Biomedical subjects
L B Ray
Publications and source records attributed to L B Ray.
Purification of mitogen-activated protein kinase from epidermal growth factor-treated 3T3-L1 fibroblasts.
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Microtubule-associated protein 1A is the fibroblast HMW MAP undergoing mitogen-stimulated serine phosphorylation.
A variety of antibodies to microtubule-associated protein (MAP) have been used to demonstrate that phosphorylation of a 350 kDa microtubule-associated protein is stimulated 2-3 fold by epidermal growth factor or serum in quiescent 3T3-L1 fibroblasts and by insulin in 3T3-L1 adipocytes. Phosphorylation occurs on serine residues, and is maximal by 15-20 min. The phosphoprotein has been identified as MAP1A by specific immunoprecipitation with a well-characterized monoclonal antibody.
Characterization of insulin-stimulated microtubule-associated protein kinase. Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3-L1 cells.
A protein kinase, termed microtubule-associated protein (MAP) kinase, which phosphorylates microtubule-associated protein 2 (MAP-2) in vitro and is stimulated 1.5-3-fold in extracts from insulin-treated 3T3-L1 cells has been identified (Ray, L.B., and Sturgill, T.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1502-1506). Here, we describe chromatographic properties of MAP kinase and provide biochemical characterization of the partially purified enzyme. Isolation of the enzyme is facilitated by its unusually high affinity for hydrophobic interaction chromatography matrices. The molecular weight of the partially purified enzyme was determined to be 35,000 by gel filtration chromatography and 37,000 by glycerol gradient centrifugation. MAP kinase activity of chromatographic fractions correlated precisely with the presence of a 40-kDa phosphoprotein detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MAP kinase has a Km of 7 microM for ATP and does not utilize GTP. Acetyl-CoA carboxylase, ATP citrate-lyase, casein, histones, phosvitin, protamine, and ribosomal protein S6 were all poor substrates relative to MAP-2. The enzyme is inhibited by fluoride and beta-glycerol phosphate but not by heparin. These properties of MAP kinase distinguish it from protein kinases previously described in the literature.
Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II.
Ribosomal protein S6 is a component of the eukaryotic 40S ribosomal subunit that becomes phosphorylated on multiple serine residues in response to a variety of mitogens, including insulin, growth factors, and transforming proteins of many oncogenic viruses. Recently, an activated S6 kinase (S6 K II) has been purified to homogeneity from Xenopus eggs, and characterized immunologically and at the molecular level. Purified S6 K II can be deactivated in vitro by incubation with either protein phosphatase 1 or protein phosphatase 2A. Reactivation and phosphorylation of S6 K II occurs in vitro with an insulin-stimulated microtubule-associated protein-2 (MAP-2) protein kinase which is itself a phosphoprotein that can be deactivated by protein phosphatase 2A. These studies suggest that a step in insulin signalling involves sequential activation by phosphorylation of at least two serine/threonine protein kinases.
Rapid stimulation by insulin of a serine/threonine kinase in 3T3-L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro.
Insulin treatment (Kact, 5 X 10(-9) M) of serum-starved 3T3-L1 adipocytes stimulates a soluble serine/threonine kinase that catalyzes phosphorylation of microtubule-associated protein 2 (MAP-2) in vitro. Maximal activation of MAP-2 kinase activity by 80 nM insulin was observed after 10 min of hormonal stimulation, prior to maximal stimulation of S6 kinase activity (20 min). The insulin-stimulatable MAP-2 kinase activity is not adsorbed to phosphocellulose, whereas the principal S6 kinase activity is retained and elutes at approximately 0.5 M NaCl. The insulin-stimulatable MAP-2 kinase is less stable during incubation at 30 degrees C than S6 kinase activity. Inclusion of phosphatase inhibitors decreases the rate at which the stimulated MAP-2 kinase activity is lost from extract supernatants incubated at 30 degrees C. p-Nitrophenyl phosphate is more effective than DL-phosphotyrosine, whereas DL-phosphoserine is without effect at the concentration used (40 mM). The difference in MAP-2 kinase activity in extract supernatants from control and insulin-treated cells is also preserved after rapid chromatography on Sephadex G-25. These results show that a soluble serine/threonine kinase is rapidly activated by insulin, possibly by phosphorylation of either the kinase itself or an interacting modulator.
Investigations on the role of cAMP in regulating resorption of the tail fin from tadpoles of Rana catesbeiana.
Conflicting reports have appeared regarding the role of cAMP in regulating resorption of the tadpole tail during anuran metamorphosis. That cyclic nucleotide has been suggested as a mediator of the effects of both the thyroid hormones and prolactin. We tested the effects of cAMP and its derivatives dibutyryl-cAMP and 8-bromo-cAMP on explants of tail fin from tadpoles of Rana catesbeiana maintained in tissue culture. Unmodified cAMP (0.1, 2 mM) did not influence resorption. Dibutyryl-cAMP (0.1, 1 mM) and 8-bromo-cAMP (1 mM) inhibited resorption of explants induced by thyroxine (T4). The phosphodiesterase inhibitor isobutylmethylxanthine similarly inhibited regression of explants cultured with T4. None of these agents affected the increase in specific activity of hexosaminidase brought about by T4. Although the effects of cAMP in antagonizing tail resorption were similar to those of prolactin, we found no direct effect of prolactin on levels of cAMP in cultured tail fin. Thus, the effects of prolactin appear not to be mediated by increased levels of cAMP. We conclude, however, that the elevation of cellular levels of cAMP does inhibit the resorptive action of T4.