Biomedical subjects

P Sherline

Publications and source records attributed to P Sherline.

At least 19 recordsLinked to original sources

PubMed1991

Nerve growth factor stimulates protein tyrosine phosphorylation in PC-12 pheochromocytoma cells.

The cellular actions of nerve growth factor (NGF) and epidermal growth factor (EGF) may be mediated by changes in protein phosphorylation. The tyrosine phosphorylation of two predominant proteins of molecular mass 40 and 42 kDa is seen in PC-12 cells treated with NGF or EGF, correlating with activation of a previously identified serine/threonine protein kinase that phosphorylates microtubule-associated protein (MAP). Stimulation of phosphoprotein (pp) 40 and 42 phosphorylation and MAP kinase activity by NGF but not EGF is selectively attenuated by staurosporine and K-252A. Moreover, the time courses of pp40/42 phosphorylation and MAP kinase activation produced by NGF or EGF are identical. Chromatography of lysates from growth factor-treated cells on ion-exchange or hydrophobic-interaction HPLC resolves MAP kinase into two peaks, neither of which precisely coelutes with pp40 or pp42. One of these peaks (II) exhibits no detectable phosphotyrosine. The other peak (I) has some overlap with pp40. However, the activity residing in both peaks is almost completely inhibited after treatment with alkaline phosphatase, suggesting that, at least, serine/threonine phosphorylation is required for the activity of these enzymes. These data indicate that while tyrosine phosphorylation appears to be a critical early event in NGF action, the role of this modification in activation of MAP kinases remains unclear.

Adrenal Gland Neoplasms↗

PubMed1990

Nerve growth factor stimulates a protein kinase in PC-12 cells that phosphorylates microtubule-associated protein-2.

Some of the effects of nerve growth factor (NGF) may be mediated by changes in protein phosphorylation. We have identified a protein kinase from PC-12 cells that catalyzes the phosphorylation of pig brain microtubule-associated protein (MAP)-2 in vitro. This activity is stimulated 2-4-fold in extracts from cells treated with NGF or epidermal growth factor (EGF). The partial purification and characterization of this MAP kinase indicate that it is distinct from previously described NGF-stimulated protein kinases. The NGF-stimulated kinase activity is unaffected by direct addition to the assay of the heat-stable cAMP-dependent kinase peptide inhibitor, staurosporine, or K-252A, is slightly stimulated by heparin and is inhibited by sodium fluoride and calcium ions. Treatment of cells with NGF increases the activity of the kinase within 2 min. The activity declines after 10 min, and a second phase of activation is observed at 20-30 min. Comparison of its behavior on gel permeation and sucrose density gradients indicates a molecular mass in the range of 40,000 daltons. The kinase activity is specific for ATP as substrate with a Km of 12 microM. Although the pathway of activation of MAP kinase by NGF is unknown, the stimulation can be reversed by treatment of the enzyme with alkaline phosphatase, suggesting that activation involves phosphorylation of the kinase itself. The properties and hormone sensitivity of the PC-12 MAP kinase suggest that it is similar to the previously identified, growth factor-sensitive MAP kinase from 3T3-L1 adipocytes.

Alkaline Phosphatase↗

PubMed1987

Purification of phosphatidylinositol kinase from bovine brain myelin.

A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.

1-Phosphatidylinositol 4-Kinase↗

PubMed1987

Insulin-stimulated diacylglycerol production results from the hydrolysis of a novel phosphatidylinositol glycan.

We recently described the insulin-dependent release of a carbohydrate substance from plasma membranes which regulated certain intracellular enzymes (Saltiel, A. R., and Cuatrecasas, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5793-5797). This enzyme-modulating substance appeared to arise from the phosphodiesterase hydrolysis of a novel inositol-containing glycolipid. This is supported by observations that insulin stimulated the rapid generation of [3H]myristate-labeled diacylglycerol in cultured BC3Hl myocytes. Myristoyl diacylglycerol production in these cells was unaffected by epinephrine, although arachidonate-labeled diacylglycerol was rapidly produced in response to stimulation by this alpha-1 adrenergic agent. The production of distinct species of diacylglycerol was apparently due to hormonally specific hydrolysis of different precursors. A novel glycolipid was identified on silica TLC or high pressure liquid chromatography which served as a substrate for the insulin-stimulated phosphodiesterase reaction. This glycolipid was metabolically labeled with radioactive inositol, glucosamine, and myristic acid, suggesting a phosphatidylinositol (PI)-glycan structure. Treatment of this glycolipid with a PI-specific phospholipase C resulted in the generation of two products: an inositol phosphate-glycan which modulated the activity of the low Km cAMP phosphodiesterase and myristoyl diacylglycerol. Insulin caused the rapid hydrolysis of the PI-glycan, which was then apparently resynthesized. These data further suggest that insulin stimulates the activity of a phospholipase C which selectively hydrolyzes a novel PI-glycan, releasing a carbohydrate enzyme modulator as well as a unique species of diacylglycerol.

