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.     

Constitutive presence of a catalytic fragment of protein kinase C epsilon in a small cell lung carcinoma cell line.

Protein kinase C (PKC) has been implicated in a variety of cellular responses such as proliferation, differentiation, and secretion. We assessed the role of PKC in the mitogenic effects of gastrin-releasing peptide (in a small cell lung cancer (SCLC) cell line. Using antisera that specifically recognize the PKC isoforms alpha, beta, gamma, delta, and epsilon, we determined that PKC epsilon is the major isoform in the SCLC cell line NCI-N417, followed by PKC alpha and delta. In addition to the 90-kDa PKC epsilon, our anti-PKC epsilon antiserum specifically detected a 40-kDa immunoreactive protein. Treatment of the cells with either 20 nM phorbol myristate acetate or 50 nM GRP enhanced significantly the level of the 40-kDa protein in a time-dependent (1-8 h), cycloheximide-sensitive fashion. Subcellular fractionation revealed that 90% of PKC epsilon was in particulate form, while the 40-kDa immunoreactive protein was cytosolic. To test the hypothesis that the 40-kDa soluble protein represented a catalytically independent PKC epsilon fragment, cytosolic extracts were assayed for kinase activity. 45-50% of the activity was apparent in the absence of the PKC activators phosphatidylserine and diacylglycerol. This effector-independent kinase activity was further purified by affinity chromatography using a synthetic peptide corresponding to the pseudosubstrate region of PKC epsilon (ERMRPRKRQGAVRRRV) coupled to Sepharose. The partially purified protein, recognized by the anti-PKC epsilon antiserum, exhibited histone kinase activity with kinetics similar to those of the tryptically generated catalytic fragment of brain PKC epsilon. This activity was inhibited by staurosporine (IC50 = 1 x 10(-8) M) and by the pseudosubstrate inhibitor peptide (IC50 = 7.7 x 10(-8) M). The SCLC kinase and the brain PKC epsilon catalytic fragment were similar as indicated by the relative sizes of the PKC epsilon immunoreactive peptides generated with protease V8 from Staphylococcus aureus (Mr approximately 37,000, 34,000, 28,000, 26,000, and 25,000). Taken together, we conclude that a variant SCLC cell line expresses a constitutively active catalytic fragment of PKC epsilon. Regulation by 12-O-tetradecanoyl-13-acetate or GRP via de novo protein synthesis suggests a novel mechanism of control of PKC diversity with implications for small cell lung cancer and possibly other malignancies.     

Developmental regulation of insulin-like growth factor-II/mannose 6-phosphate receptor mRNA in the rat.

This study examined levels of insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M6PR) mRNA in tissues of rats at different stages of growth. Northern blot analysis of total RNA from tissues of rats aged 2, 9, 21 and 42 days and from 21 day fetal rats was carried out using a cDNA probe to the IGF-II/M6PR. Northern blots showed this probe hybridized to a single 9kb band in all tissues tested. Highest hybridization signals were detected in fetal and neonatal tissues with levels rapidly decreasing after birth. For all age groups tested the highest signal was obtained with heart followed by muscle, lung, and kidney, with liver and brain showing lower levels of message. These results indicate that IGF-II/M6PR mRNA is developmentally regulated, and suggest a role for the IGF-II/M6PR in fetal and neonatal growth.     

A study of methods for in situ X-ray energy dispersive analysis of polyphosphate bodies in Plectonema boryanum

A variety of preparative methods for in situ X-ray energy dispersive analysis were tested to determine their effects on the elemental composition of polyphosphate bodies in P. boryanum. The bodies were found to contain large amounts of P and K and small amounts of Ca and Mg. Air drying, freeze-drying and freeze-drying from a liquid nitrogen slush all gave similar results. Fixation of the cells in glutaraldehyde and/or OsO₄ resulted in loss of the K and enhancement of the Ca peak. Magnesium was lost during embedding in epoxy.     

Parallel activities of fatty acid methyl esters and analogous phorbol diesters toward mouse lymphocytes

Linear saturated fatty acid methyl esters were comitogenic with lectins for mouse lymphocytes, the degree of comitogenicity being strongly dependent on the length of the acyl group, and maximal for methyl tetradecanoate. Lesser effects were found for analogs with 10, 12 or 16 acyl carbon atoms, whereas those with fewer than 10 or more than 16 were inactive. Analogous structure-function relationships have been described for various membrane-active and tumor-promoting phorbol diesters, where there is a similar dependence on ester acyl group length for many activities. The fatty acid esters may therefore represent simple model compounds for studying mechanistic aspects of phorbol diester activity.     

