Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone.

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1-7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of Gq/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer.

Stimulation of Cell Migration by Flagellin Through the p38 MAP Kinase Pathway in Cultured Intestinal Epithelial Cells.

Toll-like receptor 5 (TLR5) is a receptor for flagellin and is present on the basolateral surface of intestinal epithelial cells. However, the pathological roles of TLR5 in intestinal epithelial cells are not clear at present. In previous reports, we demonstrated that treatment of cultured alveolar epithelial cells with flagellin activated the p38 mitogen-activated protein kinase (MAPK) pathway and enhanced epithelial-mesenchymal transition induced by transforming growth factor beta 1 (TGF-ß1). In translating our findings in alveolar epithelial cells to intestinal epithelial cells, we found that both flagellin and TGF-ß1 activated p38 MAPK and its downstream protein kinase, MAPK-activated protein kinase-2 (MAPKAPK-2) in an IEC-6 intestinal epithelial cell line. The phosphorylation of HSP27, one of the substrates for MAPKAPK-2, was also increased. TGF-ß1 increased the protein level of α-smooth muscle actin (αSMA), and flagellin enhanced the effect of TGF-ß1. A wound healing assay revealed that flagellin and TGF-ß1 stimulated the migration of cells. SB203580, an inhibitor of p38 MAPK, and an inhibitor of MAPKAPK-2 inhibited flagellin-stimulated migration. These results suggested that TLR5 is involved in the migration of intestinal epithelial cells through activation of the p38 MAPK pathway.

Desensitization by different strategies of epidermal growth factor receptor and ErbB4.

Four transmembrane tyrosine kinases constitute the ErbB protein family: epidermal growth factor receptor (EGFR) or ErbB1, ErbB2, ErbB3, and ErbB4. In general, the structure and mechanism of the activation of these members are similar. However, significant differences in homologous desensitization are known between EGFR and ErbB4. Desensitization of ligand-occupied EGFR occurs by endocytosis, while that of ErbB4 occurs by selective cleavage at the cell surface. Because ErbB4 is abundantly expressed in neurons from fetal to adult brains, elucidation of the desensitization mechanism is important to understand neuronal development and synaptic functions. Recently, it has become clear that heterologous desensitization of EGFR and ErbB4 are induced by endocytosis and cleavage, respectively, similar to homologous desensitization. It has been reported that heterologous desensitization of EGFR is induced by serine phosphorylation of EGFR via the p38 mitogen-activated protein kinase (p38 MAP kinase) pathway in various cell lines, including alveolar epithelial cells. In contrast, the protein kinase C pathway is involved in ErbB4 cleavage. In this review, we will describe recent advances in the desensitization mechanisms of EGFR and ErbB4, mainly in alveolar epithelial cells and hypothalamic neurons, respectively.

Phosphorylation of epidermal growth factor receptor at serine 1047 by MAP kinase-activated protein kinase-2 in cultured lung epithelial cells treated with flagellin.

It has been reported that tumor necrosis factor α (TNFα) activated the p38 MAP kinase pathway, followed by phosphorylation of epidermal growth factor receptor (EGFR) at serine 1047 (Ser1047). Although the phosphorylation of Ser1047 reportedly induced an internalization of EGFR, a protein kinase responsible for the phosphorylation has not been elucidated. In the present study, we found that treatment with flagellin of A549 cells, an alveolar epithelial cell line, induced the activation of p38 MAP kinase, followed by phosphorylation of EGFR at Ser1047. The phosphorylation was strongly inhibited by SB203580, an inhibitor of p38 MAP kinase. The flagellin treatment activated MAP kinase-activated protein kinase-2 (MAPKAPK-2), a protein kinase downstream of p38 MAP kinase, and MK2a inhibitor, an inhibitor of MAPKAPK-2, inhibited the flagellin-induced phosphorylation of EGFR at Ser1047. Unlike the flagellin treatment, the TNFα treatment induced the phosphorylation of EGFR at both Ser1047 and Tyr1173. SB203580 and MK2a inhibitor strongly inhibited the phosphorylation of Ser1047 but not Tyr1173 in EGFR. Finally, bacterially expressed and activated MAPKAPK-2 phosphorylated EGFR at Ser1047 in vitro. These results suggest that flagellin regulates the residence time of EGFR on the plasma membrane and thus the signaling of EGFR through phosphorylation of Ser1047 by MAPKAPK-2.

