Marit Nilsen-Hamilton

Selective stimulation by mitogens of incorporation of 35S-methionine into a family of proteins released into the medium by 3T3 cells

Abstract Serum and three mitogens for mouse embryo 3T3 cells—fibroblast growth factor from brain, fibroblast growth factor from pituitary, and epidermal growth factor—specifically stimulate the synthesis and release into the medium by these cells of a group of proteins that travel together on SDS gel electrophoresis and that are detected by 35 S-methionine labeling. These proteins, designated mitogen-releasable proteins (MRPs), have a median, monomer molecular weight on SDS polyacrylamide gel electrophoresis of 34,000 daltons (30,000–38,000 daltons). Our evidence indicates that these proteins comprise a family of glycoproteins, probably with a common polypeptide backbone. The observations supporting this conclusion are that MRPs give a diffuse pattern of bands upon SDS gel electrophoresis; travel as a single, diffuse band when resolved by electrophoresis in the absence of SDS; adsorb to a pea-lectin-sepharose column and can be eluted with α-methyl mannose; and can be labeled metabolically with 3 H-mannose. In addition, in the presence of tunicamycin, MRPs are not made—instead, a smaller molecular weight (22,000 dalton), and apparently homogeneous, protein appears. We believe this 22,000 dalton protein to be the unglycosylated form of MRP. Further support for this idea comes from our observation that treatment of MRPs with endoglycosidase H produces a protein with a molecular weight slightly greater than 22,000 daltons. The effect of mitogens on DNA synthesis and MRP release are correlated in the following ways. First, serum factors are required for both responses. Second, in 3T3 cells transformed by SV40, Moloney and Kirsten viruses that do not synthesize DNA in response to FGF, MRPs are not released in response to FGF. Third, in untransformed 3T3 cells, the dose-response curves for fibroblast growth factor on MRP release and thymidine incorporation are closely correlated. Fourth, insulin, a poor mitogen for 3T3 cells, does not enhance MRP release. Fifth, stimulation of MRP release by epidermal growth factor or fibroblast growth factor is inhibited by hydroxyurea and butyrate, both inhibitors of DNA synthesis in these cells. Sixth, if the mitogen is removed at any time during the 20 hr preincubation period, the effect on MRP release observed between 20 and 24 hr is severely diminished.

Stimulation of the release of two glycoproteins from mouse 3T3 cells by growth factors and by agents that increase intralysosomal pH.

Abstract Peptide growth factors selectively increase the amount of mitogen-regulated protein (MRP) and major excreted protein (MEP) released by mouse 3T3 cells. Balb c 3T3 cells release mainly MEP and Swiss 3T3 cells release mainly MRP. Fibroblast growth factor, epidermal growth factor, nerve growth factor, serum, and concanavalin A increase the extracellular appearance of both MEP and MRP, but to different extents. Several agents that have been shown to, or would be expected to increase, intralysosomal pH also selectively increase the release of MEP and MRP from both Balb c and Swiss 3T3 cells. The effective agents are monensin, nigericin, ammonium chloride, methylamine, chloroquine, and high extracellular pH.

Rapid selective stimulation by growth factors of the incorporation by Balbc 3T3 cells of [35S]methionine into a glycoprotein and five superinducible proteins

Within four hours of adding fibroblast growth factor, epidermal growth factor, prostaglandin F2α, or serum to quiescent Balbc 3T3 cells we observe selective increases in the incorporation of [35S]methionine into six proteins; “major excreted protein” (MEP) and five “superinducible proteins” (SIPs). The mechanisms regulating the extracellular expression of MEP and the SIPs differ. 1) The levels of MEP but not SIPs are increased by NH4Cl; and 2) Cycloheximide increase SIP and decreases MEP production. These results suggest that production of MEP and the SIPs are controlled by other proteins; MEP by a positive, and the SIPs by a negative effector.

Synergistic stimulation of S6 ribosomal protein phosphorylation and DNA synthesis by epidermal growth factor and insulin in quiescent 3T3 cells

Using an improved method to quantify the level of phosphorylation of the S6 ribosomal protein, we have analyzed the effect of growth stimuli on S6 phosphorylation in quiescent murine Swiss/3T3 cells to see if it can be dissociated from the later increase in DNA synthesis. Saturating concentrations of epidermal growth factor (EGF), insulin and serum each stimulate phosphorylation of the S6 ribosomal protein to the same maximal level; this is not so for DNA synthesis. Subsaturating concentrations of EGF and insulin act synergistically to stimulate both S6 phosphorylation and DNA synthesis, but qualitatively the two synergistic interactions are expressed differently. Insulin increases the maximal response of DNA synthesis to EGF, whereas it decreases the concentration of EGF required for half-maximal stimulation of S6 phosphorylation. We conclude that S6 phosphorylation is not a principal regulator of DNA synthesis, and that insulin and EGF regulate both S6 phosphorylation and DNA synthesis through different, but interacting, pathways of action.

