In vitro translation of the intermediate filament proteins desmin and vimentin.

O’Connor, Clare M.; Asai, David J.; Flytzanis, Constantin N.; Lazarides, Elias

doi:10.1128/mcb.1.4.303

Cited by 16 publications

(18 citation statements)

References 20 publications

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“…Although mature (7-to 8-day-old) myotubes showed increases in phosphorylation, myoblasts showed no changes in phosphorylation when exposed to either agonists or cAMP analogs. These data, in conjunction with our previous studies of cAMP-dependent phosphorylation of desmin and vimentin in vitro (18,19), suggest that the IF proteins desmin and vimentin are phosphorylated by the cAMP-dependent protein kinase in vivo and that changes in intracellular cAMP levels during differentiation may modulate IF protein phosphorylation and thereby regulate IF function.…”

Section: Discussionsupporting

confidence: 68%

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Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

Gard¹,

Lazarides²

1982

Mol. Cell. Biol.

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The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two-to threefold increase in incorporation of 32 o4 into both desmin and vimentin, whereas no changes in the incorporation of 32p04 into tropomyosin or other cellular proteins were observed. The BrcAMP-or hormonally induced increase in 32P04 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32P04 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7-to 10-fold increase in 32Po4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP-and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation.

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Section: Discussionsupporting

confidence: 68%

“…Two-dimensional tryptic mapping of '2PO4-labeled desmin and vimentin was performed essentially as described previously (19). Myotubes (8 days old; 106 cells per 100-mm plate) were labeled as described above for 4 h with 100 LCi of 32P04 per ml.…”

Section: Methodsmentioning

confidence: 99%

Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

Gard¹,

Lazarides²

1982

Mol. Cell. Biol.

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“…One-dimensional peptide mapping has indicated that thermin A and B are not identical polypeptides. This difference is further strengthened by the fact that both forms of thermin are translated in a reticulocyte cell-free system using poly(A)+mRNA obtained from chicken embryonic fibroblasts treated with so- (7) or from chicken smooth and skeletal muscle (14). In This argument is strengthened by the observation that, under these conditions, the 'normal level of methylation of thermin is not.inhibited.…”

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confidence: 66%

Heat shock proteins are methylated in avian and mammalian cells.

Wang¹,

Gomer²,

Lazarides³

1981

Proc. Natl. Acad. Sci. U.S.A.

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148

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Exposure of chicken cells grown in tissue culture to heat shock or sodium arsenite results in a dramatic increase in the synthesis of three major polypeptides with molecular weights of 83,000 (HSP 83), 68,000 (HSP 68; referred to here as "thermin"), and 25,000 (HSP 25). Incubation of BHK-21 or HeLa cells under the same conditions results in induction of HSP 68 and a 66,000-dalton polypeptide (HSP 66). Chicken thermin is resolved by isoelectric focusing into a major acidic and a more-basic component; mammalian thermin is resolved only into one major acidic component. HSP 83 and the acidic form of thermin are highly conserved in all avian and mammalian cells examined as judged by their electrophoretic mobilities, isoelectric points, and one-dimensional peptide maps. In addition, the acidic form of thermin is indistinguishable from a protein that copurifies with brain microtubules and that remains associated with the intermediate filament-enriched Triton/KCl cytoskeletons of cells grown in tissue culture. Thermin is also a component of skeletal myofibrils. HSP 83 and thermin are methylated in cells cultured under normal growth conditions. Induction of heat shock proteins by incubation of cells in the presence of sodium arsenite results in a marked methylation of the newly synthesized thermin. Under the same experimental conditions, no significant increase in methylation of the HSP 83 is observed. HSP 25 is not methylated in untreated cells or in cells treated with sodium arsenite. These results suggest that methylation of heat shock proteins may have an important role in regulating their function.

