JAK2 Tyrosine Kinase Phosphorylates and Is Negatively Regulated by Centrosomal Protein Ninein

JAK2 is a cytoplasmic tyrosine kinase critical for cytokine signaling. In this study, we have identified a novel centrosome-associated complex containing ninein and JAK2. We have found that active JAK2 localizes around the mother centrioles, where it partly colocalizes with ninein, a protein involved in microtubule (MT) nucleation and anchoring. We demonstrated that JAK2 is an important regulator of centrosome function. Depletion of JAK2 or use of JAK2-null cells causes defects in MT anchoring and increased numbers of cells with mitotic defects; however, MT nucleation is unaffected. We showed that JAK2 directly phosphorylates the N terminus of ninein while the C terminus of ninein inhibits JAK2 kinase activity in vitro. Overexpressed wild-type (WT) or C-terminal (amino acids 1179 to 1931) ninein inhibits JAK2. This ninein-dependent inhibition of JAK2 significantly decreases prolactin- and interferon gamma (IFN-γ)-induced tyrosyl phosphorylation of STAT1 and STAT5. Downregulation of ninein enhances JAK2 activation. These results indicate that JAK2 is a novel member of centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways.     

The Cellular Prion Protein Negatively Regulates Phagocytosis and Cytokine Expression in Murine Bone Marrow-Derived Macrophages

The cellular prion protein (PrP^C) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein on the cell surface. Previous studies have demonstrated contradictory roles for PrP^C in connection with the phagocytic ability of macrophages. In the present work, we investigated the function of PrP^C in phagocytosis and cytokine expression in bone marrow-derived macrophages infected with Escherichia coli. E. coli infection induced an increase in the PRNP mRNA level. Knockout of PrP^C promoted bacterial uptake; upregulated Rab5, Rab7, and Eea1 mRNA expression; and increased the recruitment of lysosomal-associated membrane protein-2 to phagosomes, suggesting enhanced microbicidal activity. Remarkably, knockout of PrP^C suppressed the proliferation of internalized bacteria and increased the expression of cytokines such as interleukin-1β. Collectively, our data reveal an important role of PrP^C as a negative regulator for phagocytosis, phagosome maturation, cytokine expression, and macrophage microbicidal activity.     

Phosphorylation of Tyrosine Hydroxylase by Cyclic GMP-Dependent Protein Kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated and activated by purified cyclic GMP-dependent protein kinase as well as by cyclic AMP-dependent protein kinase catalytic subunit. The extent of activation was correlated with the degree of phosphate incorporated into the enzyme. Comparable stoichiometric ratios (0.6 mol phosphate/mol tyrosine hydroxylase subunit) were obtained at maximal concentrations of either cyclic AMP-dependent or cyclic GMP-dependent protein kinases. The enzymes appeared to mediate the phosphorylation of the same residue based on the observation that incorporation was not increased when both enzymes were present. The major tryptic phosphopeptide obtained from tyrosine hydroxylase phosphorylated by each protein kinase exhibited an identical retention time following HPLC. The purified phosphopeptides also exhibited identical isoelectric points. These data provide support for the notion that the protein kinases are phosphorylating the same residue of tyrosine hydroxylase.     

Tyrosine Hydroxylase Activation and Inactivation by Protein Phosphorylation Conditions

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, catalyzes the conversion of tyrosine to DOPA, Cyclic AMP-dependent protein phosphorylation conditions alter tyrosine hydroxylase activity in rat striatal homogenates. In agreement with other laboratories, we find that short-term pre-incubation (3 min) of extracts under phosphorylating conditions (Mg . ATP, cAMP) increases enzyme activity two- to tenfold over control as measured during a subsequent 15-min assay. We now report that preincubation under phosphorylating conditions for longer periods (30 min) results in a loss of activity to levels equal to or below that of the control enzyme. Addition of purified bovine brain protein kinase catalytic subunit and Mg . ATP enhances activation and increases the rate of inactivation. To demonstrate that inactivation is not associated with proteolytic degradation or irreversible denaturation, the inactivated form of the enzyme can be reactivated. The protein kinase inhibitor protein decreases the activation process and prevents inactivation of the enzyme to below control values. The sedimentation coefficient is not changed by phosphorylation conditions (S = 8.8 +/- 0.1). Although the apparent Km of the enzyme for the 6-methyltetrahydropterine (6-MPH4) cofactor is reduced (0.86 mM, control; 0.32 mM, activated), it is also reduced in the inactivated form (0.38 mM). The Ki for dopamine is increased from 4.5 microM for the control to 28 microM for the activated enzyme, whereas the inactivated form of the enzyme exhibits a Ki of 10 microM. Removal of catecholamines by gel filtration fails to alter activity and the apparent cofactor Km. Moreover, both the activated and the inactivated states persist following gel filtration. It therefore appears that the activation-inactivation process is not mediated solely by the modulation of enzyme feedback inhibition or changes in the Km for 6-MPH4. We also describe a coupled decarboxylase assay in which labeled dopamine is resolved from the precursors tyrosine and DOPA by low-voltage paper electrophoresis.     