Rapid and efficient method for analyzing phosphorylation of the S6 ribosomal protein in 32Pi-labeled, tissue culture cells

We describe a method for studying the phosphorylation of the S6 ribosomal protein in intact cells. The procedure has the advantage of using few cells, little 32Pi, and by using an air-driven centrifuge, many samples can be processed in a short time. Metabolically labeling the ribosomes with [3H]uridine before the experiment provides a measure of ribosome yield. The amount of 32Pi incorporated into proteins other than S6, which cosediment with the ribosomes, increases by the same amount as the specific activity of [32P]ATP increases, when the cells are stimulated by prostaglandin F2α, insulin, epidermal, or fibroblast growth factor, or serum; whereas the 32Pi incorporated into S6 increases by a factor greater than the increase in the specific activity of [32P]ATP. We show that the phosphate on S6 turns over at least as rapidly as does the phosphate on ATP. This last observation allows us to use a procedure, which we have outlined for determining the absolute amount of phosphate added to S6 due to a stimulus.

Inhibition of α-aminoisobutyric acid transport in membrane vesicles from mouse fibroblasts after phosphorylation by cyclic AMP-dependent protein kinase

Cyclic AMP-dependent protein kinases from several mammalian sources inhibit Na+-dependent α-aminoisobutyric acid transport by membrane vesicles isolated from 3T3 cells. Evidence is provided that phosphorylation of membrane proteins by the enzyme is responsible for the inhibition. Lysis of the vesicles, or a reduction in the intravesicular volume is not the cause of reduced transport. The cyclic AMP-dependent protein kinase and its catalytic subunit phosphorylate a number of membrane proteins. Most of these proteins are phosphorylated, but to a lesser extent in the absence of protein kinase or cyclic AMP. The phosphorylated proteins remain associated with the membranes during hypotonic lysis treatments, which would be expected to release intra-vesicular contents and loosely associated membrane proteins. 32P-labeled bands detected on sodium dodecyl sulfate polyacrylamide gels after phosphorylation of membranes by the catalytic subunit of the cyclic AMP-dependent kinase are eliminated by treatment with either pronase or 1 N NaOH, but not by ribonuclease nor by phospholipase C. The stability of the incorporated radioactivity to hot acid and hydroxylamine relative to hot base suggests that most of the 32P from [γ-32P]ATP is incorporated into protein phosphomonoester linkages.

Conversion of monensin from an ionophore to an inhibitor of Na+ uptake by SV3T3 membrane vesicles as a function of Na+ concentration

Monensin is a Na+ ionophore in membrane vesicles from SV3T3 cells; but its ability to stimulate Na+ flux is inhibited by increasing concentrations of Na+. At greater than 20-mM Na+, monensin inhibits Na+ uptake by the vesicles. Cs+ and NH4+ also cause monensin to inhibit Na+ uptake, but general alterations in ionic strength do not convert the ionophore to an inhibitor. Monensin does not cause Na+ loss during collection of the vesicles on filters; nor is inhibition the result of the vesicle lumen being made alkaline by H+ loss in exchange for Na+. The specificity for cation and ionophore indicates that a precise interaction between the cation, ionophore, and membrane is required for inhibition.

Synergistic Interactions of Specific Prostaglandins in Regulating the Rate of Initiation of DNA Synthesis in Swiss 3T3 Cells

Prostaglandins (PG) constitute a family of structurally related molecules with diverse functions. They are produced by various animal cells in response to physiological and pathological changes, as e.g. inflammation or cancer. The function of a prostaglandin depends on the target cell and the local presence of other prostaglandins and hormones (1, 2).

SELECTIVE INDUCTION BY PEPTIDE GROWTH FACTORS OF THE SYNTHESIS OF SECRETED PROTEINS IN 3T3 CELLS

Publisher Summary The changes in cellular metabolism needed for the initiation of DNA synthesis in quiescent cells require the synthesis of new proteins and probably involve the activation of genes. It has been found that peptide mitogens, such as epidermal growth factor and fibroblast growth factor, selectively increase the extracellular appearance of two glycoproteins secreted by 3T3 cells. These proteins are mitogen-regulated protein (MRP) and major excreted protein (MEP). The mitogens regulate at transcription or translation, not at glycosylation or secretion. It is speculated that these mitogen-induced proteins could participate in the regulation of cell growth in one of several ways. MEP or MRP could be internal growth regulating proteins. These proteins, being secreted, could be communicatory molecules for coordinating the growth of a tissue.