PubMed Health⌕ Search

Biomedical subjects

J L Maller

Publications and source records attributed to J L Maller.

121 records · Page 7Linked to original sources
PubMed1982

Identification of a steroid receptor on the surface of Xenopus oocytes by photoaffinity labeling.

The synthetic progestin, 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione (R5020), was used to photoaffinity label a steroid-binding protein on the Xenopus oocyte plasma membrane. R5020 effectively induced oocyte maturation with half-maximal response at a concentration of 1.4 X 10(-6) M. Polyacrylamide gel electrophoretic analysis of plasma membranes following photolysis with tritiated R5020 resulted in the identification of a single labeled protein with a Mr = 110,000. The specificity of this steroid receptor interaction for R5020 was demonstrated by the competitive inhibition of R5020 photolabeling with nonradioactive R5020 and the lack of inhibition by 17 beta-estradiol. Covalent labeling of the 110,000-dalton protein was saturable with both time of photolysis and concentration of R5020 with the maximum number of photolabeled binding sites equal to 0.7 pmol/oocyte, and kinetic analysis of the photolabeling of the 110,000-dalton receptor protein yielded an apparent KD of 1 X 10(-6) M R5020. Progesterone had a biphasic effect on the kinetics of photolabeling with concentrations of progesterone below 5 microM increasing photolabeling by elevating Vmax up to 5-fold and higher concentrations of progesterone reducing the rate of photolabeling. Membrane-associated adenylate cyclase measured in the presence of guanyl-5'-yl imidodiphosphate was inhibited up to 70% after photolysis with [3H]R5020. Inhibition was proportional to the level of [3H]R5020 covalently bound to the 110,000-dalton protein, and significant inhibition was observed at 1 X 10(-6) M R5020.

Adenylyl Cyclases↗
PubMed1982

Increased phosphorylation of ribosomal protein S6 during meiotic maturation of Xenopus oocytes.

A single ribosomal protein (Mr, 32,000) becomes phosphorylated during progesterone-induced in vitro maturation of Xenopus laevis oocytes. The protein is identified as 40S ribosomal protein S6. Phosphorylation of S6 is monitored by incorporation of 32Pi and by two-dimensional polyacrylamide gel electrophoresis. S6 is minimally phosphorylated in unstimulated oocytes. After progesterone treatment, phosphorylation of S6 precedes germinal vesicle breakdown (GVBD) and is maximal at the time when 50% of the oocytes have undergone GVBD. S6, when maximally phosphorylated, exists in derivatives that correspond to the most highly phosphorylated forms observed in other systems, and the increase in S6 phosphorylation occurs at approximately the same time as the increase in the overall protein synthesis rate reported to occur during oocyte maturation. S6 is also maximally phosphorylated in unfertilized eggs following maturation in vivo. Injection of a partially purified preparation of maturation-promoting factor into immature oocytes induces immediate phosphorylation of S6 and rapidly increases the rate of protein synthesis. Moreover, incubation of ribosomes with this factor and radiolabeled ATP results in labeling of S6. These findings suggest that S6 phosphorylation may be important in the control of protein synthesis during maturation and may also play a role in the mechanism of action of maturation-promoting factor.

Animals↗
PubMed1979

Early effect of progesterone on levels of cyclic adenosine 3':5'-monophosphate in Xenopus oocytes.

Progesterone treatment of Xenopus oocytes in vitro causes progression through meiotic cell division. The role of altered intracellular levels of cAMP on the initiation of meiotic cell division has been studied. Basal levels of cAMP averaged 1.5 pmol in oocytes from eight females, and exposure to progesterone caused a rapid drop in cAMP to about 40 to 60% of basal. Half-maximal decreases occurred within 15 to 60 s, and cAMP returned to near basal values by 20 min after progesterone. Theophylline inhibition of progesterone-induced cell division was characterized by a small increase in basal levels of cAMP and a reduced drop in cAMP due to the hormone. Cholera toxin, an activator of adenylate cyclase, was found to be a potent inhibitor of progesterone-induced meiosis, with half-maximal inhibition at 8 times 10(-12) M. In addition, the purified A subunit of cholera toxin was an effective inhibitor of progesterone action when microinjected into oocytes, with half-maximal inhibition occurring at an approximate internal concentration of 1 X 10(-7) M. Cholera toxin alone increased cAMP levels by 20%, but upon addition of progesterone, the level increased transiently to 200% of basal, indicating that the inhibition was due to elevated levels of cAMP. The results support a model in which the initiation of meiotic cell division is regulated by cAMP and protein phosphorylation.

Animals↗
PubMed1978

In vivo phosphorylation of a synthetic peptide substrate of cyclic AMP-dependent protein kinase.