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“…If other co-or post-translational modifications are involved, such modifications would have to be brought about in both the living Xenopus oocyte and the rabbit reticulocyte lysate (for specific acetylation and phosphorylation changes in both systems, see refs. [29][30][31][32]. Our experiments further show that proteins N1-N4 as synthesized in vitro are readily accumulated in nuclei not only ofthe same species but also ofanother, taxonomically distant, amphibian species.…”

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confidence: 56%

“…5i) Results of this study show that four of the most prominent karyophilic proteins (Ni-N4) of the Xenopus oocyte nucleus appear, on synthesis by translation in vitro, in the form of molecules indistinguishable from the specific polypeptides present in nuclei of living oocytes, as judged from two-dimensional gel electrophoresis and-in the case ofproteins Ni/N2-also from peptide maps and reaction with antibodies. In view of the high sensitivity of the gel electrophoretic procedure used, which allows the detection of single phosphorylation and acetylation differences in polypeptides (29,31), we conclude that these proteins are not synthesized in the form ofprecursor molecules considerably different, by size and charge, from the molecules present in the nucleus. Polypeptides Ni and N2 seem to be phosphorylated to the same degree both in vivo and in vitro.…”

mentioning

confidence: 90%

Karyophilic proteins: polypeptides synthesized in vitro accumulate in the nucleus on microinjection into the cytoplasm of amphibian oocytes.

Dabauvalle

Franke²

1982

Proc. Natl. Acad. Sci. U.S.A.

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The specific nucleocytoplasmic compartmentalization of proteins has been examined for some major soluble acidic nuclear proteins in oocytes of different amphibia. Proteins synthesized and radioactively labeled by translation in vitro, by using mRNA from ovaries ofthe frog Xenopus laevis, were injected into the cytoplasm of living oocytes of Xenopus or of the salamander Pleurodeles waltlii At various times after injection, nucleus and cytoplasm were manually separated and endogenous and injected proteins were analyzed by two-dimensional gel electrophoresis. We show that several major nucleus-specific proteins of different sizes and electrical charges, including the very acidic proteins Ni and N2 (Mr, 110,000 and 100,000) and N4 (Mr, 34,000), are identical in both forms-i.e., as translation products in vitro and as present in the nucleoplasm. We conclude that significantly different cytoplasmic precursor forms to these nuclear proteins do not exist. The experiments indicate that (i) the translation products contain the signal(s) directing the specific sequestration of these proteins within the nucleus, (ii) post-translational processing is not required for the accumulation of these proteins in the nucleoplasm, and (iii) the signals and the mechanisms involved are not species specific.One of the fundamental determinants of function and order in the eukaryotic cell is the compartmentalization ofdiverse kinds of molecules, including proteins. The sequestration of certain proteins in specific compartments has been studied with special intensity for secretory and the membrane-bound proteins. These studies have led to the concept that the specific location of such proteins is directed by "signal sequences" that are permanent in some cases but are proteolytically removed, on transport into the specific compartment, in many others (for reviews, see refs. 1-3). Observations that polypeptide chains can be coor post-translationally removed on correct translocation into the target structure have come from assays of translation of mRNA in vitro showing that newly synthesized polypeptides are larger by a number of amino acid residues than the endogenous compartmentalized form of the molecule (1-3). Alternatively, it is possible that the specific location ofa given protein is controlled by conformational changes and co-or post-translational modifications (for review, see ref.3).The cell nucleus contains a number of proteins that are synthesized in the cytoplasm but are translocated into the nucleus with remarkable specificity and efficiency so that they are usually not detected in significant amounts in the compartment of their origin-i.e., the cytoplasm. Although the nuclear accumulation of many such proteins can be explained by their association with nuclear structures (4-7), it is more difficult to explain the accumulation of those "karyophilic" proteins that are dispersed throughout the nuclear sap and appear in soluble complexes when examined in isolated nuclei. The latter category includes some of the most abundan...

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In vitro translation of the intermediate filament proteins desmin and vimentin.

Abstract: Polyadenylated ribonucleic acid (RNA) was isolated from chicken skeletal and smooth muscle and translated in a cell-free rabbit reticulocycle system.

Cited by 16 publications

References 20 publications

Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

Heat shock proteins are methylated in avian and mammalian cells.

Karyophilic proteins: polypeptides synthesized in vitro accumulate in the nucleus on microinjection into the cytoplasm of amphibian oocytes.

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