[Capillary gel electrophoresis in clinical molecular biology].
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Animals↗
Biomedical subjects
J Liao
Publications and source records attributed to J Liao.
[High performance liquid chromatography for analysis of reverse transcription polymerase chain reaction (RT-PCR) products].
The polymerase chain reaction is a powerful method for amplifying specific DNA sequences in vitro. Reverse transcribing mRNA into cDNA expands the use of PCR to monitor mRNA expression in biological system. A method for the analysis of RT-PCR products by HPLC was developed. The separation was performed on a nonporous ion exchange resin column with gradient elution of sodium chloride in 20 mmol/L Tris-HCl buffer (pH 9.0) at a flow rate of 1.0 mL/min and the detection wavelength was 260 nm. lambda-DNA-Hind III digest and a series of RT-PCR products were analyzed for studying the mRNA expression of secreted phospholipase A2 after being injured.
Animals↗
Growth hormone stimulates phosphorylation and activation of elk-1 and expression of c-fos, egr-1, and junB through activation of extracellular signal-regulated kinases 1 and 2.
Growth hormone (GH), a major regulator of normal body growth and metabolism, regulates cellular gene expression. The transcription factors Elk-1 and Serum Response Factor are necessary for GH-stimulated transcription of c-fos through the Serum Response Element (SRE). GH stimulates the serine phosphorylation of Elk-1, thereby enabling Elk-1 to mediate transcriptional activation. The contribution of the Ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway to Elk-1-mediated transcriptional activation of the c-fos SRE in response to GH was examined. The MEK inhibitor PD098059 attenuated GH-induced expression of the endogenous SRE-regulated genes c-fos, egr-1, and junB as well as transcriptional activation mediated by the c-fos promoter. The MEK inhibitor blocked GH-stimulated activation of MEK, phosphorylation of ERK1/ERK2, and MAP kinase activity in 3T3-F442A cells. Blocking MEK activation prevented GH-induced phosphorylation of Elk-1, as well as the ability of Elk-1 to mediate transcriptional activation in response to GH. Overexpression of dominant-negative Ras or the ERK-specific phosphatase, mitogen-activated protein kinase phosphatase-1, blocked the Ras/MEK/ERK pathway and abrogated GH-induced phosphorylation of Elk-1. GH failed to stimulate phosphorylation or activation of Jun N-terminal kinase under the conditions used. GH slightly increased p38-mediated mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2 activity, but the p38 inhibitor SB203580 did not attenuate GH-promoted Elk-1 phosphorylation. Wortmannin, which inhibited GH-induced ERK phosphorylation, also attenuated transcriptional activation of c-fos by GH. Taken together, these data suggest that GH-dependent activation of the Ras/MEK/ERK pathway and subsequent serine phosphorylation of Elk-1 contribute to GH-stimulated c-fos expression through the SRE.
3T3 Cells↗
Activation of protein kinase B/Akt is sufficient to repress the glucocorticoid and cAMP induction of phosphoenolpyruvate carboxykinase gene.
A rat hepatoma cell line, H4IIE, was stably transfected with a tamoxifen regulatable Akt-1 construct. Treatment of these cells with tamoxifen caused a rapid stimulation of Akt enzymatic activity that was comparable with the activity observed with the endogenous Akt after insulin stimulation. Prior studies have extensively documented that insulin can repress the glucocorticoid and cAMP-stimulated increase in phosphoenolpyruvate carboxykinase (PEPCK) gene transcription. Activation of this regulatable Akt with tamoxifen was found to mimic the dominant inhibitory effect of insulin on PEPCK gene transcription. Dose response curves to insulin and tamoxifen demonstrated that this response was very sensitive to Akt activation although the maximal response observed with tamoxifen activation was slightly less than that observed with insulin, indicating that the response to insulin may also involve other signaling cascades. The regulation of PEPCK transcription via Akt was, like that previously described for insulin, not dependent upon 70 kDa S6 kinase activity in that it was not inhibited by rapamycin. Finally, the expression of a kinase dead Akt was able to partially inhibit the ability of insulin to stimulate this response. In summary, the present results indicate that activation of Akt alone is sufficient to repress the glucocorticoid and cAMP-stimulated increase in PEPCK gene transcription.
Animals↗
Inhibition of Stat1-mediated gene activation by PIAS1.
