Biomedical subjects

F A Gonzalez

Publications and source records attributed to F A Gonzalez.

At least 19 recordsLinked to original sources

PubMed1992

Signal transduction within the nucleus by mitogen-activated protein kinase.

The nucleus is an important target of signal transduction by growth factor receptors that stimulate mitogen-activated protein (MAP) kinases. We tested the hypothesis that MAP kinases have a signaling role within the nucleus by examining the effect of the expression of a human MAP kinase isoform (p41mapk) in tissue culture cells. The expressed p41mapk was found to be localized in both the cytoplasmic and nuclear compartments of the cells. Significantly, the expression of p41mapk caused an increase in the phosphorylation of a nuclear substrate: Ser62 of c-Myc. Phosphorylation at Ser62 stimulated the activity of the NH2-terminal transactivation domain of c-Myc. Thus, p41mapk causes the phosphorylation and regulation of a physiologically significant nuclear target of signal transduction. These data establish that at least one MAP kinase isoform has a nuclear role during signal transduction.

Amino Acid Sequence↗

PubMed1992

Heterogeneous expression of four MAP kinase isoforms in human tissues.

Mitogen-activated protein kinases (MAP kinases) are a group of closely related enzymes implicated in signal transduction pathways. We report the molecular cloning of four human proteins (p40mapk, p41mapk, p44mapk and p63mapk) with high homology to members of the MAP kinase family. Sequence analysis demonstrated that p44mapk and p63mapk were the products of distinct genes. However, the p40mapk and p41mapk were found to be related, and are likely to result from alternative processing of transcripts from a single gene. The heterogeneous expression of these human MAP kinase isoforms in different tissues may reflect the diversity of signal transduction pathways in differentiated cells.

Amino Acid Sequence↗

PubMed1991

Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases.

Two epidermal growth factor-stimulated protein kinases that correspond to ERK1 and ERK2 have been purified from human epidermoid carcinoma cells (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). A consensus primary sequence for substrates of ERK1 has been identified as -Pro-Leu-Ser/Thr-Pro- (Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C., Curran, T., and Davis, R. J. (1991) J. Biol. Chem. 266, 15277-15285). However, the structural determinants for substrate recognition are not understood. We performed a systematic analysis of the effect of point mutations in the primary sequence of peptide substrates on the rate of phosphorylation by ERK1 and ERK2. The results of this investigation demonstrate that the substrate specificities of the ERK1 and ERK2 protein kinases are very similar. We propose that the primary sequence of substrates for ERK1 and ERK2 protein kinases can be generalized as -Pro-Xaan-Ser/Thr-Pro- (where Xaa is a neutral or basic amino acid and n = 1 or 2).

Amino Acid Sequence↗

PubMed1991

Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669.

A growth factor-stimulated protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669 has been described (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Anion-exchange chromatography demonstrated that this protein kinase activity was accounted for by two enzymes. The first peak of activity eluted from the column corresponded to the microtubule-associated protein 2 (MAP2) kinase. However, the second peak of activity was found to be a distinct enzyme. We present here the purification of this enzyme from human tumor KB cells by sequential ion-exchange chromatography. The isolated protein kinase was identified as a 46-kDa protein by polyacrylamide gel electrophoresis and silver staining. Gel filtration chromatography demonstrated that the enzyme was functional in a monomeric state. A kinetic analysis of the purified enzyme was performed at 22 degrees C using a synthetic peptide substrate based on the primary sequence of the EGF receptor (KREL VEPLT669PSGEAPNQALLR). The Km(app) for ATP was 40 +/- 5 microM (mean +/- S.D., n = 3). GTP was not found to be a substrate for the purified enzyme. The Km(app) for the synthetic peptide substrate was 260 +/- 40 microM (mean +/- S.D., n = 3). The Vmax(app) for the isolated protein kinase was determined to be 400-900 nmol/mg/min. The purified enzyme was designated EGF receptor Thr669 (ERT) kinase. It is likely that the MAP2 and ERT kinases account for the phosphorylation of the EGF receptor at Thr669 observed in cultured cells. The marked stimulation of protein kinase activity caused by growth factors indicates that these enzymes may have an important function during signal transduction.