Selective cleavage of ErbB4 by G-protein-coupled gonadotropin-releasing hormone receptor in cultured hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons). GnRH neurons have a GnRH receptor belonging to the G-protein-coupled receptors. The stimulation of this receptor activates extracellular signal-regulated kinase (ERK). In the present study, we found that epidermal growth factor receptor (EGFR) and ErbB4 were expressed in immortalized GnRH neurons (GT1-7 cells). AG1478, a relatively specific inhibitor of the ErbB family, and small interfering RNA (siRNA) for ErbB4 inhibited the GnRH-induced activation of ERK in GT1-7 cells, suggesting that EGFR and ErbB4 were necessary for the activation. In addition, GnRH induced the cleavage of ErbB4 and accumulation of an 80-kDa fragment. After treatment of the cells with 50 nM GnRH for 5 min, about 80% of ErbB4 was cleaved. Biotinylation of cell surface proteins revealed that more than 70% of the cell surface ErbB4 was cleaved by GnRH treatment. A higher concentration and longer treatment were necessary for GnRH to induce ErbB4 cleavage than ERK activation. TAPI-2, an inhibitor of tumor necrosis factor-α-converting enzyme (TACE), and siRNA for TACE inhibited the cleavage of ErbB4, suggesting that TACE was involved. After ErbB4 cleavage, the activation of ERK by neuregulin 1 was almost completely inhibited. These results suggest that the down-regulation of ErbB4 expression is induced by G-protein-coupled receptor stimulation.

Induction of epithelial-mesenchymal transition by flagellin in cultured lung epithelial cells.

Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and activates host inflammatory responses, mainly through activation of the NF-κB pathway. Although pulmonary fibrosis occurs in some cases of lung infection by flagellated bacteria, the pathological roles of TLR5 stimulation in pulmonary fibrosis have yet to be elucidated. In the present study, we first confirmed that flagellin activated the NF-κB pathway in cultured A549 alveolar epithelial cells. Next, we examined the types of genes whose expression was modulated by flagellin in the cells. Microarray analysis of gene expression indicated that flagellin induced a change in gene expression that had a similar trend to transforming growth factor-ß1 (TGF-ß(1)), a key factor in the induction of epithelial-mesenchymal transition (EMT). Biochemical analysis revealed that TGF-ß(1) and flagellin increased the level of fibronectin protein, while they reduced the level of E-cadherin protein after 30 h of treatment. Interestingly, simultaneous treatment with TGF-ß(1) and flagellin significantly augmented these EMT-related changes. Flagellin strongly activated p38 MAP kinase, and the activation was sustained for longer than 30 h. SB203580, an inhibitor of p38 MAP kinase, inhibited the upregulation of fibronectin by both flagellin and TGF-ß(1). Simultaneous treatment with TGF-ß(1) and flagellin augmented the activation of p38 MAP kinase by TGF-ß(1) or flagellin alone. These results strongly suggest that flagellin cooperates with TGF-ß(1) in the induction of EMT in alveolar epithelial cells.

Inhibition by ethyl pyruvate of the nuclear translocation of nuclear factor-kappaB in cultured lung epithelial cells.