STAT (signal transducer and activator of transcription) proteins are latent cytoplasmic transcription factors that become activated by tyrosine phosphorylation in response to cytokine stimulation. Tyrosine phosphorylated STATs dimerize and translocate into the nucleus to activate specific genes. Different members of the STAT protein family have distinct functions in cytokine signaling. Biochemical and genetic analysis has demonstrated that Stat1 is essential for gene activation in response to interferon stimulation. Although progress has been made toward understanding STAT activation, little is known about how STAT signals are down-regulated. We report here the isolation of a family of PIAS (protein inhibitor of activated STAT) proteins. PIAS1, but not other PIAS proteins, blocked the DNA binding activity of Stat1 and inhibited Stat1-mediated gene activation in response to interferon. Coimmunoprecipitation analysis showed that PIAS1 was associated with Stat1 but not Stat2 or Stat3 after ligand stimulation. The in vivo PIAS1-Stat1 interaction requires phosphorylation of Stat1 on Tyr-701. These results identify PIAS1 as a specific inhibitor of Stat1-mediated gene activation and suggest that there may exist a specific PIAS inhibitor in every STAT signaling pathway.
Amino Acid Sequence↗
Characterization of genes which exhibit reduced expression during the retinoic acid-induced differentiation of F9 teratocarcinoma cells: involvement of cyclin D3 in RA-mediated growth arrest.
In the presence of retinoic acid (RA), F9 murine teratocarcinoma cells differentiate into cells resembling the extra-embryonic endoderm of the early mouse embryo. Using differential hybridization, we have cloned and characterized six cDNAs corresponding to mRNAs that exhibit reduced expression in F9 cells following RA treatment. Two of these cDNAs encode novel genes (REX-2 and REX-3). The other isolated cDNAs encode genes that have been previously described in other contexts: 1-4 (cyclin D3); 2-10 (pyruvate kinase); 2-12 (glutathione S-transferase); and 2-17 (GLUT 3). The mRNA levels of these genes are reduced by RA or RA plus theophylline and cAMP (RACT) only after 48 h of treatment, and continue to decrease at 96 h. The half-lives of these mRNAs are not changed by RA treatment, indicating that these mRNAs may be regulated through a transcriptional mechanism. In isoleucine-deprived cells, which are growth arrested but do not differentiate, the steady state mRNA levels of genes Rex 2, Rex 3, pyruvate kinase and GLUT 3 are not reduced, in contrast to cyclin D3 and glutathione S-transferase. The expression of the REX-2, REX-3, pyruvate kinase, glutathione S-transferase and GLUT 3 genes is reduced by RACT to the same extent in F9 RARgamma-/- and RARalpha-/- lines as in F9-Wt. In contrast, cyclin D3 exhibits lower mRNA expression in F9 RARgamma-/- and RARalpha-/- stem cells, and this mRNA is not decreased by RACT treatment. Overexpression of cyclin D3 blocks the RA-induced growth arrest of F9 cells, indicating that the downregulation of this gene following RA treatment may constitute a necessary step in the cascade of events leading to growth inhibition by RA.
Amino Acid Sequence↗
Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins.
Members of the 14-3-3 protein family bind the human intermediate filament protein keratin 18 (K18) in vivo, in a cell-cycle- and phosphorylation-dependent manner. We identified K18 Ser33 as an interphase phosphorylation site, which increases its phosphorylation during mitosis in cultured cells and regenerating liver, and as an in vitro cdc2 kinase phosphorylation site. Comparison of wild-type versus K18 Ser33-->Ala/Asp transfected cells showed that K18 Ser33 phosphorylation is essential for the association of K18 with 14-3-3 proteins, and plays a role in keratin organization and distribution. Mutation of another K18 major phosphorylation site (Ser52) or K18 glycosylation sites had no effect on the binding of K18 to 14-3-3 proteins. The K18 phospho-Ser33 motif is different from several 14-3-3-binding phosphomotifs already described. Antibodies that are specific to K18 phospho-Ser33 or phospho-Ser52 show that although Ser52 and Ser33 phosphorylated K18 molecules manifest partial colocalization, these phosphorylation events reside predominantly on distinct K18 molecules. Our results demonstrate a unique K18 phosphorylation site that is necessary but not sufficient for K18 binding to 14-3-3 proteins. This binding is likely to involve one or more mitotic events coupled to K18 Ser33 phosphorylation, and plays a role in keratin subcellular distribution. Physiological Ser52 or Ser33 phosphorylation on distinct K18 molecules suggests functional compartmentalization of these modifications.
