Differential phosphorylation of CK8 and CK18 by 12-O-tetradecanoyl-phorbol-13-acetate in primary cultures of mouse hepatocytes.

The phosphorylation of cytokeratin was investigated in primary cultures of hepatocytes. The two hepatocyte cytokeratins CK8 and CK18 (55,000 and 49,000 M_r, respectively) were phosphorylated, CK8 being more phosphorylated than CK18. Treatment of the hepatocytes with 150 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA) an activator of protein kinase C induced a transient increase in the level of phosphorylation of CK8 but not CK18. This effect was maximal after 15 min of TPA treatment and was maintained for up to 3 h. After 22 h of treatment with TPA, which down-regulates protein kinase C, CK8 phosphorylation was returned to the basal level. Further addition of TPA to the 22-h treated cells did not cause an increase in CK8 phosphorylation. Indirect immunofluorescence microscopy with a monoclonal antibody to CK8 indicated that while the addition of TPA induced the formation of granular cytokeratin aggregates in some hepatocytes, in most hepatocytes no major changes in the intermediate filament network were observed. Staining for actin showed that actin microfilaments were rapidly reorganized after the treatment and a loss of stress fibres were observed. We propose that CK8 is an in vitro substrate for protein kinase C and that the specific phosphorylation of CK8 plays a role in protein kinase C signal transduction.     

Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae

A 40-kD protein kinase C (PKC)epsilon related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18. The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKC epsilon specific antibody showed that the 40-kD species, and not native PKC epsilon (90 kD) associated with the cytokeratins. Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKC epsilon, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. A peptide pseudosubstrate specific for PKC epsilon inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates. Tryptic peptide map analysis of the cytokeratins that were phosphorylated by purified rat brain PKC epsilon or as immunoprecipitates by the associated kinase showed similar phosphopeptides. Furthermore, PKC epsilon immunoreactive species and CK8/18 colocalized using immunofluorescent double staining. We propose that a kinase related to the catalytic fragment of PKC epsilon physically associates with and phosphorylates cytokeratins 8 and 18.     

Modulation of cytokeratin and actin gene expression and fibrillar organization in cultured rat hepatocytes.

Intermediate filaments of rat hepatocytes are composed of cytokeratins 8 and 18 (CK8 and CK18, respectively). Recent work from our laboratory has indicated a close relationship between the synthesis of these cytokeratins, their organization into intermediate filaments, and the promotion of growth and differentiation of cultured rat hepatocytes by insulin, epidermal growth factor, and dexamethasone. In the present study, we examined the mRNA expression, level of protein synthesis, and fibrillar distribution of cytokeratins 8 and 18 and actin in hepatocytes, isolated from normal and dexamethasone-injected rats and cultured as monolayers or spheroids in the presence of insulin, or from normal rat hepatocytes, cultured as monolayers in the presence of dexamethasone, insulin, and dimethyl sulfoxide. The CK8 mRNA level was lower in hepatocytes isolated from noninjected rats and cultured as either monolayers or spheroids, than in those from dexamethasone-injected rats. However, the CK18 mRNA level varied in a manner that was different from that of CK8 mRNA, showing that the modes of expression of the two genes were independent. The various changes in hepatocyte culture conditions led to variations in albumin mRNA levels that largely followed those observed in CK8 mRNA levels. In the case of actin, the amount of mRNAs varied from relatively high levels in hepatocyte monolayers to extremely low levels in hepatocyte spheroids, even though in both cases the cells were isolated from dexamethasone-injected rats. These changes in mRNA levels did not necessarily correlate with changes in the synthesis of cytokeratins 8 and 18, and actin. Changes in culture conditions induced a major reorganization in the distribution of cytokeratin intermediate filaments and actin filament between the region near the surface membrane and the cytoplasm. The most divergent patterns in cytokeratin intermediate filaments and actin filament distributions were observed between hepatocytes cultured as spheroidal aggregates and as monolayers in the presence of dimethyl sulfoxide. The former condition resulted in patterns of cytokeratin and actin gene expression and fibrillar organization that best matched those in situ. In the latter condition, inappropriate patterns were obtained, in spite of the fact that dimethyl sulfoxide treated hepatocytes are known to exhibit survival and functional activities equivalent to that of hepatocyte spheroids. These results demonstrate for the first time that the survival and functional activity (i.e., albumin production) of rat hepatocytes in vitro is not necessarily correlated with a particular pattern of cytokeratin and actin gene expression and fibrillar arrangement.     