Cell Line↗

PubMed1987

Glyburide modulates insulin-mediated locomotor response of confluent cell cultures to wounding.

We determined whether the sulfonylurea glyburide could modify the insulin-induced locomotor response to wounding of bovine aortic, pulmonary artery endothelial, and gerbil fibroma cells. Serum-deprived cells were preincubated in medium containing glyburide (10(-4) M) or diluent, then exposed to insulin (10(-6) to 10(-12) M) at the time of wounding, after which the centrosomal-orientation and migratory responses of the cells bordering the experimental wound were monitored by immunofluorescence microscopy and time-lapse cinemicrophotography, respectively. Insulin (10(-6) M) stimulated the centrosomal-orientation and locomotor responses, resulting in the narrowing of the linear wound. Glyburide enhanced the effect of insulin on both parameters of the locomotor response and also sensitized the cells to a previously ineffective concentration of insulin (10(-12) M). The action of glyburide depended on a preincubation period of greater than or equal to 60 min and was blocked by cycloheximide and actinomycin D. The relevance of this novel action of glyburide to the impaired healing process observed in diabetic patients requires further study.

Animals↗

PubMed1986

Insulin-stimulated hydrolysis of a novel glycolipid generates modulators of cAMP phosphodiesterase.

Insulin action may involve the intracellular generation of low molecular weight substances that modulate certain key enzymes. The production of two substances that regulate the activity of adenosine 3',5'-monophosphate phosphodiesterase was evaluated in cultured myocytes by incorporation of radiolabeled precursors. Insulin caused the rapid hydrolysis of a chemically undefined membrane glycolipid, resulting in the production of two related complex carbohydrates as well as diacylglycerol. Both the glycolipid precursor and the aqueous products were monitored by labeling with radioactive inositol and glucosamine. Depletion of the labeled precursor and the appearance of labeled water-soluble products and diacylglycerol occurred within 30 seconds after hormone treatment and was followed by rapid resynthesis of the precursor. The aqueous products that were radioactively labeled appeared chromatographically and electrophoretically identical to phosphodiesterase modulating activities produced by insulin from the same cells. The purified radiolabeled and bioactive substances had similar chemical properties. Hydrolysis of the glycolipid precursor and subsequent generation of products could be reproduced by incubation of extracted lipids with a phosphatidylinositol-specific phospholipase C. These studies suggest that insulin stimulates an endogenous, selective phospholipase C activity that hydrolyzes a novel glycolipid, resulting in the generation of a complex carbohydrate-phosphate substance containing inositol and glucosamine that may mediate some of the actions of the hormone.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1986

Brain-specific expression of MAP2 detected using a cloned cDNA probe.

We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low stringency, the mouse MAP2 cDNA probe cross-hybridizes with genomic sequences from rat, human, and (weakly) chicken, but not with sequences in frog, Drosophila, or sea urchin DNA. Thus, there is significant interspecies divergence of MAP2 sequences. The implications of the above observations are discussed in relationship to the potential biological function of MAP2.

Animals↗

PubMed1986

A cloned cDNA encoding MAP1 detects a single copy gene in mouse and a brain-abundant RNA whose level decreases during development.