14-3-3 Proteins↗
Poly(A)- and poly(U)-specific RNA 3' tail shortening by E. coli ribonuclease E.
Ribonuclease (RNase) E is an extensively studied enzyme from Escherichia coli whose site-specific endoribonuclease activity on single-stranded RNA has a central role in the processing of ribosomal RNA, the degradation of messenger RNA and the control of replication of ColE1-type plasmids. Here we report a previously undetected activity of RNase E: the ability to shorten 3' poly(A)- and poly(U)-homopolymer tails on RNA molecules. This activity, which leaves a 6-nucleotide adenylate or a 1-nucleotide uridylate remnant on primary transcripts, resides in the amino-terminal region of RNase E and does not require other protein cofactors. Addition of a 3'-terminal phosphate group prevents both removal of the poly(A) tail and endonucleolytic cleavage within primary transcripts, but has no effect on the cleavage of transcripts with tails that have already been truncated. The ability of RNase E to shorten poly(A) tails, together with the effect of tail length on endonucleolytic cleavage within primary transcripts, suggests a mechanism by which RNase E may exercise overall control over RNA decay.
Base Sequence↗
Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation.
Two zebrafish bHLH genes, neurogenin-related gene I (ngn1) and neuroD (nrd), have been isolated. ngn1 expression is initiated at the end of gastrulation in the neural plate and defines broad domains of cells that probably possess an ability to develop as neurons. This finding suggests that ngn1 may play a role during determination of cell fate in neuroblasts. ngn1 and pax-b are expressed in a mutually exclusive manner. nrd expression follows that of ngn1 in restricted populations of cells selected from ngn1-positive clusters of cells. The earliest nrd-positive cells in the brain and the trunk are a subset of the primary neurons. ngn1 is not expressed in the eye. Here, nrd transcription is activated at 25 hours postfertilization in the ventral retina. Expression of islet-1 occurs in nrd-positive cells after expression of nrd, and the expression of the two genes partially overlaps in time. These observations suggest that during eye development nrd expression may follow expression of some other neurodetermination gene(s). This supports the idea that expression of nrd is a necessary step leading toward overt neuronal differentiation.
Amino Acid Sequence↗
Structures of murine carbonic anhydrase IV and human carbonic anhydrase II complexed with brinzolamide: molecular basis of isozyme-drug discrimination.
Carbonic anhydrase IV (CAIV) is a membrane-associated enzyme anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage. We have determined the 2.8-angstroms resolution crystal structure of a truncated, soluble form of recombinant murine CAIV. We have also determined the structure of its complex with a drug used for glaucoma therapy, the sulfonamide inhibitor brinzolamide (Azopt). The overall structure of murine CAIV is generally similar to that of human CAIV; however, some local structural differences are found in the active site resulting from amino acid sequence differences in the "130's segment" and the residue-63 loop (these may affect the nearby catalytic proton shuttle, His-64). Similar to human CAIV, the C-terminus of murine CAIV is surrounded by a substantial electropositive surface potential that may stabilize the interaction with the phospholipid membrane. Binding interactions observed for brinzolamide rationalize the generally weaker affinity of inhibitors used in glaucoma therapy toward CAIV compared with CAII.
Amino Acid Sequence↗
Inhibition of growth and induction of apoptosis in human cancer cell lines by tea polyphenols.
In order to study the biological activities of tea preparations and purified tea polyphenols, their growth inhibitory effects were investigated using four human cancer cell lines. Growth inhibition was measured by [3H]thymidine incorporation after 48 h of treatment. The green tea catechins (-)-epigallocatechin-3-gallate (EGCG) and (-)-epigallocatechin (EGC) displayed strong growth inhibitory effects against lung tumor cell lines H661 and H1299, with estimated IC50 values of 22 microM, but were less effective against lung cancer cell line H441 and colon cancer cell line HT-29 with IC50 values 2- to 3-fold higher. (-)-Epicatechin-3-gallate, had lower activities, and (-)-epicatechin was even less effective. Preparations of green tea polyphenols and theaflavins had higher activities than extracts of green tea and decaffeinated green tea. The results suggest that the growth inhibitory activity of tea extracts is caused by the activities of different tea polyphenols. Exposure of H661 cells to 30 microM EGCG, EGC or theaflavins for 24 h led to the induction of apoptosis as determined by an annexin V apoptosis assay, showing apoptosis indices of 23, 26 and 8%, respectively; with 100 microM of these compounds, the apoptosis indices were 82, 76 and 78%, respectively. Incubation of H661 cells with EGCG also induced a dose-dependent formation of H2O2. Addition of H2O2 to H661 cells caused apoptosis in a manner similar to that caused by EGCG. The EGCG-induced apoptosis in H661 cells was completely inhibited by exogenously added catalase (50 units/ml). These results suggest that tea polyphenol-induced production of H2O2 may mediate apoptosis and that this may contribute to the growth inhibitory activities of tea polyphenols in vitro.
