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J E Ferrell

Publications and source records attributed to J E Ferrell.

16 recordsLinked to original sources
PubMed1992

Inhibition of c-Jun DNA binding by mitogen-activated protein kinase.

Here we demonstrate that partially purified Xenopus p42 mitogen-activated protein (MAP) kinase phosphorylates bacterially expressed human c-Jun at a single site, serine 243. Several lines of evidence argue that this phosphorylation is due to p42 MAP kinase itself rather than some contaminating species. Phosphorylation of serine 243 markedly decreases the binding of c-Jun to oligonucleotides containing the 12-O-tetradecanoylphorbol-13-acetate response element. These findings suggest that MAP kinase may play a role in the down-regulation of c-Jun or in the cycle of transcriptional initiation and elongation.

Amino Acid Sequence↗
PubMed1991

Cell cycle tyrosine phosphorylation of p34cdc2 and a microtubule-associated protein kinase homolog in Xenopus oocytes and eggs.

We have examined the time course of protein tyrosine phosphorylation in the meiotic cell cycles of Xenopus laevis oocytes and the mitotic cell cycles of Xenopus eggs. We have identified two proteins that undergo marked changes in tyrosine phosphorylation during these processes: a 42-kDa protein related to mitogen-activated protein kinase or microtubule-associated protein-2 kinase (MAP kinase) and a 34-kDa protein identical or related to p34cdc2. p42 undergoes an abrupt increase in its tyrosine phosphorylation at the onset of meiosis 1 and remains tyrosine phosphorylated until 30 min after fertilization, at which point it is dephosphorylated. p42 also becomes tyrosine phosphorylated after microinjection of oocytes with partially purified M-phase-promoting factor, even in the presence of cycloheximide. These findings suggest that MAP kinase, previously implicated in the early responses of somatic cells to mitogens, is also activated at the onset of meiotic M phase and that MAP kinase can become tyrosine phosphorylated downstream from M-phase-promoting factor activation. We have also found that p34 goes through a cycle of tyrosine phosphorylation and dephosphorylation prior to meiosis 1 and mitosis 1 but is not detectable as a phosphotyrosyl protein during the 2nd through 12th mitotic cell cycles. It may be that the delay between assembly and activation of the cyclin-p34cdc2 complex that p34cdc2 tyrosine phosphorylation provides is not needed in cell cycles that lack G2 phases. Finally, an unidentified protein or group of proteins migrating at 100 to 116 kDa increase in tyrosine phosphorylation throughout maturation, are dephosphorylated or degraded within 10 min of fertilization, and appear to cycle between low-molecular-weight forms and high-molecular-weight forms during early embryogenesis.

Animals↗
PubMed1990

Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation.

We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.

Animals↗
PubMed1990

Intracellular localization of pp60c-src in human platelets.

We have studied the intracellular distribution of the proto-oncogene product pp60c-src in human platelets by immunofluorescence microscopy and by immunogold electron microscopy on frozen thin sections. Virtually all of the immunoreactive pp60c-src was found to be associated with the plasma membrane and with membranes of the surface-connected canalicular system. This localization suggests that pp60c-src may relay signals from an as yet unidentified cell surface protein to intracellular targets.

Antibodies, Monoclonal↗
PubMed1989

Thrombin stimulates the activities of multiple previously unidentified protein kinases in platelets.

We have used a renaturation method to search for previously unidentified protein kinases in human platelets. The method involves subjecting lysates to denaturing gel electrophoresis, transferring the proteins to blotting membranes, and treating the blotted proteins with guanidine. The guanidine is then removed to allow the proteins to renature, and the blots are overlaid with [gamma-32P]ATP. We have identified 14 electrophoretically distinct, serine/threonine-specific protein kinases. Eleven of the kinases clearly differ in molecular weight from all previously described platelet serine/threonine kinases. Ten of these novel kinases (PK220, PK200, PK170, PK150, PK64, PK60, PK56, PK52, PK48, and PK40) were found to possess markedly increased in vitro activity when isolated from thrombin-stimulated platelets, presumably as a result of thrombin-stimulated covalent modification. Treatment of intact platelets with the calcium ionophore ionomycin and phorbol 12-myristate 13-acetate also increased the in vitro activity of these kinases. The agonist-stimulated kinases could be divided into three classes: 1) one kinase whose activity was increased by in vivo phorbol ester treatment but not by ionomycin (PK150); 2) two kinases whose activity was increased by ionomycin but not phorbol ester (PK48 and PK40); 3) seven kinases whose activity was markedly increased by combinations of phorbol ester and ionomycin, but not by either agent alone (PK220, PK200, PK170, PK64, PK60, PK56, and PK52). This third mode of regulation is what would be expected of enzymes that mediate the biological effects of inositide-mobilizing stimuli.

Amino Acids↗
PubMed1989

Tyrosine-specific protein phosphorylation is regulated by glycoprotein IIb-IIIa in platelets.