Screening of a bacteriophage lambda gt11 cDNA expression library with a polyclonal anti-microtubule associated protein (MAP) antiserum resulted in the isolation of two non-cross-hybridizing sets of cDNA clones. One set was shown to encode MAP2 (Lewis, S. A., A. Villasante, P. Sherline, and N. J. Cowan, 1986, J. Cell Biol., 102:2098-2105). To determine the specificity of the second set, three non-overlapping fragments cloned from the same mRNA molecule via a series of "walking" experiments were separately subcloned into inducible plasmid expression vectors in the appropriate orientation and reading frame. Upon induction and analysis by immunoblotting, two of the fusion proteins synthesized were shown to be immunoreactive with an anti-MAP1-specific antibody, but not with an anti-MAP2-specific antibody. Since these MAP1-specific epitopes are encoded in non-overlapping cDNAs cloned from a single contiguous mRNA, these clones cannot encode polypeptides that contain adventitiously cross-reactive epitopes. Furthermore, these cDNA clones detected an abundant mRNA species of greater than 10 kb in mouse brain, consistent with the coding requirement of a 350,000-D polypeptide and the known abundance of MAP1 in that tissue. The MAP1-specific cDNA probes were used in blot transfer experiments with RNA prepared from brain, liver, kidney, stomach, spleen, and thymus. While detectable quantities of MAP1-specific mRNA were observed in these tissues, the level of MAP1 expression was approximately 500-fold lower than in brain. The levels of both MAP1-specific and MAP2-specific mRNAs decline in the postnatal developing brain; the level of MAP1-specific mRNA also increases slightly in rat PC12 cells upon exposure to nerve growth factor. These surprising results contrast sharply with reported dramatic developmental increases in the amount of MAP1 in brain and in nerve growth factor-induced PC12 cells. The cDNA clones encoding MAP1 detect a single copy sequence in mouse DNA, even under conditions of low stringency that would allow the detection of related but mismatched sequences. The cDNAs cross-hybridize with genomic sequences in rat, human, and chicken DNA, but not with DNA from frog, Drosophila, or sea urchin. These data are discussed in terms of the evolution and possible biological role of MAP1.

Animals↗

PubMed1984

Co-ordinate control of centrosomal separation and DNA synthesis by growth regulators.

We have tested the effects of various mitogens and growth inhibitors on centrosomal separation (CS) in serum-deprived HeLa, gerbil fibroma (GF) and A431 cells. All of the agents which were mitogenic in a given cell type also stimulated CS. No agent was found which stimulated CS but failed to stimulate DNA synthesis. Inhibitors of DNA synthesis, including somatostatin, hydrocortisone, 8-bromo-cAMP, and epidermal growth factor (EGF) in A431 cells, also inhibited CS in response to mitogens. In GF cells (blocked at the G1/S interface with hydroxyurea) centrosomal re-association and the decay in commitment to DNA synthesis upon serum withdrawal occurred with a similar t1/2 (8.8 h). These results demonstrate that CS and DNA synthesis are co-ordinately regulated by a variety of stimulators and inhibitors of cell proliferation. Separation of the centrosomes, or an underlying event with which it is tightly coupled, may represent the point of cellular commitment to enter S phase.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1984

Somatostatin has an antiproliferative effect on concanavalin A-activated rat thymocytes.

Somatostatin, a cyclic tetradecapeptide which is widely distributed in different tissues of the body, exerts an inhibitory effect on numerous cellular processes. It has been observed recently that somatostatin and its analogs were antimitogenic in several established cultured cell lines and in rat tumors. To determine whether this ubiquitous peptide had an antiproliferative effect on a primary culture of rat thymocytes, we observed its effect on the separation of the centrosome (a cell maker of the G1 to S traverse) and DNA synthesis of rat thymocytes activated by concanavalin A. Somatostatin inhibited both indices of proliferation in rat thymocytes at a concentration of 10(-8)M. This observation suggests that somatostatin, which has been localized in the thymus, may play a regulatory role in the growth and development of the cells found in the thymus gland, and may affect thymocyte function in disease states characterized by elevated circulating concentrations of somatostatin.

Animals↗

PubMed1984

Catecholamines are mitogenic in 3T3 and bovine aortic endothelial cells.

We have found that catecholamines stimulate DNA synthesis and centrosomal separation in 3T3 and bovine aortic endothelial cells cultured in the absence of serum or added growth factors. The mitogenic effect is mediated by an alpha 1-adrenergic receptor, as it is inhibited by phentolamine and prazosin but not by propranolol or yohimbine. The physiological and pathological consequences of this effect remain to be determined.