Amino Acid Sequence↗

PubMed1991

Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase.

A growth factor-stimulated (MAP2-related) protein kinase, ERT, that phosphorylates the epidermal growth factor receptor at Thr669 has been purified from KB human tumor cells by Northwood and co-workers (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). The ERT protein kinase has a restricted substrate specificity, and the structural determinants employed for substrate recognition by this enzyme have not been defined. As an approach toward understanding the specificity of substrate phosphorylation, we have used an in vitro assay to identify additional substrates for the ERT protein kinase. In this report we describe two novel substrates: (a) the human c-myc protein at Ser62 and (b) the rat c-jun protein at Ser246. Alignment of the primary sequences surrounding the phosphorylation sites located within the epidermal growth factor receptor (Thr669), Myc (Ser62), and Jun (Ser246) demonstrated a marked similarity. The observed consensus sequence was Pro-Leu-Ser/Thr-Pro. We propose that this sequence forms part of a substrate structure that is recognized by the ERT protein kinase.

Amino Acid Sequence↗

PubMed1991

Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells: I. Involvement of protein kinase C-dependent and -independent pathways.

We recently reported that extracellular ATP was mitogenic for Swiss 3T3, 3T6, and A431 cells (Huang et al.: Proc. Natl. Acad. Sci. USA, 86:7904-7908, 1989). Here we examined the possible involvement of activation of the protein kinase C (PKC) signal transduction pathway in the mechanism of action of extracellular ATP. A potent synergistic stimulation of DNA synthesis in quiescent cultures of 3T3 and 3T6 cells was observed when ATP was presented in combination with growth factors that activate PKC, such as bombesin, vasopressin, or tumor-promoting phorbol esters. This finding suggests that ATP and these mitogens do not act through a common mechanism. In contrast, ATP was unable to show synergism with phorbol esters in A431 cells. We discovered striking differences when we examined the kinetics of formation of diacylglycerol (DAG) stimulated by ATP among these cell lines. Thus, ATP stimulated a sustained biphasic increase of DAG in A431 cells, but only a rapid transient increase of DAG formation was observed in 3T3 and 3T6 cells. The breakdown of phosphatidylcholine was stimulated by ATP in A431 cells; however, a significantly reduced effect was displayed in 3T6 cells. Furthermore, we found that the diacylglycerol-kinase inhibitor, 1-monooleoylglycerol, greatly potentiated ATP-stimulated DNA synthesis in A431 cells. Finally, down-regulation of PKC by long-term exposure to phorbol dibutyrate (PDBu) prevented stimulation of DNA synthesis induced by bombesin, vasopressin, or phorbol esters in 3T3 or 3T6 cells, while it had no such effect on ATP-stimulated mitogenesis in the presence of insulin or epidermal growth factor. On the other hand, PDBu-mediated down-regulation of PKC partially inhibited [3H [thymidine incorporation stimulated by ATP in A431 cells. Taken together, we conclude that a protein kinase C-dependent pathway is partially involved in ATP-stimulated DNA synthesis in A431 cells, but a protein kinase C-independent pathway exists in 3T3 and 3T6 cells. Pertussis toxin (PTX) inhibited the sustained phase of DAG formation and the breakdown of phosphatidylcholine stimulated by ATP in A431 cells. This suggests involvement of a PTX-sensitive G protein.

Adenosine Triphosphate↗

PubMed1991

Extracellular ATP and some of its analogs induce transient rises in cytosolic free calcium in individual canine keratinocytes.