We have previously shown that a number of platelet proteins become phosphorylated at tyrosine residues in response to platelet-activating agents. Here we present two lines of evidence implicating a platelet integrin, glycoprotein IIb-IIIa, in the regulation of a specific subset of these tyrosine phosphorylations. (i) Two peptides that inhibit the binding of fibrinogen and other ligands to gpIIb-IIIa, Arg-Gly-Asp-Ser and His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val, also inhibited the thrombin-induced tyrosine phosphorylation of this subset of proteins. The tetrapeptide Arg-Gly-Glu-Ser, which does not inhibit fibrinogen binding, did not inhibit thrombin-stimulated tyrosine phosphorylation. (ii) Platelets lacking gpIIb-IIIa (from a subject with Glanzmann thrombasthenia) did not undergo this subset of tyrosine phosphorylation in response to thrombin, although other serine, threonine, and tyrosine phosphorylations proceeded normally. These findings suggest a role for tyrosine-specific protein phosphorylation in integrin-mediated cell-matrix recognition.

Adenosine Diphosphate↗
PubMed1988

Membrane bilayer balance and platelet shape: morphological and biochemical responses to amphipathic compounds.

Activated platelets adopt a characteristic spiculate morphology. A wide variety of anionic and zwitterionic amphipathic compounds were found to effect a similar shape change and to cause the open canalicular system to become less prominent. Several cationic amphipaths reversed thrombin-, PAF-, and amphipath-induced spiculation and restored the discoid shape. Higher concentrations of cationic amphipaths caused the cells to assume spheroid and indented forms, and caused the canalicular system to appear more prominent. Three amphipaths were studied further to address possible mechanisms underlying their morphological effects. Dilauroylphosphatidylcholine was found to induce spiculation without causing the changes in protein phosphorylation and inositide metabolism generally associated with platelet activation. Two other amphipaths, chlorpromazine (which induced sphering) and dilauroylphosphatidylserine (which caused spiculation followed by sphering) caused specific changes in protein and/or lipid phosphorylation, which may be responsible for some, but not all, of the morphological effects of these compounds. To account for these findings, we propose that platelet shape can be influenced by changes in the plasma membrane bilayer balance. Agents that bind to the membrane outer monolayer are accommodated by spiculation; those that bind to the inner monolayer are accommodated by sphering.

Adult↗
PubMed1988

Platelet tyrosine-specific protein phosphorylation is regulated by thrombin.

Intact human platelets, terminally differentiated cells with no growth potential, were found to possess unusually high levels of tyrosine-specific protein phosphorylation. The physiological platelet activator thrombin transiently elevated platelet phosphotyrosine content, apparently through stimulation of one or more tyrosine-specific protein kinases. Immunoblotting with antiphosphotyrosine antiserum showed that thrombin caused dramatic changes in the tyrosine phosphorylation of a number of individual protein bands and that these changes occurred in three distinct temporal waves. Most but not all of the protein bands phosphorylated at tyrosine in response to thrombin were also tyrosine phosphorylated in response to chilling or the combination of ionophore A23187 and tetradecanoylphorbol acetate. Thrombin stimulated the phosphorylation of the tyrosine kinase pp60c-src, primarily at Ser-12 and Tyr-527, although the effects of these phosphorylations on platelet pp60c-src function were not apparent. Together, these results suggest that tyrosine-specific protein kinases of uncertain identity are involved in signal transduction in platelets.

Adult↗
PubMed1986

Separation of phosphoinositides and other phospholipids by two-dimensional thin-layer chromatography.

A simple, rapid, two-dimensional TLC system is presented which resolves the four phosphoinositide cycle phospholipids as well as all commonly encountered major and minor phospholipids. Ca2+-free lipid samples are loaded onto silica gel HL plates and developed first in 48:40:7:5 chloroform:methanol:water:concentrated ammonia, and then in 55:25:5 chloroform:methanol:formic acid. The method was applied successfully to human erythrocytes, human platelets, and BL/VL3 murine lymphoma cells.

Blood Platelets↗
PubMed1986

Sulfhydryl reducing agents and shape regulation in human erythrocytes.

Metabolic crenation of red cells is reversible; on addition of nutrients, echinocytes recover the normal discoid shape. When the shape recovery takes place in the presence of reducing agents such as dithiothreitol (DTT), morphological change continues until the cells are stomatocytic. The degree of stomatocytosis varies, depending on the cell morphology when the nutrients and reducing agent are added. DTT has minimal effect on the shape of normal discocytes, but in its presence, mildly echinocytic cells become slightly cupped and advanced-stage echinocytes become severely stomatocytic. DTT must be present continuously for development and retention of stomatocytosis; echinocytes preincubated with or metabolically depleted in DTT do not become stomatocytic when supplemented in the absence of DTT, and DTT-induced stomatocytes revert to discocytes when the reducing agent is removed. DTT has no effect on adenosine triphosphate synthesis or equilibrium cell glutathione levels, and the induced stomatocytosis is not inhibited by excluding oxygen from cells during depletion. Spectrin phosphorylation and phosphate turnover are not affected by DTT. The echinocyte-to-discocyte transformation coincides with phosphorylation of membrane inner monolayer lipids (diacylglycerol to phosphatidic acid and phosphatidylinositol to phosphatidylinositol-4,5-bisphosphate). Overphosphorylation of these phospholipids is not responsible for the exaggerated shape recovery seen with reducing agents; phosphorylation of inner monolayer lipids proceeds identically in the presence and absence of DTT.

