Selective depletion of mouse kidney proximal straight tubule cells causes acute kidney injury.

The proximal straight tubule (S3 segment) of the kidney is highly susceptible to ischemia and toxic insults but has a remarkable capacity to repair its structure and function. In response to such injuries, complex processes take place to regenerate the epithelial cells of the S3 segment; however, the precise molecular mechanisms of this regeneration are still being investigated. By applying the "toxin receptor mediated cell knockout" method under the control of the S3 segment-specific promoter/enhancer, Gsl5, which drives core 2 ß-1,6-N-acetylglucosaminyltransferase gene expression, we established a transgenic mouse line expressing the human diphtheria toxin (DT) receptor only in the S3 segment. The administration of DT to these transgenic mice caused the selective ablation of S3 segment cells in a dose-dependent manner, and transgenic mice exhibited polyuria containing serum albumin and subsequently developed oliguria. An increase in the concentration of blood urea nitrogen was also observed, and the peak BUN levels occurred 3-7 days after DT administration. Histological analysis revealed that the most severe injury occurred in the S3 segments of the proximal tubule, in which tubular cells were exfoliated into the tubular lumen. In addition, aquaporin 7, which is localized exclusively to the S3 segment, was diminished. These results indicate that this transgenic mouse can suffer acute kidney injury (AKI) caused by S3 segment-specific damage after DT administration. This transgenic line offers an excellent model to uncover the mechanisms of AKI and its rapid recovery.

Protein kinase Cepsilon: the mitochondria-mediated signaling pathway.

Mitochondria, which are the cellular energy plants, also act as the integration center of cellular signaling pathways. Apoptosis is a well-known pathway in which mitochondria are involved. Protein kinase Cepsilon has been classified as a novel type of protein kinase C and is involved in many cellular events regulating mitochondrial function. Much evidence has accumulated regarding the relationships between mitochondria-mediated apoptosis and protein kinase Cepsilon. Therefore, by focusing on these relationships, in particular the anti-apoptotic effects of protein kinase Cepsilon on mitochondrial function, we highlight the importance and significance of protein kinase Cepsilon in cell survival and death.

Protein kinase Cepsilon: multiple roles in the function of, and signaling mediated by, the cytoskeleton.

Recent studies have established essential roles of protein kinase Cepsilon in signaling pathways controlling various functions of microfilaments and intermediate filaments by modulating multiple cytoskeletal proteins. This review summarizes recent progress in our understanding of the roles of protein kinase Cepsilon in the functions and signaling of microfilaments and intermediate filaments, with a focus mainly on cell-matrix and cell-cell interactions, migrations and contraction, in addition to its relevance in the development of several diseases, such as malignant tumors or cardiac disease.

Protein kinase C epsilon phosphorylates keratin 8 at Ser8 and Ser23 in GH4C1 cells stimulated by thyrotropin-releasing hormone.

Protein kinase C epsilon (PKCepsilon) is activated by thyrotropin-releasing hormone (TRH), a regulator of pituitary function in rat pituitary GH(4)C(1) cells. We analyzed the downstream mechanism after PKCepsilon activation. Exposure of GH(4)C(1) cells to TRH or a phorbol ester increased the phosphorylation of three p52 proteins (p52a, p52b and p52c) and decreased the phosphorylation of destrin and cofilin. GF109203X, an inhibitor of protein kinases including PKC, inhibited phosphorylation of the p52 proteins by TRH stimulation. Peptide mapping, amino-acid sequencing, and immunochemical studies indicated that p52a, p52b, and p52c are the differentially phosphorylated isoforms of keratin 8 (K8), an intermediate filament protein. The unphosphorylated K8 (p52n) localized exclusively to the cytoskeleton, whereas the phosphorylated forms (especially p52c), which are increased in TRH-stimulated cells, localized mainly to the cytosol. K8 phosphorylation was enhanced in PKCepsilon-overexpressing clones, and purified recombinant PKCepsilon directly phosphorylated K8 with a profile similar to that observed in TRH-stimulated cells. PKCepsilon and K8 colocalized near the nucleus under basal conditions and were concentrated in the cell periphery and cell-cell contact area after TRH stimulation. MS analyses of phospho-K8 and K8-synthesized peptide (amino acids 1-53) showed that PKCepsilon phosphorylates Ser8 and Ser23 of K8. Phosphorylation of these sites is enhanced in TRH-stimulated cells and PKCepsilon-overexpressing cells, as assessed by immunoblotting using antibodies to phospho-K8. These results suggest that K8 is a physiological substrate for PKCepsilon, and the phosphorylation at Ser8 and Ser23 transduces, at least in part, TRH-PKCepsilon signaling in pituitary cells.

Identification of phosphoproteins associated with maintenance of transformed state in temperature-sensitive Rous sarcoma-virus infected cells by proteomic analysis.

To identify phosphotyrosine-containing proteins essential for maintaining the transformed state, we studied the tyrosine phosphorylation profile of temperature-sensitive mutant of Rous sarcoma virus, tsNY68, infected cells (68N7). Shifting the temperature from 39 degrees C (nonpermissive) to 32 degrees C (permissive) markedly increased the expression of phosphotyrosine-containing cell membrane proteins of approximately 40kDa, as assessed by SDS-PAGE. Membrane and nuclear proteins were separated by two-dimensional gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Proteins showing temperature-dependent changes in phosphorylation profile were subjected to in-gel digestion with trypsin and analyzed by mass spectrometry. Five proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A3, hnRNP A2, annexin II, phosphoglycerate mutase 1, and triosephosphate isomerase 1. hnRNP A3 was phosphorylated at serine residues and had both serine and tyrosine phosphorylated sites. These results suggest an important complementary role for proteomics in identifying molecular abnormalities associated with tumor progression that may be attractive candidates for tumor diagnosis.