Changes in intracellular free calcium ([Ca++]i) play an important role in a variety of biochemical reactions that lead to cellular responses such as proliferation and differentiation. The response of [Ca++]i to extracellular nucleotides (ATP, UTP, ITP, and AMP-PNP) was determined in individual canine keratinocytes using the fluorescent probe fura-2 and digital video fluorescence imaging microscopy. In the presence of 1.8 mM extracellular Ca++, 100 and 500 microM ATP caused a rapid (less than 9 sec) three- to twelvefold rise in [Ca++]i above resting levels of 50-150 nM followed by occasional fluctuations. Small responses were elicited with doses as low as 0.1 microM ATP. The response of cells stimulated with 500 microM ATP in Ca(++)-free medium was characterized by 1.5 to 3 times rapid initial peak followed by a decrease of [Ca++]i below resting levels. Loss of response occurred in the majority of keratinocytes preincubated for 30 min in Ca(++)-free medium. UTP was as effective as ATP in stimulating rises in [Ca++]i in keratinocytes. Smaller elevations in [Ca++]i up to four- to fivefold resting levels were noted with 100 microM AMP-PNP or 500 microM ITP. Desensitization of cells was demonstrated when a second stimulation followed the primary ATP or UTP treatment. These results are suggestive of the presence of purinergic receptors in the cytoplasmic membrane of canine keratinocytes. Experiments using the calcium channel blocker lanthanum suggest that ATP-induced initial rises and sustained levels of [Ca++]i are dependent on the release of Ca++ from intracellular stores. These intracellular Ca++ stores appear to be rapidly depleted after removal of extracellular calcium ([Ca++]e), thereby abolishing ATP-induced [Ca++]i increases.

Adenosine Triphosphate↗

PubMed1990

Covalent incorporation of 3'-O-(4-benzoyl)benzoyl-ATP into a P2 purinoceptor in transformed mouse fibroblasts.

ATP, 3'-O-(4-benzoyl)benzoyl-ATP (BzATP), a photoaffinity analog of ATP, and several other ATP analogs induced an increase in plasma membrane permeability to monovalent ions and normally impermeant metabolites, including nucleotides, in transformed 3T6 mouse fibroblasts. The rank order of agonist potency for induction of nucleotide channels was BzATP (EC50 = 15 microM) greater than ATP (EC50 = 50 microM) approximately adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) greater than 2-methylthio-ATP (EC50 = 75 microM) approximately 3'-amino-3'-deoxy-ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) (EC50 = 175 microM). Long wavelength UV illumination of 3T6 cells in the presence of greater than or equal to 20 microM BzATP at 4 degrees C, a nonpermeabilizing temperature, followed by removal of unbound BzATP, resulted in the efflux of 86Rb+ and the release of a prelabeled pool of cytoplasmic nucleotides when the temperature was shifted to 37 degrees C. Photoincorporation of BzATP was inhibited by ATP, ATP alpha S, ATP gamma S, and other ATP analogs that induced an increase in plasma membrane permeability to nucleotides in 3T6 cells under nonphotoactivating conditions. GTP, ITP, UTP, adenosine, and ATP analogs that did not alter plasma membrane permeability to nucleotides under nonphotoactivating conditions also had no effect on BzATP photoincorporation. Photoincorporation of BzATP occurred optimally between pH 6.6 and pH 8.2 but was inhibited at pH 6.0. Photoincorporation of BzATP was also modulated by the osmolarity and the divalent cation concentration of the assay medium. The increase in plasma membrane permeability to nucleotides induced by photoincorporated BzATP occurred at the same rate and had the same temperature, pH, ionic strength, and divalent cation requirements as the increase in plasma membrane permeability to nucleotides induced by ATP and BzATP under nonphotoactivating conditions. These findings support the hypothesis that BzATP can be covalently incorporated into a P2 purinoceptor in 3T6 cells that is coupled to plasma membrane channels for ions and other metabolites.

Adenine Nucleotides↗

PubMed1990

Activation of early events of the mitogenic response by a P2Y purinoceptor with covalently bound 3'-O-(4-benzoyl)-benzoyladenosine 5'-triphosphate.