Tumor necrosis factor alpha (TNFalpha) is a cytokine inducing inflammatory responses. It has been reported that ethyl pyruvate has anti-inflammatory actions through inhibition of the transcription mediated by nuclear factor-kappa B (NF-kappaB). By reporter gene assay, we first confirmed that TNFalpha activated the NF-kappaB pathway in cultured alveolar epithelial cells, A549 cells. This activation was strongly inhibited by ethyl pyruvate in a concentration-dependent manner. Treatment of the cells with TNFalpha-induced phosphorylation and degradation of IkappaBalpha within 15 min. The level of IkappaBalpha protein was increased from 30 min, suggesting an increase in the NF-kappaB-mediated transcription of IkappaBalpha. Ethyl pyruvate did not affect the changes in IkappaBalpha within 15 min, but strongly inhibited the increase in the IkappaBalpha protein level from 30 min. An immunoblot analysis revealed that ethyl pyruvate inhibited the nuclear translocation of RelA from 5 min of the treatment with TNFalpha. These results strongly suggested that ethyl pyruvate inhibited the NF-kappaB pathway through inhibition of the nuclear translocation of RelA. Ethyl pyruvate may be a good therapeutic drug for inflammation in which activation of the NF-kappaB pathway is involved.

Phosphorylation of ribosomal protein S19 at Ser59 by CaM kinase I alpha.

In order to examine the possible involvements of Ca(2+)/calmodulin-dependent protein kinases (CaM kinases) in the regulation of ribosomal functions, we tested the phosphorylation of rat ribosomal protein S19 (RPS19) by various CaM kinases in vitro. We found that CaM kinase Ialpha, but not CaM kinase Ibeta1, Ibeta2, II, or IV, robustly phosphorylated RPS19. From the consensus phosphorylation site sequence, Ser59, Ser90, and Thr124 were likely to be phosphorylated; therefore, we mutated each amino acid to alanine and found that the mutation of Ser59 to alanine strongly attenuated phosphorylation by CaM kinase Ialpha, suggesting that Ser59 was a major phosphorylation site. Furthermore, we produced a specific antibody against RPS19 phosphorylated at Ser59, and found that Ser59 was phosphorylated both in GT1-7 cells and rat brain. Phosphorylation of RPS19 in GT1-7 cells was inhibited by KN93, an inhibitor of CaM kinases. Immunoblot analysis after subcellular fractionation of rat brain demonstrated that phosphorylated RPS19 was present in 80S ribosomes. Phosphorylation of RPS19 by CaM kinase Ialpha augmented the interaction of RPS19 with the previously identified S19 binding protein. These results suggest that CaM kinase Ialpha regulates the functions of RPS19 through phosphorylation of Ser59.

Isolation and characterization of chicken GA-binding protein.

We have purified and characterized chicken liver nuclear proteins that bind to Ets binding sites (EBSs) of ribosomal protein (r-protein) gene promoters. We employed supershift assays and antibodies to mouse GA binding protein (GABP), to show that the proteins were similar to alpha and beta subunits of GABP. Western blot analysis identified 54- and 38-kDa proteins as the alpha type, and a 46-kDa protein as the beta type. When compared with nuclear extracts (NEs) of other species, we observed that the 38-kDa protein was unique to chicken, and appears to be derived from the 54-kDa protein. The 54- and 46-kDa proteins were highly expressed in chicken tissues and were major components through higher animals, indicating that both proteins have a conserved role.

Differential regulation of epidermal growth factor receptor by hydrogen peroxide and flagellin in cultured lung alveolar epithelial cells.

In previous studies, we found that stimulation of Toll-like receptor 5 (TLR5) by flagellin induced the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2) through activation of the p38 MAPK pathway in cultured alveolar epithelial A549 cells. Our studies strongly suggested that MAPKAPK-2 phosphorylated epidermal growth factor receptor (EGFR) at Ser1047. It has been reported that phosphorylation of Ser1047 after treatment with tumor necrosis factor α (TNFα) induced the internalization of EGFR. In the present study, we first found that treatment of A549 cells with hydrogen peroxide induced the activation of MAPKAPK-2 and phosphorylation of EGFR at Ser1047 within 30 min. This was different from flagellin treatment because hydrogen peroxide treatment induced the phosphorylation of EGFR at Tyr1173 as well as Ser1047, indicating the activation of EGFR. We also found that KN93, an inhibitor of CaM kinase II, inhibited the hydrogen peroxide-induced phosphorylation of EGFR at Ser1047 through inhibition of the activation of the p38 MAPK pathway. Furthermore, we examined the internalization of EGFR by three different methods. Flow cytometry with an antibody against the extracellular domain of EGFR and biotinylation of cell surface proteins revealed that flagellin, but not hydrogen peroxide, decreased the amount of cell-surface EGFR. In addition, activation of extracellular signal-regulated kinase by EGF treatment was reduced by flagellin pre-treatment. These results strongly suggested that hydrogen peroxide activated the p38 MAPK pathway via activation of CaM kinase II and that flagellin and hydrogen peroxide regulate the functions of EGFR by different mechanisms.

