ and HPV- HNSCC cell lines were grown in cell culture and treated with selective and non-selective PiK3Ca inhibitors, in combination with interferon-γ. After 72 hours, cells were harvested and flow cytometry was used to measure the expression of PD-L1 expression. Protein expression of various pathway intermediaries were evaluated via Western blot to better delineate the mechanism of PD-L1 upregulation. Results: Treatment with selective PiK3Ca inhibitors in combination with interferon-γ in several cell lines was noted to statistically significantly increase expression of PD-L1, beyond treatment with interferon-γ alone. Maintenance of STAT1 phosphorylation correlated with upregulation of PD-L1 expression, while total STAT1 expression remained stable. The majority of the cell lines which were able to maintain STAT1 phosphorylation were HPV+, but a few HPV- cell lines were noted to also maintain this phosphorylation with a correlating upregulation in PDL1 expression. Additionally, mutation in PiK3Ca despite HPV-negative status was noted to maintain phosphorylation of STAT1 with upregulation of PD-L1 expression. Conclusions: Pik3Ca inhibition in combination with interferon-γ was noted to significantly upregulate PD-L1 expression in several cell lines, suggesting a possible synergistic effect of PiK3Ca inhibition with interferon-γ. This was noted to correlate with maintenance of phosphorylation of STAT1, suggesting a pivotal link between PIK3CA signaling, STAT1 activity and PDL1 expression in PIK3CA aberrant HNSCC. Citation Format: Rebecca C. Hoesli, Nicole L. Michmerhuizen, Vivek Nair, Chloe Matovina, Elizabeth Leonard, Mark EP Prince, Matthew E. Spector, J. Chad Brenner. Mechanistic link between phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) activity and PDL1 expression in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 29.

Abstract 3712: Mechanistic link between phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) activity and PDL1 expression in head and neck squamous cell carcinoma (HNSCC)

Abstract Introduction: HNSCC is an immunosuppressive disease, with multiple functional and quantitative defects contributing to immune evasion and tumor escape. One of the identified areas for therapy development is the Programmed Death-1 (PD-1)/Programmed Death Ligand 1 (PD-L1) pathway, as this pathway has been hypothesized to allow cancer cells to evade the immune system by promoting T cell anergy and apoptosis. Pembrolizumab, a PD-L1 inhibitor, has recently been approved for the treatment of recurrent/metastatic HNSCC. As regulation of PD-L1 expression could play an important role in the effectiveness of therapy, we further explored the regulation of PD-L1 expression in HNSCC cells. Specifically, we targeted our investigation by evaluating the effects on expression of one of the most frequently mutated genes in HNSCC, PIK3CA. We evaluated this with and without interferon-γ, which has previously been shown to affect PD-L1 expression, possibly through activation of the STAT1 pathway. Methods: HPV+ and HPV- HNSCC cell lines were grown in cell culture and treated with selective and non-selective PI3K inhibitors, in combination with interferon-γ. After 72 hours, cells were harvested and flow cytometry was used to measure the expression of PD-L1. Protein expression of various pathway intermediaries was evaluated via western blot to better delineate the mechanism of PD-L1 upregulation. Results: Treatment with selective PI3K inhibitors in combination with interferon-γ in several cell lines significantly increased expression of PD-L1, beyond the increase noted after treatment with interferon-γ alone. Maintenance of STAT1 phosphorylation correlated with upregulation of PD-L1 expression, while total STAT1 expression remained stable. The majority of the cell lines maintaining STAT1 phosphorylation were HPV+, but a few HPV- cell lines also maintained this phosphorylation with a correlating upregulation in PD-L1 expression. Additionally, mutation in PIK3Ca despite HPV- status was noted to maintain phosphorylation of STAT1 with upregulation of PD-L1 expression. Conclusions: Treatment with PI3K inhibitors in combination with interferon-γ significantly upregulated PD-L1 expression in several cell lines, suggesting a possible synergistic effect. Since PD-L1 expression correlated with maintenance of phosphorylation of STAT1, these results suggest a pivotal link between PIK3CA signaling, STAT1 activity and PD-L1 expression in PIK3CA aberrant HNSCC. Citation Format: Rebecca C. Hoesli, Nicole L. Michmerhuizen, Vivek Nair, Chloe Matovina, Elizabeth Leonard, Matthew E. Spector, Carol R. Bradford, Mark E. Prince, Andrew C. Birkeland, J Chad Brenner. Mechanistic link between phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) activity and PDL1 expression in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3712. doi:10.1158/1538-7445.AM2017-3712