3'-O-(4-Benzoyl)benzoyl-ATP (BzATP), a photoaffinity analog of ATP, was used as a ligand for a P2Y purinoceptor (adenine nucleotide receptor) present in intact Swiss 3T3 and 3T6 cells and A-431 epidermoid carcinoma cells. Photolysis of serum-starved cells in the presence of 10-50 microM BzATP, followed by extensive washing to remove unincorporated BzATP, induced the release of arachidonic acid. A trace (less than 0.01%) of photoincorporated BzATP was as effective as when 50 microM BzATP or ATP was contained in the incubation medium during the assay. Photoincorporated BzATP also stimulated the production of prostaglandin E2 and the accumulation of cyclic AMP. In previous studies, we demonstrated that these three events are obligatory early steps in a pathway leading to DNA synthesis in the above cell lines. The evidence indicated that the purinoceptor activated by extracellular ATP or BzATP was linked to a pertussis toxin-sensitive GTP-binding protein. Consistent with these observations, we now find that pertussis toxin inhibits the effect of photoincorporated BzATP on arachidonic acid release. These results indicate that BzATP is an effective agonist for the P2Y purinoceptor concerned with stimulation of DNA synthesis in 3T3, 3T6, and A-431 cells. Furthermore, after photolysis it becomes irreversibly associated with intact cells and promotes the activation of early events required for DNA synthesis.

Adenosine Triphosphate↗

PubMed1989

Two distinct receptors for ATP can be distinguished in Swiss 3T6 mouse fibroblasts by their desensitization.

The stimulation of calcium efflux from Swiss 3T6 mouse fibroblasts by extracellular ATP was studied. It was found that the cells could be desensitized to ATP by a previous exposure to the nucleotide, lending support to the theory that this is a receptor mediated process. Another ATP-receptor mediated process in Swiss 3T6 cells, that is also subject to desensitization, causes the permeabilization of the plasma membrane to nucleotides and other normally impermeant compounds [Gonzalez et al., J. Cell. Physiol. 139:109 (1989)]. Here we demonstrate that selective desensitization of the ATP-dependent calcium mobilization pathway can be achieved without affecting ATP-induced permeabilization. Data are presented in support of the existence of multiple ATP-receptors (purinoceptors) in Swiss 3T6 cells.

Adenosine Triphosphate↗

PubMed1989

Permeabilization of transformed mouse fibroblasts by 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate and the desensitization of the process.

A photoreactive analogue of ATP, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP) altered the plasma membrane permeability of transformed 3T6 mouse fibroblasts to normally impermeant molecules as previously reported for ATP, but at lower concentrations. BzATP-induced permeabilization was modulated by pH, temperature, and the ionic composition of the medium similar to the permeabilizing effects of ATP. Conditions known to enhance ATP-induced permeabilization, such as treatment with the mitochondrial uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP) or the Ca2+-calmodulin antagonist trifluoperazine also enhanced BzATP-induced permeabilization. Conditions inhibitory to ATP-induced permeabilization, including chloride replacement or treatment with furosemide or dithiothreitol (DTT), inhibited permeabilization induced by BzATP. The ionic strength of the medium modulated the responsiveness of the cells to ATP and BzATP; a decrease in the ionic strength below isotonicity increased the sensitivity of the cells to the nucleotides, whereas an increase in ionic strength above isotonicity inhibited permeabilization. Prolonged exposure to ATP under non-permeabilizing conditions caused the cells to become insensitive to ATP and BzATP. The densensitization phenomenon provides support for the theory that the permeabilization process is mediated by a receptor for ATP.

Adenosine Triphosphate↗

PubMed1989

Receptor specific for certain nucleotides stimulates inositol phosphate metabolism and Ca2+ fluxes in A431 cells.