Interaction of ethyl pyruvate in vitro with NF-κB subunits, RelA and p50.

Ethyl pyruvate, an aliphatic ester derived from pyruvate, reportedly has anti-inflammatory actions through inhibition of the transcription mediated by nuclear factor-kappa B (NF-κB). It was suggested that ethyl pyruvate inhibited NF-κB/DNA-binding activity through the covalent modification of RelA. However, the interaction of ethyl pyruvate with RelA in vitro has not been reported. In the present study, we confirmed that treatment of cultured alveolar epithelial cells, A549 cells, with tumor necrosis factor α (TNFα) increased the NF-κB/DNA-binding activity. When the nuclear extract of the cells was incubated with ethyl pyruvate, the NF-κB/DNA-binding activity was strongly inhibited. Because we previously found that the NF-κB/DNA complex included RelA and p50, we bacterially expressed a deletion mutant of RelA, RelA (1-220), and a full-length form of p50. Incubation of RelA (1-220) or p50 with ethyl pyruvate induced dramatic changes in mobility in two types of nondenaturing gel electrophoresis. Electrophoretic mobility shift assays revealed that incubation of RelA (1-220) or p50 with ethyl pyruvate inhibited the DNA-binding activity. Furthermore, immunostaining of A549 cells revealed that ethyl pyruvate inhibited the nuclear association of RelA after TNFα treatment. These results suggest that ethyl pyruvate interacts with RelA and p50 to inhibit their functions at multiple points.

Involvement of CaM kinase II in gonadotropin-releasing hormone-induced activation of MAP kinase in cultured hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic GnRH neurons. There is accumulating evidence that GnRH neurons have GnRH receptors and that the autocrine action of GnRH activates MAP kinase. In this study, we found that KN93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinases (CaM kinases), inhibited the GnRH-induced activation of MAP kinase in immortalized GnRH neurons (GT1-7 cells). Immunoblot analysis indicated that the CaM kinase IIdelta2 isoform (CaM kinase IIdelta2) and synapsin I were expressed in GT1-7 cells. GnRH treatment rapidly increased phosphorylation of synapsin I at serine 603, a specific phosphorylation site for CaM kinase II, suggesting that GnRH treatment rapidly activated CaM kinase IIdelta2. In addition, when we stably overexpressed CaM kinase IIdelta2 in GT1-7 cells, the activation of MAP kinase was strongly enhanced. These results suggest that CaM kinase IIdelta2 was involved in the GnRH-induced activation of MAP kinase in GT1-7 cells.

A novel nucleolar protein interacts with ribosomal protein S19.

The gene encoding ribosomal protein S19 (RPS19) is mutated in approximately 25% of patients with Diamond-Blackfan anemia (DBA), which is a rare congenital erythroblastopenia. DBA patients have a variety of clinical characteristics, and the role of the RPS19 gene in the pathogenesis of the disease is presently unknown. To investigate a possible role for RPS19 in erythropoiesis, we looked for proteins associated with mouse RPS19 using a yeast two-hybrid system and identified a novel protein, which we named S19 binding protein (S19BP). The deduced amino acid sequence of S19BP derived from cDNA defines a calculated mass of 15,849 and an isoelectric point of 11.3. No known functional motifs were found in S19BP except a short polylysine tract embedded in a putative nucleolar localization signal. Immunolocalization experiments revealed that S19BP was highly concentrated in nucleoli after 6 h of transfection in Cos-7 cells. S19BP was expressed ubiquitously at a basal level but a significantly high level of expression was observed in some tissues.