A model synthetic peptide substrate of the cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase; EC 2.7.1.37), Leu-Arg-Arg-Ala-Ser-Leu-Gly, closely resembling the local phosphorylation site sequence in porcine hepatic pyruvate kinase, was shown to be phosphorylated in vivo after microinjection into Xenopus oocytes. This result demonstrates that the microinjection technique, utilizing a synthetic peptide substrate, or possibly a synthetic substrate analog inhibitor [Kemp, B. E., Benjamini, E. & Krebs, E. G. (1976) Proc. Natl. Acad. Sci. USA 73, 1038--1042], can be used to study protein phosphorylation-dephosphorylation reactions in living oocytes. This follows, since it is clear that the injected peptide was accessible to the cellular compartment containing the protein kinase.

Animals↗
PubMed1977

Progesterone-stimulated meiotic cell division in Xenopus oocytes. Induction by regulatory subunit and inhibition by catalytic subunit of adenosine 3':5'-monophosphate-dependent protein kinase.

Ripe Xenopus oocytes in first meiotic prophase when incubated with progesterone in vitro progress synchronously in 3 to 5 h without interphase to second meiotic metaphase where they remain until fertilization or activation. Using highly purified preparations of regulatory and catalytic subunits of adenosine 3':5'-monophosphate-dependent protein kinase from muscle, this progesterone-stimulated cell division sequence was found to be inhibited by microinjection of the catalytic subunit and induced directly in the absence of progesterone after microinjection of regulatory subunit. Dose-response curves revealed that half-maximal effects of regulatory and catalytic subunits occurred at an internal concentration of approximately 0.1 muM. These results indicate that the catalytic subunit is necessary and sufficient to block progesterone-stimulated meiotic cell division. Other experiments revealed that the catalytic subunit was inhibitory only during the first hour after progesterone exposure, suggesting that initial steps in meiotic cell division are affected. Control experiments demonstrate that the muscle cAMP-dependent protein kinase subunits may interact with the endogenous oocyte protein kinase. The results support a model in which meiotic cell division is regulated by a phosphoprotein subject to control by cAMP-dependent protein kinase.

Animals↗
PubMedDate not supplied

Increased phosphorylation of ribosomal protein S6 following microinjection of insulin receptor-kinase into Xenopus oocytes.

The protein products of several transforming retroviruses as well as the receptors for several hormones and growth factors, including insulin, have been shown to possess a protein kinase activity in vitro specific for tyrosine residues in protein substrates, including themselves. In the case of pp60src and the insulin receptor, autophosphorylation activates the tyrosine kinase activity towards exogenous substrates. Experiments indicate that, in vivo, many of these viruses or growth factors induce an increase in cellular phosphotyrosine, as well as an increase in the phosphorylation of serine residues on proteins, including ribosomal protein S6. It seems likely that some of the effects of insulin might be mediated by phosphorylation of intracellular substrates by its receptor. As the beta subunit of the receptor is a transmembrane protein, such phosphorylation could occur either while the receptor is still in the membrane or after its internalization. In various cell systems, internalized receptors are degraded, reshuttled back to the plasmalemma or maintained in a separate compartment before reinsertion in the membrane; shuttling of the insulin receptor could provide the opportunity for it to phosphorylate various intracellular components as part of its mechanism of signal transduction. To approach directly the question of whether the receptor can elicit a signal while acting at an intracellular location, we have microinjected Xenopus oocytes with the insulin receptor kinase. The results indicate that an S6 protein-serine kinase is stimulated or an S6 protein-serine phosphatase inhibited by the activity of the insulin receptor, supporting the concept that the insulin receptor acting within the cell can elicit a biological response.

Animals↗
PubMedDate not supplied

Mitogenic signalling and protein phosphorylation in Xenopus oocytes.

Xenopus oocytes are stimulated to undergo meiotic cell division in response to several types of mitogenic stimuli. Agents that reduce cAMP levels induce cell division in oocytes, and this occurs due to inhibition of adenylate cyclase with progesterone as well as by activation of phosphodiesterase with insulin. Phorbol esters and microinjected protein kinase C also promote cell division, implicating phospholipid breakdown as another signalling pathway competent to induce proliferation in this system. A third signalling pathway is via the tyrosine kinase activity of the insulin receptor. A proximal activation of a ribosomal protein S6 kinase by insulin has provided insight into the regulation of this pathway. All three of these signal transduction pathways lead to the activation of a cytoplasmic protein able to induce nuclear breakdown, chromosome condensation and spindle formation in vivo and in vitro. This protein, known as maturation-promoting factor, is associated with changes in protein phosphorylation on both serine and tyrosine residues. These results support a model in which signal transduction by different pathways activates a common cell cycle control element that regulates the G2----M transition via changes in protein phosphorylation.

Animals↗