Animals↗

PubMed1984

Tumor promoters stimulate hyperplasia of microtubule organizing center and inhibit DNA synthesis in cultured cells.

Chemical tumor promoters induce significant morphologic changes in several cultured cell models. In this article we describe a new effect of two potent, chemically different tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and dihydroteleocidin B (DHTB) on cultured human HeLa and melanoma cells. Using immunofluorescence microscopy, we observed that TPA and DHTB induced a dramatic increase in the size (greater than or equal to 3X normal diameter) of the centrosome, a microtubule-organizing center, within 24 h of incubation. In HeLa cells the effect was serum- and dose-dependent, was observed in 76-92% of cells within 72 h of incubation, and was associated with an increase in cytoplasm-nucleus ratio and proliferation of microtubules from the centrosome. The tumor promoters inhibited serum-induced DNA synthesis in both cell lines. Electron microscopy revealed the presence of clumps of microcentriole bodies or fragments adjacent to the intact centriole.

DNA↗

PubMed1984

Insulin and multiplication-stimulating activity induce a very rapid centrosomal orientation response to wounding in endothelial cell monolayers.

Using serum-deprived monolayers of bovine aortic endothelial cells that were disrupted experimentally with a linear wound, we observed, by immunofluorescence microscopy, centrosomal perinuclear movement that is associated closely with cell orientation and locomotion. In the presence of the growth factors fetal bovine serum, insulin, or multiplication-stimulating activity (MSA), the centrosome rapidly (within 10 s) translocated and positioned itself between the nucleus and wound track in greater than or equal to 70% of cells bordering the wound. Random centrosomal orientation was observed in control border cells (50%). This growth factor-stimulated centrosomal orientation response to wounding depended on growth factor concentration, high-energy phosphates, functional microtubules and microfilaments, and calcium-calmodulin interaction. In the absence of fetal bovine serum, insulin, or MSA, the border cells of the wounded endothelial cell monolayer exhibited a significant centrosomal orientation response 5-6 h after wounding.

Animals↗

PubMed1984

In vivo and in vitro studies on the role of HMW-MAPs in taxol-induced microtubule bundling.

The involvement of high molecular weight microtubule-associated proteins (HMW-MAPs) in the process of taxol-induced microtubule bundling has been studied using immunofluorescence and electron microscopy. Immunofluorescence microscopy shows that HMW-MAPs are released from microtubules in granulosa cells which have been extracted in a Triton X-100 microtubule-stabilizing buffer (T-MTSB), unless the cells are pretreated with taxol. 1.0 microM taxol treatment for 48 h results in microtubule bundle formation and the retention of HMW-MAPs in these cells upon extraction with T-MTSB. Electron microscopy demonstrates that microtubules in control cytoskeletons are devoid of surface structures whereas the microtubules in taxol-treated cytoskeletons are decorated by globular particles of a mean diameter of 19.5 nm. The assembly of 3 X cycled whole microtubule protein (tubulin plus associated proteins) in vitro in the presence of 1.0 microM taxol, results in the formation of closely packed microtubules decorated with irregularly spaced globular particles, similar in size to those observed in cytoskeletons of taxol-treated granulosa cells. Microtubules assembled in vitro in the absence of taxol display prominent filamentous extensions from the microtubule surface and center-to-center spacings greater than that observed for microtubules assembled in the presence of taxol. Brain microtubule protein was purified into 6 s and HMW-MAP-enriched fractions, and the effects of taxol on the assembly and morphology of these fractions, separately or in combination, were examined. Microtubules assembled from 6 s tubulin alone or 6 s tubulin plus taxol (without HMW-MAPs) were short, free structures whereas those formed in the presence of taxol from 6 s tubulin and a HMW-MAP-enriched fraction were extensively crosslinked into aggregates. These data suggest that taxol induces microtubule bundling by stabilizing the association of HMW-MAPs with the microtubule surface which promotes lateral aggregation.

Alkaloids↗

PubMed1982

Microtubule-associated proteins of HeLa cells: heat stability of the 200,000 mol wt HeLa MAPs and detection of the presence of MAP-2 in HeLa cell extracts and cycled microtubules.