Comparative effects of hepatocyte growth factor and epidermal growth factor on motility,morphology, mitogenesis,and signal transduction of primary rat hepatocytes

Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are major hepatacyte mitogens, but HGF, also known as scatter factor (SF), has also been shown as a potent motogen for epithelial and endothelial cells. The mechanisms by which HGF is a stronger motogen compared to other mitogens are not understood. Here we report a comparative study of the effect of the two growth factors on cultured primary rat hepatocytes regarding their differential effects on morphology, mitogenicity, and motility as well as the phosphorylation of cytoskeletal-associated proteins. Using three different motility assays, both HGF and EGF increased the motility of hepatocytes, but HGF consistently elicited a significantly greater motility response than EGF. Additionally, HGF induced a more flattened, highly spread morphology compared to EGF. To examine if HGF and EGF phosphorylated different cytoskeletal elements as signal transduction targets in view of the observed variation in morphology and motility, primary cultures of ³²P-loaded rat hepatocytes were stimulated by either HGF or EGF for up to 60 min. Both mitogens rapidly stimulated four isoforms of MAP kinase with similar kinetics and also rapidly facilitated the phosphorylation of cytoskeletal-associated F-actin. Two cytoskeletal-associated proteins, however, were observed to undergo rapid phosphorylation by HGF and not EGF during the time points described. One protein of 28 kDa was observed to become phosphorylated fivefold over controls, while the EGF-stimulated cells showed only a slight increase in the phosphorylation of this protein. Another protein with an apparent mwt of 42 kDa was phosphorylated 20-fold at 1 min and remained phosphorylated over 50-fold over control up to the 60 min time point. This protein was observed to become phosphorylated by EGF only after 10 min, and to a lesser extent (20-fold). Taken together, the data suggest that HGF and EGF stimulate divergent as well as redundant signal transduction pathways in the hepatocyte cytoskeleton, and this may result in unique HGF- or EGF-specific motility, morphology, and mitogenicity in hepatocytes. © 1994 Wiley-Liss, Inc.     

Association of the tyrosine phosphorylated epidermal growth factor receptor with a 55-kD tyrosine phosphorylated protein at the cell surface and in endosomes

After the intraportal injection of EGF, the EGF receptor (EGFR) is rapidly internalized into hepatic endosomes where it remains largely receptor bound (Lai et al., 1989. J. Cell Biol. 109:2751-2760). In the present study, we evaluated the phosphotyrosine content of EGFRs at the cell surface and in endosomes in order to assess the consequences of internalization. Quantitative estimates of specific radioactivity of the EGFR in these two compartments revealed that tyrosine phosphorylation of the EGFR was observed at the cell surface within 30 s of ligand administration. However, the EGFR was also highly phosphorylated in endosomes reaching levels of tyrosine phosphorylation significantly higher than those of the cell surface receptor at 5 and 15 min after EGF injection. A 55-kD tyrosine phosphorylated polypeptide (pyp55) was observed in association with the EGFR at the cell surface within 30 s of EGF injection. The protein was also found in association with the EGFR in endosomes as evidenced by coprecipitation studies using a mAb to the EGFR as well as by coelution with the EGR in gel permeation chromatography. Limited proteolysis of isolated endosomes indicated that the tyrosine phosphorylated domains of the EGFR and associated pyp55 were cytosolically oriented while internalized EGF was intraluminal. The identification of pyp55 in association with EGFR in both hepatic plasma membranes and endosomes may be relevant to EGFR function and/or trafficking of the EGFR.     

Cytoskeletal changes in hepatocytes induced by Microcystis toxins and their relation to hyperphosphorylation of cell proteins.

