Functional identification of protein phosphatase 1-binding consensus residues in NBCe1-B

Protein phosphatase 1 (PP1) is involved in various signal transduction mechanisms as an extensive regulator. The PP1 catalytic subunit (PP1c) recognizes and binds to PP1-binding consensus residues (FxxR/KxR/K) in NBCe1-B. Consequently, we focused on identifying the function of the PP1-binding consensus residue, ⁹²²FMDRLK⁹²⁷ , in NBCe1-B. Using site-directed mutagenesis and co-immunoprecipitation assays, we revealed that in cases where the residues were substituted (F922A, R925A, and K927A) or deleted (deletion of amino acids 922–927), NBCe1-B mutants inhibited PP1 binding to NBCe1-B. Additionally, by recording the intracellular pH, we found that PP1-binding consensus residues in NBCe1-B were not only critical for NBCe1-B activity, but also relevant to its surface expression level. Therefore, we reported that NBCe1-B, as a substrate of PP1, contains these residues in the C-terminal region and that the direct interaction between NBCe1-B and PP1 is functionally critical in controlling the regulation of the HCO₃⁻ transport. These results suggested that like IRBIT, PP1 was another novel regulator of HCO₃⁻ secretion in several types of epithelia.

Role of wild-type p53-induced phosphatase 1 in cancer / 国际外科学杂志

Wild-type p53-induced phosphatase 1 is a member of the protein phosphatase family and is an established oncogene due to its dephosphorylation of key protein in pathways and negative control of the DNA damage response system.Wild-type p53-induced phosphatase 1 serves a major role in tumorigenesis,progression,invasion,distant metastasis and chemotherapy resistance in various types of human cancer.Therefore,it may be a potential biomarker and therapeutic target in the diagnosis and treatment of cancer.In the paper,the current knowledge on the role of wild-type p53-induced phosphatase 1 in cancer is discussed.

The Role of PP1 in the Cochlea of Mimetic Aging Mice Induced by D -galactose / 听力学及言语疾病杂志

Objective This study was designed to investigate the expression and potential role of the protein phosphatase 1 (PP1) impairment in D -galactose-induced inner ear aging mouse model .Methods Forty Kunming mice were randomly divided into two groups :the control group and D -galactose group ,20 mice for each .The D-galactose group mice were treated with a daily subcutaneous injection of the D -galactose solutions (800 mg · kg -1 · d-1 ) or an equal volume of normal saline(for the control group) in the nape back for 8 weeks .Eight weeks after D-galactose administration ,the effects of were measured by total Superoxide Dismutase (SOD) activity and Malon-dialdehyde (MDA) level in plasma .Immunofluoresence was performed to detect the location of the PP1 expression in the cochlea .Real-time PCR was performed to detect the level of PP1 mRNA in cochlea .A Western blot analysis was performed to analyze the protein levels of protein phosphates 1 nuclear targeting subunit (PNUTS) ,PP1 and caspase-3 in the inner ear .Results The MDA level was more significantly increased in the D -galactose group than in the control group ;however ,the total SOD activity was significantly decreased in the plasma of D -galactose- induced aging mice(P<0 .01) .The results showed that PP1 was predominantly localized in the nucleus and cyto-plasm of the hair cell ,spiral ganglion cell and stria vascularis cell .And the protein levels of PP1 and caspase-3 sig-nificantly increased ,and the level of PNUTS was decreased in the cochlea of the D -galactose group when compared to the control group .Conclusion PP1 contributes to the development of D -galactose-induced aging mice .

The expression of PNUTS in the cochlea of D-galactose induced ageing mice / 天津医药

Objective To observe the expression of protein phosphates 1 nuclear targeting subunit (PNUTS) in the cochlea of D-galactose induced ageing mice. Methods Twenty Kunming mice, six weeks old, cleaning degree, were randomly divided into two groups, control group and D-galactose group, ten mice for each group. Mice in D-galactose group were administrated with D-galactose at a dose of 800 mg/(kg · d) by subcutaneous injection for eight weeks. Mice in control group were injected with the same volume of saline. After eight weeks, auditory brainstem responses (ABR) were collected to test the hearing thresholds of mice. Western blot assay was used to detect expressions of PNUTS and p53 protein. The expression and distribution of PNUTS in the cochlear Corti, spiral ganglion and striavascularis cells were observed by immunohistochemical (IHC) staining. Results There were no significant differences in ABRs at 8, 12 and 24 kHz between two groups. Protein expressions of PNUTS were located in the cochlear hair cells, spiral ganglion cells and striavascularis cells, and the expression level of cochlea was significantly decreased in D-galactose group than that in control group ( P<0.05). The expression level of p53 protein was significantly increased in D-galactose group than that in control group (P<0.01). Conclusion PNUTS is expressed in the normal mouse cochlea, and which is down-regulated in the cochlea of ageing mice induced by D-galactose.

Upregulation of PP1 Expression in Hippocampus Impairs Long-term Spatial Memory in Rats.