Tyrosine Hydroxylase mRNA Concentration in Midbrain Dopaminergic Neurons Is Differentially Regulated by Reserpine

Tyrosine hydroxylase (TH)-mRNA, assayed by in situ hybridization combined with TH immunocytochemistry, showed a selective increase in the ventral tegmental area (A-10) but not in the substantia nigra (A-9) midbrain dopaminergic (DAergic) neurons 3 days after reserpine treatment. TH-mRNA in locus ceruleus noradrenergic (A-4) neurons was increased by reserpine, as confirmed by RNA blot hybridization. These findings show that TH-mRNA is differentially regulated in midbrain DAergic neurons in response to reserpine.     

Expression of Tyrosine Hydroxylase Gene in Cultured Hypothalamic Cells: Roles of Protein Kinase A and C

Abstract: In hypothalamic cells cultured in serum-free medium, the quantity of tyrosine hydroxylase mRNA increases after treatment with an activator of the protein kinase A pathway (8-bromoadenosine cyclic AMP, 3-isobutyl-1-methylxanthine, or forskolin) or an activator of protein kinase C (12- O -tetradecanoylphorbol 13-acetate or sn -1,2-diacylglycerol). The tyrosine hydroxylase mRNA level decreases in the cells after inhibition of protein kinase C with calphostin C or after depletion of protein kinase C by extended phorbol ester treatment. These data suggest that both protein kinase pathways regulate tyrosine hydroxylase gene expression in hypothalamic cells. As simultaneous activation of both pathways has less than an additive effect on the tyrosine hydroxylase mRNA level, they appear to be interrelated. Compared with the rapid and dramatic increase of the tyrosine hydroxylase mRNA level in pheochromocytoma cells, activation of the protein kinase A or protein kinase C pathway in the cultured hypothalamic cells induces slow changes of a small magnitude in the amount of tyrosine hydroxylase mRNA. The slow regulation of tyrosine hydroxylase gene expression in hypothalamic dopaminergic neurons corresponds to the relatively high stability of tyrosine hydroxylase mRNA (half-life = 14 ± 1 h) in these cells.     

Gene Expression of Tyrosine Hydroxylase in the Developing Fetal Brain

Tyrosine hydroxylase, aromatic L-amino-acid decarboxylase, and dopamine beta-hydroxylase activities were studied in the developing fetal rat brain. A delay of 2-3 days between the detection of the tyrosine hydroxylase and the aromatic L-amino-acid decarboxylase and dopamine beta-hydroxylase activities was observed. For this reason, the expression of tyrosine hydroxylase mRNA was studied. Tyrosine hydroxylase mRNA was visualized in the whole brain from 13 days of gestation, but the largest increase of the expression was observed in the hypothalamus. These results are discussed in terms of the relative gene expressions of the three enzymes involved in the biosynthesis of catecholamines and phenolamines in nervous tissues.     

Protein Tyrosine Phosphatases Are Regulated by Mononuclear Iron Dicitrate

The involvement of macrophages (Mφs) as host, accessory, and effector cells in the development of infectious diseases, together with their central role in iron homeostasis, place these immune cells as key players in the interface between iron and infection. Having previously shown that the functional expression of NRAMP-1 results in increased protein phosphorylation mediated in part by an iron-dependent inhibition of Mφ protein-tyrosine phosphatase (PTP) activity, we sought to study the mechanism(s) underlying this specific event. Herein we have identified the mononuclear dicitrate iron complex [Fe(cit)₂H_4-x]^(1+x)− as the species responsible for the specific inhibition of Mφ PTP activity. By using biochemical and computational approaches, we show that [Fe(cit)₂]⁵⁻ targets the catalytic pocket of the PTP SHP-1, competitively inhibiting its interaction with an incoming phosphosubstrate. In vitro and in vivo inhibition of PTP activity by iron-citrate results in protein hyperphosphorylation and enhanced MAPK signaling in response to LPS stimulation. We propose that iron-citrate-mediated PTP inhibition represents a novel and biologically relevant regulatory mechanism of signal transduction.     