The heptapeptide toxins produced by the blue-green alga (cyanobacterium) Microcystis aeruginosa are selectively hepatotoxic in mammals. The characteristic post-mortem pathology of the liver is extensive lobular disruption due to sinusoidal breakdown, leakage of blood into the tissue and hepatocyte disintegration. Isolated hepatocytes incubated with toxin show severe structural deformity and surface blebbing. This paper demonstrates the effects of Microcystis toxins on the contraction and aggregation of actin microfilaments, and on the relocation and breakdown of cytokeratin intermediate filaments, in cultured hepatocytes. Earlier work did not show changes in the assembly/disassembly of actin; however, this paper demonstrates the change in cytokeratin from intermediate filaments to distributed granules in the cytoplasm of toxin-affected cells. Acrylamide gel electrophoresis of cytoskeletal fractions from hepatocytes did not show changes in total cytokeratins; however, marked changes in the immunogenicity of cytokeratins at 52 and 58 kDa were seen on toxin exposure of cells. Measurement of 32P-phosphorylation of proteins in toxin-affected cells incubated with [32P]orthophosphate showed a dramatic increase compared to control incubations. This is in agreement with research elsewhere describing phosphatase inhibition in vitro by Microcystis toxins. The data indicate that phosphorylated cytokeratin is a major component of cytoplasmic fraction phosphorylated protein after toxin exposure to hepatocytes. It is concluded that the mechanism of Microcystis toxicity to the hepatocyte is through cytoskeletal damage leading to loss of cell morphology, cell to cell adhesion and finally cellular necrosis. The underlying biochemical lesion is likely to be phosphatase inhibition causing hyperphosphorylation of a number of hepatocyte proteins, including those cytokeratins responsible for microfilament orientation and intermediate filament integrity.     

Vimentin Filaments Follow the Preexisting Cytokeratin Network during Epithelial–Mesenchymal Transition of Cultured Neonatal Rat Hepatocytes

Changes in cell cytoskeleton are known to play an important role in differentiation and embryogenesis and also in carcinogenesis. Previous studies indicated that neonatal hepatocytes undergo an epithelial–mesenchymal transition when cultured in a serum-free medium for several days. Here we show by Western blotting of neonatal rat liver cells cultured for 3 days that vimentin and cytokeratin were expressed by these cells. Epidermal growth factor treatment induced high coexpression of vimentin and cytokeratin filaments in hepatocytes from neonatal livers, as detected by double immunofluorescence microscopy. Confocal scanning laser microscopy was used to determine the spatial and cell distribution of cytokeratin and vimentin intermediate filament networks. Vimentin-expressing hepatocytes were mainly located on the periphery of epithelial clusters and presented a migratory morphology, suggesting that vimentin expression was related to the loss of cell–cell contact. Short vimentin filaments were mainly located at the cytoplasmic sites behind the extending lamella. Horizontal and vertical dual imaging of double immunofluorescence with anti-vimentin and anti-cytokeratin antibodies indicated that both filaments colocalize strongly. Three-dimensional reconstruction of serial optical sections revealed that newly synthesized vimentin distributed following the preexisting cytokeratin network and, when present, both filament scaffolds codistributed inside cultured hepatocytes. Immunoelectron microscopy performed in whole-mount-extracted cultured cells revealed that both filaments are closely interrelated but independent. However, a high degree of immunogold colocalization was found in the knots of the filament network. Further experiments with colce- mide and cytochalasin treatment indicated that vimentin filament distribution, but not cytokeratin, was dependent on an intact microtubule network. These results are consistent with a mechanism of vimentin assembly, whereby growth of vimentin intermediate filaments is dependent on microtubules in topographically restricted cytoplasmic sites, in close relation to the cytokeratin cytoskeleton and to changes in cell–cell contact and cell shape.     

Expression of exogenous c-myc oncogene does not initiate DNA synthesis in primary rat hepatocyte cultures.

Cultured hepatocytes from adult rats stimulated with combinations of growth factors enter into S phase but do not undergo multiple rounds of DNA synthesis nor mitosis. We have examined the potential of an introduced oncogene to induce alterations in the DNA synthetic activity of the cultured hepatocytes in response to epidermal growth factor (EGF). Overexpression of c-myc did not initiate significant DNA synthesis in rat hepatocyte cultures alone, although it cooperated with added EGF to super-induce thymidine incorporation into DNA. From our results, it is suggested that EGF is also necessary to initiate hepatocyte DNA synthesis probably by inducing a battery of cell cycle-related genes if incubated with c-myc transfected cultures for only 5 hours. Hepatocyte polypeptides reacting with anti-MYC antisera were found to migrate between 55-67 KDa in SDS-PAGE; only the 64-67 KDa species were found to be phosphorylated, and the observed size heterogeneity may be due to proteolytic degradation or may reflect presently unknown posttranslational modifications.     

