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The effects of insulin, cycloheximide and phalloidin on the content of actin and p35 in extracts prepared from the nuclear fraction of Krebs II ascites cells

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Abstract

The nuclear fraction isolated from Krebs II ascites cells following cell disruption by nitrogen cavitation was separated into four fractions by salt/detergent extraction: NP-40 soluble fraction, 130 mM KCl extract, DOC/Triton × 100 soluble fraction and salt/detergent treated nuclei. The protein composition of the individual fractions was studied by SDS-PAGE and the relative amounts of actin and a 35 kDa protein (p35) were measured from gel scans. There was a time-dependent shift of actin from the 130 mM KCl extract to the NP-40 soluble fraction upon storage of the nuclear fraction on ice, indicating a progressive depolymerization of microfilaments. Compared with actin there was a slower release of p35 into the NP-40 soluble fraction. The results suggest that p35 is not integrated in the microfilament network. Phalloidin, which stabilizes the microfilaments, enriched the amount of both proteins in the 130 mM KCl extracts, together with a series of other proteins in the range 50–205 kDa. The presence of phalloidin also resulted in a large increase in the actin content in both the DOC/Triton × 100 extract and the fraction containing salt/detergent treated nuclei. Incubation of cells with insulin and/or cycloheximide enriched the amount of actin in the 130 mM KCl fraction. The results show that short term incubation of cells with phalloidin, insulin or cycloheximide increases the actin content of the nuclear fraction and also affects the presence of several other proteins.

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References

  1. Almås B, Pryme IF, Vedeler A, Hesketh JE: Insulin: Signal transmission and short term effect on the cytoskeleton and protein synthesis. Int J Biochem 24: 183–191, 1992

    Google Scholar 

  2. Vedeler A, Pryme IF, Hesketh JE: Insulin induces changes in the subcellular distribution of actin and 5′-nucleotidase. Mol Cell Biochem 108: 67–74, 1991

    Google Scholar 

  3. Hesketh JE, Campbell GP, Reeds PJ: Rapid response of protein synthesis to insulin in 3T3 cells: Effects of protein kinase C depletion and differences from the response to serum repletion. Biosci Repts 6: 797–804, 1986

    Google Scholar 

  4. Ong JM, Kirchgessner TG, Schotz MC, Kern PA: Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocytes. J Biol Chem 263: 12933–12938,1988

    Google Scholar 

  5. Vedeler A, Pryme IF, Hesketh JE: Insulin and step-up conditions cause a redistribution of polysomes among free, cytoskeletal-bound and membrane-bound fractions in Krebs II ascites cells. Cell Biol Int Repts 14: 211–218, 1990

    Google Scholar 

  6. Hesketh JE, Campbell GP: Effects of insulin, pertussis toxin and cholera toxin on protein synthesis and diacylglycerol production in 3T3 fibroblasts: Evidence for a G-protein mediated activation of phospholipase C in the insulin signal mechanism. Biosci Repts 7:533–543,1987

    Google Scholar 

  7. Hesketh JE, Pryme IF: Evidence that insulin increases the proportion of polysomes that are bound to the cytoskeleton in 3T3 fibroblasts. FEBS Lett 231: 62–66, 1988

    Google Scholar 

  8. Nanhua C, Masters C: The influence of insulin and glucagon on the interactions between glycolytic enzymes and cellular structure. Biochem Int 16: 903–911, 1988

    Google Scholar 

  9. Rao KMK, Betschart JM, Virji MA: Hormone-induced actin polymerization in rat hepatoma cells and human leucocytes. Biochem J 230: 709–714, 1985

    Google Scholar 

  10. Weeds AG, Gooch J, Hawkins M, Pope B, Way M: Role of actin-binding proteins in cytoskeletal dynamics. Biochem Soc Trans 19:1016–1020,1991

    Google Scholar 

  11. Craig SW, Pollard TD: Actin binding proteins. Trends Biochem Sci 7:88–92, 1982

    Google Scholar 

  12. Kirkeeide E-K, Pryme IF, Vedeler A: Changes in amounts of polysomes in free, cytoskeletal-bound and membrane-bound populations in Krebs II ascites cells subjected to different growth conditions. Biochem Soc Trans 19: 1136–1137, 1991

    Google Scholar 

  13. Vedeler A, Pryme IF, Hesketh JE: The characterization of free, cytoskeletal and membrane-bound polysomes in Krebs II ascites and 3T3 cells. Mol Cell Biochem 100: 183–193, 1991

    Google Scholar 

  14. Vedeler A, Pryme IF, Hesketh JE: Compartmentalization of polysomes into free, cytoskeletal-bound and membrane-bound populations. Biochem Soc Trans 19: 1108–1111, 1991

    Google Scholar 

  15. Hesketh JE, Campbell GP, Whitelaw PF: c-myc mRNA in cytoskeletal-bound polysomes in fibroblasts. Biochem J 274: 607–609,1991

    Google Scholar 

  16. Moss R, Pryme IF, Vedeler A: Difference in patterns of proteins isolated from polysomes in free, cytoskeleton-bound and membrane-bound fractions in MPC-11 cells incubated with insulin. Biochem Soc Trans 19: 1138–1139, 1991