大鼠酪氨酸羟化酶基因在肌母细胞中稳定表达

用电穿孔法将大鼠酪氨酸羟化酶（Ｔｙｒｏｓｉｎｅｈｙｄｒｏｘｙｌａｓｅ,ＴＨ）基因转染大鼠Ｌ－６ＴＧ成肌细胞株,经ＰＣＲ检测、免疫组织化学和荧光组织化学检测证明,ＴＨ基因能在细胞内稳定整合和表达,并在辅因子存在时将酪氨酸转化为多巴．移植于大鼠纹状体后可成活并表达ＴＨ。     

Histone Demethylase Retinoblastoma Binding Protein 2 is Overexpressed in Hepatocellular Carcinoma and Negatively Regulated by hsa-miR-212

Background

The H3K4 demethylase retinoblastoma binding protein 2 (RBP2) is involved in the pathogenesis of gastric cancer, but its role and regulation in hepatocellular carcinoma (HCC) is unknown. We determined the function of RBP2 and its regulation in HCC in vitro and in human tissues.

Methods

We analyzed gene expression in 20 specimens each of human HCC and normal liver tissue by quantitative real-time PCR and immunohistochemistry. Proliferation was analyzed by foci formation and senescence by β-galactosidase staining. Promoter activity was detected by luciferase reporter assay.

Results

The expression of RBP2 was stronger in cancerous than non-cancerous tissues, but that of its binding microRNA, Homo sapiens miR-212 (hsa-miR-212), showed an opposite pattern. SiRNA knockdown of RBP2 significantly upregulated cyclin-dependent kinase inhibitors (CDKIs), with suppression of HCC cell proliferation and induction of senescence. Overexpression of hsa-miR-212 suppressed RBP2 expression, with inhibited cell proliferation and induced cellular senescence, which coincided with upregulated CDKIs; with low hsa-miR-212 expression, CDKIs were downregulated in HCC tissue. Inhibition of hsa-miR-212 expression upregulated RBP2 expression. Luciferase reporter assay detected the direct binding of hsa-miR-212 to the RBP2 3′ UTR.

Conclusions

RBP2 is overexpressed in HCC and negatively regulated by hsa-miR-212. The hsa-miR-212–RBP2–CDKI pathway may be important in the pathogenesis of HCC.     

Protein Tyrosine Phosphatase Receptor Type Z Negatively Regulates Oligodendrocyte Differentiation and Myelination

Background

Fyn tyrosine kinase-mediated down-regulation of Rho activity through activation of p190RhoGAP is crucial for oligodendrocyte differentiation and myelination. Therefore, the loss of function of its counterpart protein tyrosine phosphatase (PTP) may enhance myelination during development and remyelination in demyelinating diseases. To test this hypothesis, we investigated whether Ptprz, a receptor-like PTP (RPTP) expressed abuntantly in oligodendrocyte lineage cells, is involved in this process, because we recently revealed that p190RhoGAP is a physiological substrate for Ptprz.

Methodology/Principal Findings

We found an early onset of the expression of myelin basic protein (MBP), a major protein of the myelin sheath, and early initiation of myelination in vivo during development of the Ptprz-deficient mouse, as compared with the wild-type. In addition, oligodendrocytes appeared earlier in primary cultures from Ptprz-deficient mice than wild-type mice. Furthermore, adult Ptprz-deficient mice were less susceptible to experimental autoimmune encephalomyelitis (EAE) induced by active immunization with myelin/oligodendrocyte glycoprotein (MOG) peptide than were wild-type mice. After EAE was induced, the tyrosine phosphorylation of p190RhoGAP increased significantly, and the EAE-induced loss of MBP was markedly suppressed in the white matter of the spinal cord in Ptprz-deficient mice. Here, the number of T-cells and macrophages/microglia infiltrating into the spinal cord did not differ between the two genotypes after MOG immunization. All these findings strongly support the validity of our hypothesis.

Conclusions/Significance

Ptprz plays a negative role in oligodendrocyte differentiation in early central nervous system (CNS) development and remyelination in demyelinating CNS diseases, through the dephosphorylation of substrates such as p190RhoGAP.     

Regulation of Tyrosine Hydroxylase Gene Expression in the Rat Carotid Body by Hypoxia

The activity (Vmax) of tyrosine hydroxylase (TH; EC 1.14.16.2), the rate limiting enzyme in the synthesis of catecholamines, is increased in carotid body, superior cervical ganglion, and the adrenal medulla during hypoxia (i.e., reduced PaO2). The present study was undertaken to determine if the increase in TH activity in these tissues during hypoxia is regulated at the level of TH mRNA. Adult rats were exposed to hypoxia (10% O2) or room air for periods lasting from 1 to 48 h. The carotid bodies, superior cervical ganglia, and adrenals were removed and processed for in situ hybridization using 35S-labeled oligonucleotide probes. The concentration of TH mRNA was increased by hypoxia at all time points in carotid body type I cells, but not in cells of either superior cervical ganglion or adrenal medulla. The increase in TH mRNA in carotid body during hypoxia did not require innervation of the carotid body or intact adrenal glands. In addition, hypercapnia, another physiological stimulus of carotid body activity, failed to induce an increase in TH mRNA in type I cells. Our findings suggest that hypoxia stimulates TH gene expression in the carotid body by a mechanism that is intrinsic to type I cells.