三种大鼠肝损伤模型中细胞角蛋白异常表达及其机制──免疫组化及免疫电镜研究

本研究应用ＡＢＣ免疫组化技术显示,奶油黄、液氮致局部冻伤及ＣＣｌ＿４所致的三种肝损伤中也有细胞角蛋白（ＣＫ）异常表达肝细胞。（１）在局部肝冻伤及奶油黄性肝损伤中表明不伴脂肪变性的肝细胞坏死不能直接引起肝细胞ＣＫ表达的改变;（２）在奶油黄性肝损伤中显示了卵圆细胞对肝细胞ＣＫ异常表达的诱导作用,表明层粘连蛋白（ＬＮ）可能是这种作用的媒介;（３）在ＣＣｌ＿４致慢性肝损伤中表明肝细胞ＣＫ异常表达和ＬＮ异常沉积无论在位相上还是在时相上都一致,提出肝小叶结构破坏可能也是通过ＬＮ异常沉积而影响肝细胞的ＣＫ表达;（４）应用电镜及免疫电镜技术表明ＣＣｌ＿４性肝损伤中肝细胞中间丝细胞骨架结构的改变伴随着ＣＫ１９阳性抗原决定簇的出现;（５）设计了一种局部肝冻伤模型,利用这种模型表明,ＣＫ１９阳性肝细胞在肝小叶结构完整性遭到破坏且伴纤维组织增生时出现,随小叶结构的恢复而消失。这是对关于肝细胞ＣＫ异常表达是肝小叶结构修复过程中局部肝细胞的修复性反应这一假说的有力支持。讨论了这种改变的意义。     

Altered cytokeratin expression and differentiation induction during neoplastic transformation of cultured rat liver cells by nickel subsulfide

Rat liver T51 B cells were maintained in the presence of low concentrations of Ni(H) derived from Ni₃S₂ for 3–I5 months in culture in order to monitor cytokeratin, differentiation, and transformation patterns. Nickel exposures caused irreversible, heritable juxtanuclear aggregates of cytokeratin CKSS, which increased in size and complexity with prolonged nickel exposure, eventually resembling Mallory bodies and expressing glutamyltransferase. Altered cytokeratin expression was accompanied by induction of differentiation, with markers of both bile ductular cells and hepatocytes, such as induction of cytokeratin polypeptides CK39 and CK49, cell morphology, and cytokeratin filament network changes, whereas control cultures similarly maintained for long periods in culture remained unchanged. Altered cytokeratin expression was also accompanied by acquisition of transformation markers—loss of density dependence, progression toward calcium independence, and (benign) growth in nude mice. Observed cytokeratin aberrations may be a factor in nickel carcinogenesis, in view of the known affinity of the metal for cellular structural proteins, especially keratin, which play a role in maintenance of cell behavior.Abbreviations CK cytokeratin - GGT gamma-glutamyltransferase - HCM standard calcium medium - LCM low calcium medium (Ca²⁺ = 0.02 mM) - MB mallory body - NI neoplastic index (ratio of growth of cells in LCM/HCM) - Ni ni(II) used as leachate from Ni₃S₂ - PBS phosphate-buffered saline - SB sodium butyrate     

Biochemical and cytochemical localization of inosine-5''-diphosphatase in rat liver microsomal fractions.