Adenosine Triphosphate↗
PubMed1985

Membrane bilayer balance and erythrocyte shape: a quantitative assessment.

When human erythrocytes are incubated with certain phospholipids, the cells become spiculate echinocytes, resembling red cells subjected to metabolic starvation or Ca2+ loading. The present study examines (1) the mode of binding of saturated phosphatidylcholines and egg lysophosphatidylcholine to erythrocytes and (2) the quantitative relationship between phospholipid incorporation and red cell shape. We find that the phospholipids studied become intercalated into erythrocyte membranes, not simply adsorbed to the cell surface. Spin-labeling and radiolabeling data show that the incorporation of (4 +/- 1) X 10(6) molecules of exogenous phosphatidylcholine per cell converts discocytes to stage 3 echinocytes with about 35 conical spicules. This amount of lipid incorporation is estimated to expand the red cell membrane outer monolayer by 1.7% +/- 0.6%. Calculations of the inner and outer monolayer surface areas of model discocytes and stage 3 echinocytes yield an estimated difference of 0.7% +/- 0.2%.

Cholesterol↗
PubMed1985

Lipid transfer between phosphatidylcholine vesicles and human erythrocytes: exponential decrease in rate with increasing acyl chain length.

The rate of phospholipid transfer from sonicated phospholipid vesicles to human erythrocytes has been studied as a function of membrane concentration and lipid acyl chain composition. Phospholipid transfer exhibits saturable first-order kinetics with respect to both cell and vesicle membrane concentrations. This kinetic behavior is consistent either with transfer during transient contact between cell and vesicle surfaces (but only if the fraction of the cell surface susceptible to such interaction is small) or with transfer of monomers through the aqueous phase. The acyl chain composition of the transferred phospholipid affects the transfer kinetics profoundly; for homologous saturated phosphatidylcholines, the rate of transfer decreases exponentially with increasing acyl chain length. This behavior is consistent with passage of phospholipid monomers through a polar phase, which might be the bulk aqueous phase( as in the monomer transfer model) or the hydrated head-group regions of a cell-vesicle complex (transient collision model). Collisional transfer also predicts that intercell transfer of phospholipids should be slow compared to cell-vesicle transfer, as surface charge and steric effects should prevent close apposition of donor and acceptor membranes. This is not found; dilauroylphosphatidylcholine transfers rapidly between red cells. Thus, the observed relationship between acyl chain length and intermembrane phospholipid transfer rates likely reflects the energetics of monomer transfer through the aqueous phase.

Erythrocytes↗
PubMed1979

Adrenergic stimulation of membrane protein phosphorylation in human erythrocytes.

Adrenergic modification of membrane protein phosphorylation was studied in intact human erythrocytes. Micromolar norepinephrine increased 32P incorporation into Band 2 by 70%, and into Band 3 by 40%. Phosphorylation levels observed with a series of specific agonists and antagonists suggest that an alpha-adrenergic receptor is involved in this effect. The mechanism of linkage between this receptor and protein phosphorylation does not appear to involve modulation of intracellular concentrations of ATP, cyclic AMP, or cyclic GMP.

1-Methyl-3-isobutylxanthine↗
PubMed1979

Quantum chemical studies of polycyclic aromatic hydrocarbons and their metabolites: correlations to carcinogenicity.

In the context of the bay region hypothesis for polycyclic aromatic hydrocarbon (PAH) carcinogenesis, molecular properties were calculated for seventeen polycyclic aromatic hydrocarbons related to (1) intrinsic substrate reactivities towards activating and detoxifying metabolism and (2) the stabilities of the putative carbocation ultimate carcinogens. All-valence electron methods were used, avoiding the inherent difficulties found in the pi-electron methods. The calculated substrate reactivities were found to predict major metabolites successfully, supporting the validity of their use in attempted correlations with observed carcinogenic potencies. Positive correlations were found between observed carcinogenic potencies and (1) the reactivities of the parent polycyclic aromatic hydrocarbons towards the initial distal bay region epoxidation and (2) the stabilities of the diol epoxide carbocations. The reactivities of the distal bay region diol epoxides, were high for both carcinogenic and non-carcinogenic compounds, implying that the second epoxidation does not determine relative carcinogenic activity. Support for a possible alternative hypothesis, that polycyclic aromatic hydrocarbons are activated by one electron oxidation, was also found.

Carcinogens↗