The alpha 7 nicotinic receptor agonist PHA-543613 hydrochloride inhibits Porphyromonas gingivalis-induced expression of interleukin-8 by oral keratinocytes

ObjectiveThe alpha 7 nicotinic receptor (α7nAChR) is expressed by oral keratinocytes. α7nAChR activation mediates anti-inflammatory responses. The objective of this study was to determine if α7nAChR activation inhibited pathogen-induced interleukin-8 (IL-8) expression by oral keratinocytes.Materials and methodsPeriodontal tissue expression of α7nAChR was determined by real-time PCR. OKF6/TERT-2 oral keratinocytes were exposed to Porphyromonas gingivalis in the presence and absence of a α7nAChR agonist (PHA-543613 hydrochloride) alone or after pre-exposure to a specific α7nAChR antagonist (α-bungarotoxin). Interleukin-8 (IL-8) expression was measured by ELISA and real-time PCR. Phosphorylation of the NF-κB p65 subunit was determined using an NF-κB p65 profiler assay and STAT-3 activation by STAT-3 in-cell ELISA. The release of ACh from oral keratinocytes in response to P. gingivalis lipopolysaccharide was determined using a GeneBLAzer M3 CHO-K1-bla cell reporter assay.ResultsExpression of α7nAChR mRNA was elevated in diseased periodontal tissue. PHA-543613 hydrochloride inhibited P. gingivalis-induced expression of IL-8 at the transcriptional level. This effect was abolished when cells were pre-exposed to a specific α7nAChR antagonist, α-bungarotoxin. PHA-543613 hydrochloride downregulated NF-κB signalling through reduced phosphorylation of the NF-κB p65-subunit. In addition, PHA-543613 hydrochloride promoted STAT-3 signalling by maintenance of phosphorylation. Furthermore, oral keratinocytes upregulated ACh release in response to P. gingivalis lipopolysaccharide.ConclusionThese data suggest that α7nAChR plays a role in regulating the innate immune responses of oral keratinocytes.

Abstract 17446: Intra-arrest Hypothermia Protection Requires Endothelial Nitric Oxide Synthase

Introduction: Intra-arrest hypothermia (IH), cooling the body to 30 - 32°C during cardiac arrest, has previously been shown as protective in a mouse model of cardiac arrest. Hypothermia has also been shown to protect cultured cardiomyocytes exposed to simulated ischemia/reperfusion (I/R) in a nitric oxide synthase-dependent manner. However, the effect of IH on NOS signaling in whole animal models of cardiac arrest has not been studied. We hypothesized that mice deficient in endothelial NOS (NOS3) would not be protected by IH following cardiac arrest. Furthermore, we hypothesized that IH would alter NOS3 signaling in WT mouse hearts. METHODS: Adult NOS3 -/- (B6.129P2-Nos3tm1Unc/J, n = 30) and WT (C57Bl/6J, n = 24) mice underwent 8 min of cardiac arrest. At 6 min, mice were randomized to normothermia (NT, 37°C) or IH (30°C) followed by re-warming to 37°C at 60 min post-ROSC. Animals were hemodynamically monitored for 2 h and then neurologically scored at regular intervals up to 48 h. In separated experiments, cardiac NOS3 signaling was evaluated in similarly randomized mice at the end of the arrest period. RESULTS: In NOS3-/- mice, IH and NT groups had similar 48 h neurologically-intact survival rates (20 % vs. 0%, p=0.80). By contrast survival rates were significantly higher in IH vs. NT treated WT mice (47% vs. 0%, p=0.03). After 8 min of arrest heart p-NOS3(Ser1177) was significantly lower in NT relative to sham controls (p<0.05) and IH treated WT mice (p<0.05). Also during arrest, phosphorylation of the metabolically-sensitive AMP activated protein kinase p-AMPK(Thr172) was similarly increased (p<0.05) following arrest in both IH and NT groups. Cardiac p-Akt(Ser474/Thr308) content was unchanged at 8 min of arrest (p>0.05) in both groups. CONCLUSIONS: Loss of NOS3 abrogates IH protection following cardiac arrest. Maintenance of cardiac NOS3 phosphorylation during cardiac arrest may play an important role in determining survival in this model. Such maintenance of phosphorylation appears to be independent of upstream kinase phosphorylation.