Golgi and endoplasmic-reticulum fractions were prepared from the lactating guinea-pig mammary gland. The endoplasmic-reticulum fraction was highly active in the processing and sequestration of milk-protein primary translation products. Explants from the lactating gland in organ culture were used to identify milk-protein intermediates present in the secretory pathway, and the timing of the events leading to their post-translational modification. With [35S]methionine, the milk proteins labelled after a short pulse (3 min) were represented by the partially processed (but not phosphorylated) caseins and alpha-lactalbumin sequestered within membrane-bound vesicles. After a 30 min labelling period, higher-Mr caseins with electrophoretic mobilities identical with those of the phosphorylated caseins isolated from milk were identified in the incubation medium, and sequestered within membrane-bound vesicles. Pulse-chase experiments established a precursor-product relationship between these forms. Secretion is apparent approx. 30 min after sequestration. Caseins are highly phosphorylated; removal of the phosphate residues with acid phosphatase results in proteins with increased electrophoretic mobility, similar to those of the partially processed early casein intermediates found sequestered in explants after a 3 min pulse with [35S]methionine, and those sequestered within microsomal membranes after mRNA-directed cell-free protein synthesis. A comparison of the proteins labelled during both short (5 min) and long (30 min) pulses with [35S]methionine and [32P]Pi shows that, in contrast with the 35S-labelled caseins, those labelled with [32P]Pi exhibit only electrophoretic mobilities identical with those of the mature caseins isolated from milk and those identified after long labelling periods with [35S]methionine. No phosphorylated early intermediate forms of caseins were identified. We conclude that the synthesis and post-translational modification of guinea-pig caseins occurs in two stages, (i) an early event involving synthesis and sequestration within the endoplasmic reticulum, an event that involves signal-peptide removal, followed (ii) 10-20 min later by phosphorylation at a different point in the secretory pathway, probably in the Golgi complex. Secretion of the phosphorylated caseins occurs 10-20 min later.     

A new negatively regulated acute-phase phosphoprotein synthesized by rat hepatocytes.

The effect of acute inflammation on the production of the major phosphorylated protein (PP63) excreted by rat hepatocytes was investigated. Both intra- and extracellular forms of the protein labelled with [32P]Pi, [3H]fucose and [35S]methionine were immunoprecipitated with monospecific polyclonal antibodies, and relative rates of PP63 synthesis were measured. The hepatocytes of acutely inflamed rats produced and excreted 85% less 32P- and 3H-labelled PP63 than did control cells. This decreased amount of PP63 did not result from an impairment in the phosphorylation or glycosylation processes or from a blockade in excretion, but rather was found to be due to extensive shut-off in biosynthesis of the protein as measured by [35S]methionine incorporation. Thus PP63 would appear to represent a new negatively regulated acute-phase protein.     

Synergistic stimulatory effect of glucocorticoid, EGF and insulin on the synthesis of ribosomal RNA and phosphorylation of nucleolin in primary cultured rat hepatocytes.

Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1 microM) and/or for 20 h with dexamethasone (1 microM) before the end of incubation. The incorporation of [3H]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2-3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [32P]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin.     

Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics

We have used biochemical and morphological techniques to demonstrate that hepatocytes in the perfused liver bind, internalize, and degrade substantial amounts of murine epidermal growth factor (EGF) via a receptor-mediated process. Before ligand exposure, about 300,000 high-affinity receptors were detectable per cell, displayed no latency, and co-distributed with conventional plasma membrane markers. Cytochemical localization using EGF coupled to horseradish peroxidase (EGF-HRP) revealed that the receptors were distributed along the entire sinusoidal and lateral surfaces of hepatocytes. When saturating concentrations of EGF were perfused through a liver at 35 degrees C, ligand clearance was biphasic with a rapid primary phase of 20,000 molecules/min per cell that dramatically changed at 15-20 min to a slower secondary phase of 2,500 molecules/min per cell. During the primary phase of uptake, approximately 250,000 molecules of EGF and 80% of the total functional receptors were internalized into endocytic vesicles which could be separated from enzyme markers for plasma membranes and lysosomes on sucrose gradients. The ligand pathway was visualized cytochemically 2-25 min after EGF-HRP internalization and a rapid transport from endosomes at the periphery to those in the Golgi apparatus-lysosome region was observed (t 1/2 approximately equal to 7 min). However, no 125I-EGF degradation was detected for at least 20 min. Within 30 min after EGF addition, a steady state was reached which lasted up to 4 h such that (a) the rate of EGF clearance equaled the rate of ligand degradation (2,500 molecules/min per cell); (b) a constant pool of undegraded ligand was maintained in endosomes; and (c) the number of accessible (i.e., cell surface) receptors remained constant at 20% of initial values. By 4 h hepatocytes had internalized and degraded 3 and 2.3 times more EGF, respectively, than the initial number of available receptors, even in the presence of cycloheximide and without substantial loss of receptors. All of these results suggest that EGF receptors are internalized and that their rate of recycling to the surface from intracellular sites is governed by the rate of entry of ligand and/or receptor into lysosomes.     