One of the major groups of microtubule-associated proteins (MAPs) found associated with the microtubules isolated from HeLa cells has a molecular weight of just over 200,000. Previous work has demonstrated that these heLa MAPs are similar in several properties to MAP-2, one of the major MAPs of mammalian neural microtubules, although the two types of proteins are immunologically distinct. The 200,000 mol wt HeLa MAPs have now been found to remain soluble after incubation in a boiling water bath and to retain the ability to promote tubulin polymerization after this treatment, two unusual properties also shown by neural MAP-2. This property of heat stability has allowed the development of a simplified procedure for purification of the 200,000 HeLa MAPs and has provided a means for detection of these proteins, even in crude cell extracts. These studies have also led to the detection of a protein in crude extracts of HeLa cells and in cycled HeLa microtubules which has been identified as MAP-2 on the basis of (a) comigration with calf brain MAP-2 on SDS PAGE, (b) presence in purified microtubules, (c) heat stability, and (d) reaction with two types of antibodies prepared against neural high molecular weight-MAPs, one of these a monoclonal antibody against hog brain MAP-2, although present in HeLa cells, is at all stages of microtubule purification a relatively minor component in comparison to the 200,000 HeLa MAP's.

Cross Reactions↗

PubMed1982

Epidermal growth factor induces rapid centrosomal separation in HeLa and 3T3 cells.

Using indirect immunofluorescence, we have found that epidermal growth factor (EGF), at 100 ng/ml, induces centrosomal separation within 20 min in HeLa and 3T3 cells. The effect was evident both in unsynchronized cultures and in HeLa cells blocked in early S phase by hydroxyurea. EGF also induced centrosomal separation in quiescent 3T3 cells blocked in G0/G1 by serum deprivation, indicating that DNA replication is not necessary for this effect. The mechanism of this rapid centrosomal separation and its role in the mitogenic effects of EGF remains to be determined.

Animals↗

PubMed1982

Epidermal growth factor-induced centrosomal separation: mechanism and relationship to mitogenesis.

Using a rabbit antibody to MAP1 to stain centrosomes we have studied the mechanism by which epidermal growth factor (EGF) induces centrosomal separation in HeLa cells. The response is rapid, being detectable within 20 min after EGF (100 ng/ml) addition and by 4 h 40% of logarithmically growing cells and greater than 70% of cells synchronized at G1/S with 1 mM hydroxyurea show centrosomes separated by more than one diameter. A concentration of 0.05 ng/ml of EGF induces significant separation in synchronized cells (5-9% control vs. 20% with EGF at 0.05 ng/ml) and 0.1 to 0.5 ng/ml induces a half maximal response. Centrosomal separation is blocked by energy inhibitors, trifluoperazine, chlorpromazine, and W-7, cytochalasins B and D, and taxol, and is stimulated or enhanced by A23187, colchicine, and oncodazole. Trifluoperazine, W-7, cytochalasin D, and taxol also block DNA synthesis in response to EGF as measured by autoradiography using [3H]thymidine. Our hypothesis based upon these results is that EGF, by raising the free calcium level, activates calmodulin, which stimulates contraction of microfilaments attached to the centrosome, pulling the daughter centrosome apart. EGF may also induce depolymerization or detachment of microtubules in the vicinity of the centrosome which ordinarily serve to maintain its position and inhibit separation. Centrosomal separation may be a key event in triggering DNA synthesis in response to EGF and colchicine.

Calcium↗

PubMed1982

Somatostatin inhibits rapid centrosomal separation and cell proliferation induced by epidermal growth factor.

The role of endogenous growth inhibitors in the regulation of cell proliferation is unclear. Although there are numerous studies on the stimulatory effect of peptide hormones such as insulin, the somatomedins and epidermal growth factor (EGF) on cell proliferation, little is known about the existence of hormones that might exert an antiproliferative effect on cells. Somatostatin (SS), a cyclic tetradecapeptide hormone that is widely distributed in the body, exerts an inhibitory effect on numerous cellular processes. We observed that SS at concentrations of 10(-8) - 10(13M), inhibited EGF-induced centrosomal separation, which recent evidence suggests is necessary for DNA synthesis in response to EGF. This SS effect was associated with inhibition of DNA synthesis and cell replication induced by EGF in gerbil fibroma and HeLa cells. Inhibition of centrosomal separation and the consequent antiproliferative effect of SS may represent a biologically significant action of this ubiquitous hormone.

Animals↗