Adenocarcinoma↗
Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis by dietary olive oil and squalene.
Epidemiological studies have suggested that frequent olive oil consumption may be a protective factor against lung cancer formation. Squalene, a characteristic compound in olive oil, is an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and has been proposed to inhibit the farnesylation of ras oncoproteins. The present study investigated the effect of dietary olive oil and squalene in a mouse lung tumorigenesis model. Female A/J mice were fed AIN-76A diets containing 5% corn oil (control), 19.6% olive oil, or 2% squalene starting at 3 weeks before a single dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (103 mg/kg, i.p.). Animals were maintained on their respective diets throughout the study. At 16 weeks after NNK administration, 100% of the mice in the control group had lung tumors with a tumor multiplicity of 16 tumors per mouse. The olive oil and squalene diets significantly (P < 0.05) decreased the lung tumor multiplicity by 46 and 58%, respectively. The squalene diet significantly (P < 0.05) decreased lung hyperplasia by 70%. In mice fed a diet containing 2% squalene for 3 weeks, the activation of NNK was increased by 1.4- and 2.0-fold in lung and liver microsomes, respectively, but its relationship to the inhibition of carcinogenesis is not clear. These results demonstrate that dietary olive oil and squalene can effectively inhibit NNK-induced lung tumorigenesis.
Animals↗
Regulation of glucose transport and c-fos and egr-1 expression in cells with mutated or endogenous growth hormone receptors.
To identify mechanisms by which GH receptors (GHR) mediate downstream events representative of growth and metabolic responses to GH, stimulation by GH of c-fos and egr-1 expression and glucose transport activity were examined in Chinese hamster ovary (CHO) cells expressing mutated GHR. In CHO cells expressing wild-type GHR(GHR(1-638)), GH stimulated the expression of c-fos and egr-1, and stimulated 2-deoxyglucose uptake, responses also mediated by endogenous GHR in 3T3-F442A cells. Deletion of the proline-rich box 1 of GHR (GHR(deltaP)) abrogated all of these responses to GH, indicating that box 1, a site of association of GHR with the tyrosine kinase JAK2, is crucial for these GH-stimulated responses. As the C-terminal half of the cytoplasmic domain of GHR is required for GH-stimulated calcium flux and for stimulation of spi-2.1 transcription, GHR lacking this sequence (GHR(1-454)) were examined. Not only did GHR(1-454) mediate stimulation of c-fos and egr-1 expression and 2-deoxyglucose uptake, but they also mediated GH-stimulated transcriptional activation via Elk-1, a transcription factor associated with the c-fos Serum Response Element. Thus, the C-terminal half of the cytoplasmic domain of GHR is not required for GH-stimulated c-fos transcription, suggesting that increased calcium is not required for GH-stimulated c-fos expression. In CHO cells lacking all but five N-terminal residues of the cytoplasmic domain (GHR(1-294)), GH did not induce c-fos or egr-1 expression or stimulate 2-deoxyglucose uptake. Further, in 3T3-F442A fibroblasts with endogenous GHR, GH-stimulated c-fos expression and 2-deoxyglucose uptake were reduced by the tyrosine kinase inhibitors herbimycin A, staurosporine, and P11. Herbimycin A and staurosporine inhibit JAK2 and tyrosyl phosphorylation of all proteins stimulated by GH, whereas P11 inhibits the GH-dependent tyrosyl phosphorylation of only some proteins, including extracellular signal regulated kinases ERK1 and -2, but not JAK2. Taken together, these results implicate association of GHR with JAK2 and GH-stimulated tyrosyl phosphorylation of an additional cellular protein in GH-stimulated glucose transport and c-fos and egr-1 expression. These studies also indicate that, in contrast to spi-2.1, the N-terminal half of the cytoplasmic domain of GHR is sufficient to mediate stimulation of c-fos and egr-1 expression and Elk-1 activation, supporting multiple mechanisms for GH signaling to the nucleus.