Binding of glucocorticoid receptors to DNA.

DNA has been implicated as the nuclear acceptor for receptor-glucocorticoid complexes. The present study concerns the interaction of these complexes, isolated from cultured rat hepatoma cells, with purified DNA. This association is rapid, reaching a maximum within a few minutes at 0 degrees, whereas dissociation requires several hours. DNA binds neither free glucocorticoids nor those complexed with transcortin or cytosol proteins different from the receptor. Receptors which are not complexed by steroid have little or no affinity for DNA. "Activation," necessary for the binding of receptor-steroid complexes to isolated nuclei, also enhances DNA binding. The capacity of DNA for binding receptor-steroid complexes is large; saturation was not observed at the complex concentrations studied, using either crude or partially purified receptor preparations. The association of complexes with DNA is inhibited by divalent cations, at increasing ionic strengths, and by mercurial reagents. Complexes bind equally well to bacterial, bacteriophage, or rat DNA; however, there was either no or substantially reduced binding by bacterial 23 S rRNA. The binding of complexes to native DNA is roughly 3-fold greater than to denatured DNA. These characteristics are consistent with the possibility that DNA is the nuclear acceptor for receptor-glucocorticoid complexes; however, the actual composition of the acceptor sites remains unknown.     

Involvement of cytosol proteins in oleate activation of rabbit liver fructose-1,6-diphosphatase.

Dialyzed rabbit liver cytosol was specifically freed of endogenous fructose-1,6-diphosphatase by immunoadsorption on a column of Sepharose-immobilized anti-fructose-1,6-diphosphatase. This material increased the specific activity of homogeneous enzyme to the maximal rate observed with EDTA and shifted the pH optimum from 8.4 to 7.4. With oleate or other fatty acids as activators, the hydrolysis of fructose-1,6-diphosphatase by enzyme, at neutral pH, showed nonlinear initial rates dropping to lower linear rates. Cytosol activator acted synergistically with oleate both to increase neutral enzyme activity and to maintain the high initial catalytic rates. After sucrose density centrifugation or gel filtration, the cytosol had no effect by itself, but still potentiated oleate activation. The factor was destroyed by treatment with subtilisin or trypsin, but all attempts to identify a unique protein component in cytosol were unsuccessful. The presence of Na dodecyl-SOJ, deoxycholate, or urea did not improve the resolution of the factor, but these compounds did lower the K50 for activation by cytosol. Since fatty acids are the only unique compounds which have been isolated from cytosol which activated fructose-1,6-diphosphatase, it appears that soluble proteins can act as natural carriers for the fatty acids. This was supported by the fact that both dialyzed rabbit alpha-globulins and muscle phosphofructokinase also acted synergistically with oleate in a manner similar to cytosol. Phosphatidic acid and phosphatidylserine activated fructose-1,6-diphosphatase, and their action was synergistic with oleate. Glutathione (1 mM) activated the enzyme 5-fold at pH 7.3 and its effects were additive with oleate and cytosol or alpha-globulins.     

Profiling of apoptotic changes in human breast cancer cells using SELDI-TOF mass spectrometry.

Apoptosis is a key process in the response of tumours to chemotherapeutic agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many tumor cells, while sparing most normal cells. Several chemotherapeutic drugs synergize with TRAIL in reducing tumor growth and inducing apoptosis. Because some tumour cells respond poorly to these treatments, biomarkers that predict clinical responsiveness are needed. This study used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify novel apoptotic markers in TRAIL and etoposide (T+E)-treated MDA-MB-231 and ZR-75-1 breast cancer cells and MCF-10A non-transformed breast cells. T+E induced apoptosis, increasing caspase-3 activity at 4-8h, in all cell lines. Protein profiles revealed two prominent peaks, m/z 10090 and 8560, which decreased significantly during apoptosis. Mass spectrometry sequencing of tryptic peptides identified these proteins as S100A6 (confirmed immunologically) and ubiquitin (confirmed against a purified standard), respectively. Caspase inhibition prevented the decrease in both proteins during T+E-induced apoptosis whereas proteasome inhibition combined with T+E further decreased ubiquitin, possibly by preventing its recycling. Using SELDI-TOF MS we have identified S100A6 and ubiquitin as potential protein markers of apoptosis. Further validation using patient samples is required to confirm their potential utility in monitoring the effectiveness of anti-cancer drugs in inducing tumour cell apoptosis.