Aims: Either down-regulation of protein kinases or up-regulation of protein phosphatases weakens the level of neuronal protein phosphorylation and affects the function of neuron. Low-function and/or low-content of protein phosphatases in the hippocampus neurons is proposed to be a possible causative factor to the impairment of spatial memory ability. We show here that upregulation of PP1(protein phosphatase1) expression in hippocampus correlates with the decline of the spatial-memory retention ability in rat brain. Study Design: In the present study, 30 adult Wistar rats were tested in a one trial stepdown test to assess normal and impaired memory retention, examining protein phosphatases expression in their hippocampi and analysing both relationship. Place and Duration of Study: Department of Pathophysiology, Medical College in Wuhan University of Science and Technology (WUST), between June 2010 and July 2011. Methodology: To examine which rats have lower ability of learning and memory, thirty Wistar rats, male, 5-7 months old (360~450g), were trained and tested in step-down inhibitory avoidance task. R129d, R126d, R123d, Cdk5 (8) & CREB antibodies were respectively used to detect the content of PP1, PP2B, PP2A, Cdk5 & CREB. Densitometric analysis was used to quantitate the blots.Equal variance T test was used to analyze the escape latency and error from the two group rats, and the immunoblotting data from two groups of rat hippocampus proteins. Results: 72h after the fourth trail, MMI (mild memory impairmemt) rats showed performance markedly worse than that of the NC (normal control) rats ( latency shorter, <150sec; error more, ≥1 vs latency, 300sec; error, 0times; Both P<<0.01). From the learning and retention curves of the both groups of rats in Fig. 1, author found that in the first step-down train, the error times in MMI rats were also more than those in NC rats (P< 0.05), indicating that the ability of learning new material in MMI rats is worse than that in NC rats. The results showed that the contents of PP1 and PP-2B in MMI rats have increased or decreased ~50% (P<0.01) or ~40% (P<0.01) respectively compared with NC rats; Whereas the expression of PP2A in both groups of rats showed no obvious difference, implying that PP1 and/or PP2B might participate in the regulation of learning and memory in the rat brain. In addition, we all found that the middle degree of adverse correlation between the expression of PP1 and the latency of MMI & NC rats (-0.618, P<0.001, two tails), and the low degree of positive correlation between the expression of PP-2B and the latency of MMI & NC rats (0.381, P<0.05, two tails), indicating that the increase of PP1 content in itself might impress in part the memory ability of rats. Conclusion: It is concluded that upregulation of PP1 content may mediate worsened memory retention in rat brain.

Arsenite Acutely Decreases Nitric Oxide Production via the ROS-Protein Phosphatase 1-Endothelial Nitric Oxide Synthase-Thr497 Signaling Cascade

Chronic (>24 h) exposure of arsenite, an environmental toxicant, has shown the decreased nitric oxide (NO) production in endothelial cells (EC) by decreasing endothelial NO synthase (eNOS) expression and/or its phosphorylation at serine 1179 (eNOS-Ser1179 in bovine sequence), which is associated with increased risk of vascular diseases. Here, we investigated the acute (<24 h) effect of arsenite on NO production using bovine aortic EC (BAEC). Arsenite acutely increased the phosphorylation of eNOS-Thr497, but not of eNOS-Ser116 or eNOS-Ser1179, which was accompanied by decreased NO production. The level of eNOS expression was unaltered under this condition. Treatment with arsenite also induced reactive oxygen species (ROS) production, and pretreatment with a ROS scavenger N-acetyl-L-cysteine (NAC) completely reversed the observed effect of arsenite on eNOS-Thr497 phosphorylation. Although protein kinase C (PKC) and protein phosphatase 1 (PP1) were reported to be involved in eNOS-Thr497 phosphorylation, treatment with PKC inhibitor, Ro318425, and overexpression of various PKC isoforms did not affect the arsenite-stimulated eNOS-Thr497 phosphorylation. In contrast, treatment with PP1 inhibitor, calyculin A, mimicked the observed effect of arsenite on eNOS-Thr497 phosphorylation. Lastly, we found decreased cellular PP1 activity in arsenite-treated cells, which was reversed by NAC. Overall, our study demonstrates firstly that arsenite acutely decreases NO production at least in part by increasing eNOS-Thr497 phosphorylation via ROS-PP1 signaling pathway, which provide the molecular mechanism underlying arsenite-induced increase in vascular disease.

Effects of IGF-1 and oxLDL on expression of phosphatase PHLPP1 in vascular smooth muscular cells / 老年心脏病学杂志（英文版）

Objective To investigate the effects of insulin-like growth factor-1 (IGF-1) and oxidized low density lipoprotein (oxLDL) on expression of phosphatase PHLPP1 in vascular smooth muscle cells (VSMCs).Methods Rabbit aortic VSMCs were cultured.VSMCs proliferation ability was determined by measuring cell number and mitochondrial dehydrogenase (MD) activity with MTT assay.Western blot was used to detect the protein expression of phosphatase PHLPP 1.Results IGF-1 (100μg/L) increased cell number and MD activity to 3.02 and 3.59 times of that in control group.oxLDL(50μg/ ml) elevated the above two parameters to 2.03 and 2.91 times respectively.Western blot showed that IGF-1 and oxLDL inhibited the expression of PHLPP 1 to 39.27% and 40.26% of the control group (P＜0.01).Conclusion IGF-1 and oxLDL may enhance the proliferation of VSMCs by decreasing the expression of phosphatase PHLPP1 .