3T3 Cells↗
[Regional differences in capillary density of children's skin--an enzyme histochemical and stereological study].
OBJECTIVE: The purpose of this study was to investigate the capillary distribution characteristics of the human skin. METHOD: The alkaline phosphatase enzyme histochemical and stereological methods were used to measure the length density (Lv) of capillaries of normal skin. One hundred and twenty specimens were taken from 20 different sites in 6 children cadavers. RESULT: The results showed: 1. The capillary Lv of the human skin varied with the body region. Statistically significantly (P < 0.01) greater capillary Lv was found in the head-face/neck region both in the papillary and reticular dermis than in the other parts of the body (excluding palm and sole). The smallest dermal capillary Lv was noted in the lower limb. 2. Statistically significantly (P < 0.001) higher capillary Lv was found in the papillary dermis than in the reticular dermis. CONCLUSION: The study confirms the regional and laminal differences in capillary density of the normal human skin, and it demonstrates more accurately rich blood supply in the head-face/neck region, which supports the hypothesis that longer random-pattern skin flaps can be safely raised in the head-face/neck than in other parts of the body.
Capillaries↗
[Optimization of non-gel sieving capillary electrophoretic separation of DNA fragments of hundreds of base pairs].
Non-gel sieving capillary electrophoresis has been employed in the biological sciences for the size-based separation of macromolecules such as nucleic acids. In this paper, four factors i.e. electric field strength, capillary length, capillary diameter and hydroxy-propylmethylcellulose (HPMC) concentration were integratively evaluated to select the optimal condition of separating DNA fragments of a few hundreds of base pairs through orthogonal analysis. Conclusion was made through comprehensive analysis: better separation could be achieved in longer capillary, smaller inside diameter of capillary and less field strength. In practical application, effective separation in short time is important. We preferred to employ 8 g/L HPMC in coated capillary (37 cm x 75 microns i.d.) and electric field strength of 324 V/cm in separating DNA fragments of hundreds of base pairs. From sampling to getting results only a few more than ten minutes were needed in capillary electrophoresis. It needs less amount of sample (a few nanoliters) and shows higher sensitivity (0.1 pmol in UV detection) than polyacrylamide gel electrophoresis (PAGE). pBR322/Msp I fragments were completely resolved in both capillary electrophoresis and PAGE, and four-bases resolution was obtained. When the concentration of the PCR product is too low (< 20 mg/L), PCR reaction system is used as a negative control to make sure of the peak generated by the unpurified PCR products, avoiding interference made by PCR buffer and polymerase.
Apolipoprotein B-100↗
Economic outcomes of antipsychotic agents in a Medicaid population: traditional agents vs. risperidone.
Clinical trials reveal that the newer atypical antipsychotic agents are more effective and have fewer side effects than traditional agents. However, these newer agents have a higher acquisition cost than traditional agents. This study assessed the differential impact of risperidone and traditional agents on the total schizophrenia-related cost of care for Medicaid patients suffering from schizophrenia. This was a retrospective longitudinal pretest-posttest analysis of Medicaid claims data covering January 1992 to August 1996. Continuously eligible patients (n = 150) with a documented diagnosis of schizophrenia were evaluated. Medical claims were analyzed for patients treated with traditional antipsychotics for at least 12 months and then switched to risperidone and followed for at least 12 months. Patients who failed on at least one traditional agent and who remained on other traditional agents throughout the study timeframe served as a control group. Monthly costs per patient were estimated using mixed model linear regression with age and gender serving as covariates. The total monthly costs per patient for the risperidone and traditional cohorts were similar ($1,050.52 and $946.24, respectively; p = .5438) during the pretest phase of the study. For patients treated with risperidone, drug costs were $177.35 higher (CL0.95 +/- $7.64; p = .0001) per patient per month in the posttest period compared with the pretest period. However inpatient hospital costs were $312.04 lower (CL0.95 +/- $146.76; p = .001) per patient per month in the posttest period compared with the pretest period. In addition, physician costs were $9.55 lower (CL0.95 +/- $5.31; p = .0004) per patient per month in the posttest period. The difference from the pretest to posttest period for outpatient mental health clinic costs was statistically similar. For those in the risperidone cohort, total estimated costs decreased by $204.87 per patient per month during treatment with risperidone (CL0.95 +/- $161.01; p = .0127). Over the same time-frame, total costs increased $160.68 per patient per month (CL0.95 +/- $196.04; n.s.; p = .1082) in the control cohort. While the mean monthly drug cost was significantly higher during treatment with risperidone, this increase was offset by cost reductions elsewhere in the system.