We have recently reported that extracellular ATP induces a transient rise in cytosolic free Ca2+ [( Ca2+]i) in individual human epidermoid carcinoma A431 cells (Gonzalez et al: Journal of Cellular Physiology 135:269-276, 1988). We have now studied nucleotide specificity and desensitization for several early responses. Extracellular ATP (5-100 microM) caused the rapid formation of inositol trisphosphate and later its metabolites, inositol bisphosphate and inositol monophosphate. ATP also induced the efflux of 45Ca2+ from pre-loaded cells. In addition, an increase in the rate of influx of 45Ca2+ stimulated by extracellular ATP was detected. Based on measurements of 45Ca2+ efflux and influx, desensitization studies, and chlortetracycline fluorimetry, we conclude that ATP mobilizes Ca2+ from internal stores and also stimulates entry across the plasma membrane. These effects were also displayed by UTP and to a lesser extent by ITP, while other nucleoside triphosphates as well as ADP, AMP, and adenosine, were inactive. Furthermore, desensitization of the response to ATP and UTP was seen after prolonged exposure to either nucleotide. This was specific for the nucleotide receptor since a response to bradykinin was not affected by the ATP pretreatment, although pretreatment with phorbol ester inhibited responses to both the nucleotides and bradykinin. Quantitative data on rate of recovery from the desensitized state and the response of desensitized cells to greatly elevated levels of ATP are presented. Extracellular ATP stimulated another early change previously reported for epidermal growth factor, namely, the phosphorylation of an 81-kDa cytoskeletal protein. The stimulation of these events involves an ATP receptor whose properties differ from other ATP receptors that have been described.

Adenosine Triphosphate↗

PubMed1989

Extracellular ATP induces the release of calcium from intracellular stores without the activation of protein kinase C in Swiss 3T6 mouse fibroblasts.

Exposure of Swiss 3T6 mouse fibroblasts to extracellular ATP stimulated the formation of inositol phosphates and mobilized intracellular calcium. The mobilization of intracellular calcium was verified by imaging of fura-2 fluorescence in individual cells and by monitoring the efflux of 45Ca2+ from preloaded cells. However, we found no activation of protein kinase C as measured by phosphorylation of an 80-kDa acidic protein and by transmodulation of the receptor for epidermal growth factor. A careful examination of the kinetics of the phosphorylation reaction (from 30 sec to 10 min) revealed no activation of protein kinase C by extracellular ATP at any time. The lack of activation of protein kinase C was demonstrated even when a concentration of ATP 10-fold higher than that required to give a strong Ca2+ signal was used. Extracellular ATP did not inhibit protein kinase C activation by fetal bovine serum, platelet-derived growth factor, or phorbol esters. The effects of ATP were also produced by UTP but not by ADP, AMP, or adenosine. These findings demonstrate that it is possible to induce the mobilization of intracellular calcium by an inositol phosphate-mediated pathway without the activation of protein kinase C.

Adenosine Triphosphate↗

PubMed1989

The development and implementation of a computerized on-line obstetric record.

We developed and implemented a completely paperless and on-line obstetric record in the high-risk obstetric clinics at Columbia Presbyterian Medical Center. Patient care and interactions are input directly into the computer by the health care providers. Laboratory information is transferred automatically from the Laboratory Information Service into the computerized prenatal record. The system has been in continuous operation for 2 years and is highly regarded. Problems such as illegible handwriting, missing records, and missing laboratory data have disappeared. Research and data query capabilities have been facilitated. The feasibility and applicability of a computerized record replacing the paper record has been demonstrated.

Computers↗

PubMed1988

The rapid desensitization of receptors for platelet derived growth factor, bradykinin and ATP: studies on individual cells using quantitative digital video fluorescence microscopy.

The rise in free cytosolic Ca2+ of individual response to growth factors was studied in serum starved cultures of 3T3 fibroblasts. Quantitative digital video fluorescence microscopy revealed that with platelet derived growth factor (PDGF) there was a lag period between stimulation and Ca2+ response, with considerable cell-to-cell variation, whereas ATP, bradykinin and fetal calf serum induced an immediate, synchronous response. A coverslip with attached cells was mounted on a small flow chamber, allowing complete change of medium in 2 sec. Using this technique, homologous desensitization to a second addition of agonist 2 min after removal of the first addition was found for all agonists. Unusual heterologous desensitization was observed in that PDGF desensitized the cells to the other agonists, yet the reverse did not occur.