The second phase activation of protein kinase C delta at late G1 is required for DNA synthesis in serum-induced cell cycle progression.

BACKGROUND: Cell lines that stably over-express protein kinase C (PKC) delta frequently show a decrease in growth rate and saturation density, leading to the hypothesis that PKC delta has a negative effect on cell proliferation. However, the mode of PKC delta activation, the cell cycle stage requiring PKC delta activity, and the exact role of PKC delta at that stage remains unknown. RESULTS: Here we show that the treatment of quiescent fibroblasts with serum activates PKC delta at two distinct time points, within 10 min after serum treatment, and for a longer duration between 6 and 10 h. This biphasic activation correlates with the phosphorylation of Thr-505 at the activation loop of PKC delta. Importantly, an inhibitor of PKC delta, rottlerin, suppresses the biphasic activation of PKC delta, and suppression of the second phase of PKC delta activation is sufficient for the suppression of DNA synthesis. Consistent with this, the transient over-expression of PKC delta mutant molecules lacking kinase activity suppresses serum-induced DNA synthesis. These results imply that PKC delta plays a positive role in cell cycle progression. While the over-expression of PKC delta enhances serum-induced DNA synthesis, this was not observed for PKC epsilon. Similar experiments using a series of PKCdelta/ epsilon chimeras showed that the carboxyl-terminal 51 amino acids of PKC delta are responsible for the stimulatory effect. On the other hand, the over-expression of PKC delta suppresses cell entry into M-phase, being consistent with the previous studies based on stable over-expressors. CONCLUSIONS: We conclude that PKC delta plays a role in the late-G1 phase through the positive regulation of cell-cycle progression, in addition to negative regulation of the entry into M-phase.

Protein kinase C-epsilon (PKC-epsilon): its unique structure and function.

Protein kinase C (PKC)-epsilon was first discovered among novel PKC isotypes by cDNA cloning, and characterized as a calcium-independent but phorbol ester/diacylglycerol-sensitive serine/threonine kinase. PKC-epsilon is targeted to a specific cellular compartment in a manner dependent on second messengers and on specific adapter proteins in response to extracellular signals that activate G-protein-coupled receptors, tyrosine kinase receptors, or tyrosine kinase-coupled receptors. PKC-epsilon then regulates various physiological functions including the activation of nervous, endocrine, exocrine, inflammatory, and immune systems. The controlled activation of PKC-epsilon plays a protective role in the development of cardiac ischemia and Alzheimer's disease, whereas its uncontrolled chronic activation results in severe diseases such as malignant tumors and diabetes. This review summarizes recent progress in our understanding of the unique structure and physiological and pathological roles of PKC-epsilon with a focus mainly on knockout, transgenic, and mutational studies.

Role of specific protein kinase C isozymes in mediating epidermal growth factor, thyrotropin-releasing hormone, and phorbol ester regulation of the rat prolactin promoter in GH4/GH4C1 pituitary cells.

Epidermal growth factor (EGF) and TRH both produce enhanced prolactin (PRL) gene transcription and PRL secretion in GH4 rat pituitary tumor cell lines. These agents also activate protein kinase C (PKC) in these cells. Previous studies have implicated the PKCepsilon isozyme in mediating TRH-induced PRL secretion. However, indirect studies using phorbol ester down-regulation to investigate the role of PKC in EGF- and TRH-induced PRL gene transcription have been inconclusive. In the present study, we examined the role of multiple PKC isozymes on EGF- and TRH-induced activation of the PRL promoter by utilizing general and selective PKC inhibitors and by expression of genes for wild-type and kinase-negative forms of the PKC isozymes. Multiple nonselective PKC inhibitors, including staurosporine, bisindolylmaleimide I, and Calphostin C, inhibited both EGF and TRH induced rat PRL promoter activity. TRH effects were more sensitive to Calphostin C, a competitive inhibitor of diacylglycerol, whereas Go 6976, a selective inhibitor of Ca(2+)-dependent PKCs, produced a modest inhibition of EGF but no inhibition of TRH effects. Rottlerin, a specific inhibitor of the novel nPKCdelta isozyme, significantly blocked both EGF and TRH effects. Overexpression of genes encoding PKCs alpha, betaI, betaII, delta, gamma, and lambda failed to enhance either EGF or TRH responses, whereas overexpression of nPKCeta enhanced the EGF response. Neither stable nor transient overexpression of nPKCepsilon produced enhancement of EGF- or TRH-induced PRL promoter activity, suggesting that different processes regulate PRL transcription and hormone secretion. Expression of a kinase inactive nPKCdelta construct produced modest inhibition of EGF-mediated rPRL promoter activity. Taken together, these data provide evidence for a role of multiple PKC isozymes in mediating both EGF and TRH stimulated PRL gene transcription. Both EGF and TRH responses appear to require the novel isozyme, nPKCdelta, whereas nPKCeta may also be able to transmit the EGF response. Inhibitor data suggest that the EGF response may also involve Ca(2+)-dependent isozymes, whereas the TRH response appears to be more dependent on diacylglycerol.