Adult↗
Keratin modifications and solubility properties in epithelial cells and in vitro.
The gains that have been made in characterizing some of the keratin posttranslational modifications have helped answer some questions regarding these modifications and have generated an information base for asking additional refined questions in future studies. Highlights of where we believe we currently stand with regard to keratin posttranslational modifications are as follows: 1. Keratin glycosylation, via O-GlcNAc, is a dynamic modification that has been conclusively identified in K13, K8, and K18. Three serine glycosylation sites in the head domain of K18 have been identified, and it is possible that all keratins are glycosylated. The function of this modification remains to be defined, but is likely to be different from phosphorylation, since the two modifications are generally segregated on different molecules and several examples exist whereby both modifications increase simultaneously. 2. Keratin phosphorylation occurs within the tail and/or head domains of all keratins that have been examined. Several serine phosphorylation sites and some of the relevant kinases have been characterized in K8, K6, and K18, and serine/threonine sites have been identified in K1. Functions of keratin phosphorylation that have significant experimental support include a role in filament solubility and reorganization and a role in regulating keratin binding with other cytoplasmic proteins. The significance of filament reorganization and increased solubility under a variety of physiologic conditions such as mitosis and cell stress are important areas of future and ongoing investigation. Other associations with keratin phosphorylation include protection against cell stress, cell signaling, apoptosis, and cell compartment-specific roles. At this stage, however, it is not known if these associations play direct or indirect roles. 3. Keratin transglutamination occurs in epidermal and simple epithelial keratins under physiologic and pathologic states, respectively. In the physiological context, the role of this modification is clear in terms of providing a compact protective structure, while in the pathologic context of liver disease the role remains ambiguous. 4. Proteolysis of K18 and K19 by caspases occurs during apoptosis, and generates stable keratin fragments that are highly enriched within the cytoskeletal compartment. Proteolysis of the type II keratins appears to be spared for reasons that remain to be defined. It is likely that this apoptosis-associated degradation involves all type I keratins. Keratin fragments are also noted in sera of patients in association with a variety of epithelial tumors. If a signal does exist for the apoptosis-associated fragmentation, aside from caspase activation, then it appears that the overall increase in keratin phosphorylation during apoptosis does not account for this signal. 5. Keratins undergo several other posttranslational modifications including disulfide bond formation (not found in K8/18 due to lack of cystienes) and acetylation of their N-terminal serines. Modification by lipids is also possible, but this modification requires further confirmation. 6. Keratin solublility is highly dynamic and varies profoundly depending on the keratin pair and the physiologic state of the cell. Within the keratin family, simple epithelial keratins are among the most soluble (approximately 5% of K8/18 is soluble at basal conditions). Phosphorylation plays an important role in modulating keratin solubility, and distinct differences occur in site-specific phosphorylation depending on the soluble versus cytoskeletal partitioning of the keratin. Keratin solubility (at least for K8/18) also appears to be regulated by 14-3-3 proteins via K18 Ser33 phosphorylation.
Acetylation↗
Apoptosis generates stable fragments of human type I keratins.
Type I and II keratins help maintain the structural integrity of epithelial cells. Since apoptosis involves progressive cell breakdown, we examined its effect on human keratin polypeptides 8, 18, and 19 (K8, K18, K19) that are expressed in simple-type epithelia as noncovalent type I (K18, K19) and type II (K8) heteropolymers. Apoptosis induces rapid hyperphosphorylation of most known K8/18 phosphorylation sites and delayed formation of K18 and K19 stable fragments. In contrast, K8 is resistant to proteolysis and remains associated with the K18 fragments. Transfection of phosphorylation/glycosylation-mutant K8 and K18 does not alter fragment formation. The protein domains of the keratin fragments were determined using epitope-defined antibodies, and microsequencing indicated that K18 cleavage occurs at a conserved caspase-specific aspartic acid. The fragments are found preferentially within the detergent-insoluble pool and can be generated, in a phosphorylation-independent manner, by incubating keratins with caspase-3 or with detergent lysates of apoptotic cells but not with lysates of nonapoptotic cells. Our results indicate that type I keratins are targets of apoptosis-activated caspases, which is likely a general feature of keratins in most if not all epithelial cells undergoing apoptosis. Keratin hyperphosphorylation occurs early but does not render the keratins better substrates of the downstream caspases.
Amino Acid Sequence↗