Synergistic stimulatory effect of glucocorticoid,EGF and insulin on the synthesis of ribosomal RNA and phosphorylation of nucleolin in primary cultured rat hepatocytes

Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1 μM) and/or for 20 h with dexamethasone (1 μM) before the end of incubation. The incorporation of [³H]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2–3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [³²P]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin.     

The effects of bestatin, a microbial aminopeptidase inhibitor, on epidermal growth factor-induced DNA synthesis and cell division in primary cultured hepatocytes of rats

We investigated the effects of microbial protease inhibitors, in particular the aminopeptidase inhibitor bestatin, on DNA synthesis and cell division induced by epidermal growth factor (EGF) in hepatocytes. Although bestatin did not significantly affect binding of EGF to hepatocytes, it inhibited EGF-induced DNA synthesis and cell division. DNA synthesis in rat hepatocytes was maximal 24-26 h after EGF addition to the medium. The time required for maximal DNA synthesis was not affected if bestatin was removed less than 12 h after addition, but synthesis was partially inhibited if bestatin was added to the medium several hours after EGF addition, depending on the time of bestatin addition. Our results suggest that bestatin arrests the new cell cycle induced by EGF at about 12 h after the initiation. Considering also our results obtained by employing other protease inhibitors, we concluded that specific proteases play important roles in hepatocyte DNA synthesis and cell division induced by EGF.     

Phosphorylation and Dephosphorylation of Guard-Cell Proteins from Vicia faba L. in Response to Light and Dark

Phosphorylation and dephosphorylation of proteins were investigated in guard-cell protoplasts from Vicia faba L. When guard-cell protoplasts were incubated with 32Pi in the dark for 80 min, several proteins, with molecular masses of 42, 40, 34, 32, 26, and 19 kD, were phosphorylated. Illumination of the dark-adapted protoplasts with red light caused dephosphorylation of the 26-kD protein, but there was no detectable change in levels of phosphorylation in other proteins. In the dephosphorylation of the 26-kD protein, far-red light of 730 nm was most effective, but when the light was turned off, the protein was phosphorylated to the original level within 10 min. Subcellular fractionation of guard-cell protoplasts indicated that the 26-kD protein was located in the chloroplast. The migration pattern of the 26-kD protein was exactly the same as the light-harvesting Chl a/b protein complex of photosystem II (LHCPII) from Vicia mesophyll cells on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dephosphorylated 26-kD protein was phosphorylated by adding sodium hydrosulfite, a strong reducing agent, under the far-red illumination of guard-cell protoplasts. The magnitude of dephosphorylation by red light (660 nm) was increased by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an electron transfer inhibitor of photosystem II (PSII). Light-induced dephosphorylation was inhibited by 1 nM okadaic acid, an inhibitor of serine/threonine protein phosphatase. From these results, it is concluded that the 26-kD protein is LHCPII and that LHCPII is present mostly in the phosphorylated form in the dark and is dephosphorylated by type 2A protein phosphatase under the light absorbed by photosystem I in Vicia guard-cell protoplasts.     

Identification of Ser-55 as a major protein kinase A phosphorylation site on the 70-kDa subunit of neurofilaments. Early turnover during axonal transport.