Developmental regulation and PKC dependence of Alzheimer's-type tau phosphorylations in cultured fetal rat hippocampal neurons

Attempts to describe a mechanism of neurofibrillary tangle formation often focus on site specific phosphorylations of tau protein. These have typically been described in both Alzheimer's disease and developing brains. Therefore, study of the developmental regulation of Alzheimer epitope tau phosphorylations may help explain their persistence or recurrence during Alzheimer's disease. Using fetal rat hippocampal cultures, we report a spatial and temporal expression of tau phosphorylation during neuronal differentiation. We have examined phosphorylation at the epitopes recognized by monoclonal antibodies, PHF-1 and Tau 1. Tau was highly phosphorylated at the PHF-1 epitope at all culture ages examined using both immunohistochemical staining and Western blots. Tau was heavily phosphorylated at the Tau 1 epitope only in older cultures. The populations of tau recognized by the two antibodies also exhibited different solubilities, suggesting different microtubule binding behaviors: tau phosphorylated at PHF-1 was retained in axons following solubilization whereas Tau 1 immunoreactive tau was not retained in any cell compartment. Finally, in this culture system, maintenance of phosphorylation at the PHF-1 epitope, but not the Tau 1 epitope, required protein kinase C activity. These results indicate unique regulatory mechanisms and roles for each of these phosphorylated tau epitopes.

Inhibition of photosynthesis by low oxygen concentrations

The effect of changing the oxygen concentration from 21 to 2% on photosynthesis of wheat, sunflower, and soybean was investigated. At low CO2 concentrations and low light intensities, a stimulation of photosynthesis was observed in 2% oxygen compared with the rate in 21% O2. At high CO2 concentrations and high light intensities, a temporary inhibition of photosynthesis was observed when the oxygen concentration was changed from 21 to 2%. In wheat and sunflower, this inhibition was observed at progressively lower CO2 concentrations as temperatures were decreased. In soybean only, a slight inhibition of photosynthesis was observed at higher temperatures. In some cases, especially in plants grown under a low light intensity, a long-lasting (> 45 min) inhibition of photosynthesis was observed. In most cases, however, the inhibition lasted only for several minutes and final rates of photosynthesis in 2% O2 were equal to or greater than the rate of photosynthesis in 21% O2. The stimulatory effect of oxygen on photosynthesis at high CO2 concentrations and high light intensities could possibly be due to a regulation of the oxidation–reduction state of the electron transport chain and the maintenance of phosphorylation.

Isolation and characterization of cyclic AMP-resistant variants from a functional adrenal cortical cell line

We have isolated a number of cyclic AMP-resistant cell lines and characterized two of them, 3B4 and 10F2s, from a functional adrenal cortical cell line, Y-1. At seeding densities above 100 cells10 cm diameter plates, the variant cells are resistant in their morphological change, plating efficiency and growth to the normal effects of dibutyryl cyclic AMP (db-cAMP). At lower seeding densities, 3B4 and 10F2 have retained a slight sensitivity to db-cAMP in their plating efficiency and in their morphology. Studies with various nucleotides and cAMP analogues show that the inhibitory effects of db-cAMP on the growth and morphology of Y-1 cells are not due to degradation products of db-cAMP. There is loss of response to ACTH in the variant cell lines such that there are no effects of ACTH on plating efficiency, growth, morphology, steroidogenesis and cAMP excretion. In addition, the variant cell lines show lowered activities of the cAMP binding receptor and cAMP-dependent protein kinase. Preliminary studies indicate that in Y-1, cAMP markedly reduces protein phosphorylation, and it inhibits phosphate uptake. In the variants, the protein phosphorylation and phosphate uptake are maintained even in the presence of db-cAMP. The maintenance of phosphorylation in the presence of db-cAMP may play an important role in the ability of the cells to survive in high concentrations of db-cAMP. The variant cell lines can be stimulated by db-cAMP to increase steroidogenesis, although the stimulated levels of steroidogenesis in 3B4 and 10F2 are less than those in Y-1. The variant phenotype is stable in vitro and in vivo.