Adenosine Triphosphate↗

PubMed1988

Studies on the increase in cytosolic free calcium induced by epidermal growth factor, serum, and nucleotides in individual A431 cells.

The response of cytosolic calcium [Ca2+]i to epidermal growth factor (EGF), fetal calf serum, and nucleotides was determined in individual A431 cells, using the fluorescent probe fura-2 and quantitative digital video fluorescence microscopy. In the presence of 1 mM external Ca2+, EGF caused a rapid rise in [Ca2+]i, followed by a slower and variable decrease. The cells responded after a lag that varied from 10 to 30 seconds, and there was considerable cell-to-cell variation in extent of the rise in [Ca2+]i. A second challenge with EGF gave negative results. No response was obtained in nominally Ca2+-free medium supplemented with 100 microM EGTA. Somewhat similar results were obtained with fetal calf serum except that a rise in [Ca2+]i was observed both in the presence and absence of external Ca2+. The A431 cells responded to external ATP with a rise in [Ca2+]i in less than 10 seconds, both in Ca2+-containing and Ca2+-free media. A coverslip with attached cells was mounted on a small chamber, allowing complete change of medium in 2 seconds. A nearly full response was obtained with only 10 seconds of contact of cells with ATP-containing medium. After washing out ATP, there was little or no response to a second addition given 100 seconds after the first. However, a second response was obtained when the concentration of agonist was increased 10-20-fold. These data favor the idea of receptor desensitization. Both homologous and heterologous receptor desensitization was observed. A transient rise in [Ca2+]i was also noted with UTP, while ITP and CTP were inactive.

Adenosine Triphosphate↗

PubMed1987

The antitumor drug 3-nitrobenzothiazolo(3,2-a)quinolinium chloride (NBQ): effects on lens regeneration and interaction with DNA of Notophthalmus viridescens.

We have studied the effect of 3-nitrobenzothiazolo(3,2-a)quinolinium (NBQ) on the regeneration of the lens in adult newt Notophthalmus viridescens. NBQ has marked cytotoxic effects in tumor cells, intercalates DNA, and was found to enhance lens regeneration. Newt liver DNA was isolated, and the thermal denaturation temperature (Tm) determined to be 76.6% +/- 0.8%. The G-C content was determined to be 44.0% +/- 0.4% and 45.0% +/- 0.1%. Parameters of NBQ binding to newt DNA were determined by spectrophotometric methods and compared with those obtained for calf thymus and Micrococcus lysodeikticus. The association constant, K(o), was found to be 1.1 x 10(+5) M-1 with a site-size parameter, n, of 8.7 nucleotides. No explanation is apparent for the paradoxical stimulation of lens regeneration.

Animals↗

PubMed1987

Fetal gender effects on maternal serum prolactin levels.

Circulating maternal prolactin (PRL) levels have been reported to be higher in term pregnancies yielding male infants. The mechanism for this gender difference is unknown, but we theorized that it was mediated through the fetal adrenal cortex. To test this theory we measured circulating PRL and estriol (E3) concentrations with radioimmunoassay in 37 pregnant women at 34 and 36 weeks' gestation. We then separated the groups by newborn gender. Maternal serum PRL levels were significantly higher in the women bearing male fetuses. There was no significant difference by gender in E3 concentrations, and there was no PRL surge corresponding to the E3 surge at 34-36 weeks' gestational age. There was no correlation between E3 and PRL levels. Transmission of the fetal gender effect on maternal PRL does not appear to be mediated through the fetal adrenal as measured by the fetoplacental production of E3. The effect probably is mediated by the fetal gonad.

Adrenal Cortex↗