The 70-kDa neurofilament protein subunit (NF-L) is phosphorylated in vivo on at least three sites (L1 to L3) (Sihag, R. K. and Nixon, R. A. (1989) J. Biol. Chem. 264, 457-464). The turnover of phosphate groups on NF-L during axonal transport was determined after the neurofilaments in retinal ganglion cells were phosphorylated in vivo by injecting mice intravitreally with [32P]orthophosphate. Two-dimensional phosphopeptide maps of NF-L from optic axons of mice 10 to 90 h after injection showed that radiolabel decreased faster from peptides L2 and L3 than from L1 as neurofilaments were transported. To identify phosphorylation sites on peptide L2, axonal cytoskeletons were phosphorylated by protein kinase A in the presence of heparin. After the isolated NF-L subunits were digested with alpha-chymotrypsin, 32P-peptides were separated by high performance liquid chromatography on a reverse-phase C8 column. Two-dimensional peptide mapping showed that the alpha-chymotrypsin 32P-peptide accepting most of the phosphates from protein kinase A migrated identically with the in vivo-labeled phosphopeptide L2. The sequence of this peptide (S-V-R-R-S-Y) analyzed by automated Edman degradation corresponded to amino acid residues 51-56 of the NF-L sequence. A synthetic 13-mer (S-L-S-V-R-R-S-Y-S-S-S-S-G) corresponding to amino acid residues 49-61 of NF-L was also phosphorylated by protein kinase A. alpha-Chymotryptic digestion of the 13-mer generated a peptide which contained most of the phosphates and co-migrated with the phosphopeptide L2 on two-dimensional phosphopeptide maps. Edman degradation of the phosphorylated 13-mer identified serine residue 55 which is located within a consensus phosphorylation sequence for protein kinase A as the major site of phosphorylation. Since protein kinase A-mediated phosphorylation influences intermediate filament assembly/disassembly in vitro, we propose that the phosphopeptide L2 region is a neurofilament-assembly domain and that the cycle of phosphorylation and dephosphorylation of Ser-55 on NF-L, which occurs relatively early after subunit synthesis in vivo, regulaaes a step in neurofilament assembly or initial interactions during axonal transport.     

Phorbol acetate enhances the phosphorylation of cytokeratins 8 and 18 in human colonic epithelial cells.

The phosphorylation of epithelial-specific cytokeratin (CK) 8 and 18 was studied in the human colonic cell line HT29. Metabolic labelling of cells with orthophosphate resulted in phosphorylation of cytokeratins 8/18 on serine residues. When phorbol acetate was added to labelled cells, a 2.2-fold increase in CK8/18 phosphate labelling was noted, whereas increasing intracellular cAMP levels using forskolin or 8-Br-cAMP showed no significant change in CK phosphorylation. CKs8/18 were also phosphorylated by added PKC in the presence of [gamma-32P]ATP. Tryptic peptide map analysis of the phosphorylated CK8 species showed that treatment of cells with 8-Br-cAMP or phorbol acetate generated a phosphopeptide not seen in control cells. In contrast, tryptic peptide maps of phosphorylated CK18 showed no discernable differences. Our results support a role for PKC in the phosphorylation of epithelial cytokeratins, with some phosphorylation sites being modulated by cAMP dependent protein kinase.     

Cytokeratin filament assembly in the preimplantation mouse embryo

The timing, spatial distribution and control of cytokeratin assembly during mouse early development has been studied using a monoclonal antibody, TROMA-1, which recognizes a 55,000 Mr trophectodermal cytokeratin (ENDO A). This protein was first detected in immunoblots at the 4-cell stage, and became more abundant at the 16-cell stage and later. Immunofluorescence analysis revealed assembled cytokeratin filaments in some 8-cell blastomeres, but not at earlier stages. At the 16-cell stage, filaments were found in both polarized (presumptive trophectoderm; TE) and apolar (presumptive inner cell mass; ICM) cells in similar proportions, although polarized cells possessed more filaments than apolar cells. By the late 32-cell, early blastocyst, stage, all polarized (TE) cells contained extensive filament networks whereas cells positioned inside the embryo tended to have lost their filaments. The presence of filaments in inside cells at the 16-cell stage and in ICM cells was confirmed by immunoelectron microscopy. Lineage tracing techniques demonstrated that those cells in the ICM of early blastocysts which did possess filaments were almost exclusively the progeny of polar 16-cell blastomeres, suggesting that these filaments were directly inherited from outside cells at the 16- to 32-cell transition. Inhibitor studies revealed that proximate protein synthesis but not mRNA synthesis is required for filament assembly at the 8-cell stage. These results demonstrate that there are quantitative rather than qualitative differences in the expression of cytokeratin filaments in the inner cell mass and trophectoderm cells of the mouse embryo.