    Google Scholar 

  17. Pryme IF: The importance of salt concentration during nitrogen cavitation of MPC-11 cells for the isolation of heavy rough endoplasmic reticulum membranes. Biochem Int 13: 287–293, 1986

    Google Scholar 

  18. Almås B, Pryme IF, Vedeler A, Hesketh JE: Differences in the content of actin-binding proteins in subcellular fractions prepared from Krebs II ascites cells. Biochem Soc Trans 19: 1140–1141, 1991

    Google Scholar 

  19. Crumpton MJ, Dedman JR: Protein terminology tangle. Nature 345:212,1990

    Google Scholar 

  20. Branton D, Cohen CM, Tyler J: Interaction of cytoskeletal proteins on the human erythrocyte membrane. Cell 24: 24–32, 1981

    Google Scholar 

  21. Burridge K, Kelly T, Mangeat P: Non-erythrocyte spectrins: Actin-membrane attachment proteins occurring in many cell types. J Cell Biol 95: 478–486, 1982

    Google Scholar 

  22. Goodman SR, Shiffer K: The spectrin membrane skeleton of normal and abnormal human erythrocytes: A review. Am J Physiol 244: C121-C141, 1983

    Google Scholar 

  23. Glenney JR Jr, Tack B, Powell MA: Calpactins: Two distinct Call-regulated phospholipid and actin-binding proteins isolated from lung and placenta. J Cell Biol 104: 503–511, 1987

    Google Scholar 

  24. Fava RA, Cohen S: Isolation of a calcium-dependent 35-kilodalton substrate for the epidermal growth factor receptor/kinase from A-431 cells. J Biol Chem 259: 2636–2645, 1984

    Google Scholar 

  25. Tavaré JM, Diggle TA, Denton RM: Epidermal growth factor, but not insulin, stimulates tyrosine phosphorylation of an endogenous protein of Mr 95000 in Triton extracts of human placental syncytiotrophoblast membranes. Biochem J 244: 769–774, 1987

    Google Scholar 

  26. Pryme IF: The nuclear-associated endoplasmic reticulum. Int J Biochem 21: 119–125, 1989

    Google Scholar 

  27. Fjose A, Pryme IF, Lillehaug JR: The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate and stimulation of 3H-choline incorporation into endoplasmic reticulum membranes and other subcellular fractions of Krebs II ascites cells during in vitro incubation. Mol Cell Biochem 56: 131–136, 1983

    Google Scholar 

  28. Fjose A, Pryme IF, Lillehaug JR, Djurhuus R: The specific appearance of a 65,000 dalton protein in the nuclear-associated endoplasmic reticulum of Krebs II ascites cells early after treatment with the phorbol ester TPA. Carcinogenesis 4: 811–815, 1983

    Google Scholar 

  29. Pryme IF, Lillehaug JR, Fjose A, Kleppe K: The nuclear-associated endoplasmic reticulum is an early target for the action of the tumour promoter 12-O-tetradecanoyl phorbol-13-acetate in C3H/1OT1/2 fibroblasts. FEBS Lett 152: 17–20, 1983

    Google Scholar 

  30. Miller M, Park MK, Hanover JA: Nuclear pore complex: Structure and regulation. Physiol Rev 71: 909–949, 1991

    Google Scholar 

  31. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976

    Article  CAS  PubMed  Google Scholar 

  32. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970

    PubMed  Google Scholar 

  33. Nelson WJ, Traub P: Properties of a Ca2+-activated protease specific for the intermediate-sized filament protein vimentin in Erlich-ascites cells. Eur J Biochem 116: 51–57, 1981

    Google Scholar 

  34. Löw I, Dancker P, Wieland T: Stabilization of F-actin by phalloidin: Reversal of the destabilizing effect of cytochalasin B. FEBS Lett 54: 263–265, 1975

    Google Scholar 

  35. Estes JE, Selden LA, Gershman LC: Mechanism of action of phalloidin on the polymerization of muscle actin. Biochemistry 20: 708–712, 1981

    Google Scholar 

  36. Clark TG, Rosenbaum JL: An actin filament matrix in handisolated nuclei of X. laevis oocytes. Cell 18: 1101–1108, 1979

    Google Scholar 

  37. Wieland T: Modification of actins by phallotoxins. Naturwissenschaften 64: 303–309, 1979

    Google Scholar 

  38. Wolosewick JJ, Porter KR: Microtrabecular lattice of the cytoplasmic ground substance. J Cell Biol 82: 114–139, 1979

    Google Scholar 

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Authors and Affiliations

  1. Department of Biochemistry, University of Bergen, Årstadveien 19, N-5009, Bergen, Norway

    Bjørg Almås, Anni Vedeler & Ian F. Pryme

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  1. Bjørg Almås
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  2. Anni Vedeler
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  3. Ian F. Pryme
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Almås, B., Vedeler, A. & Pryme, I.F. The effects of insulin, cycloheximide and phalloidin on the content of actin and p35 in extracts prepared from the nuclear fraction of Krebs II ascites cells. Mol Cell Biochem 115, 187–194 (1992). https://doi.org/10.1007/BF00230330

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  • DOI: https://doi.org/10.1007/BF00230330

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