Phosphorylation <i>in vivo</i> of four basic proteins of rat brain myelin

Agrawal, Harish C.; O'Connell, Keith M.; Randle, Charlotte L.; Agrawal, Daya

doi:10.1042/bj2010039

Cited by 54 publications

(28 citation statements)

References 33 publications

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“…The linkage between the bound fatty acid and protein molecules appeared to involve an ester bond, based on the sensitivity to mild alkali. The presence of an ester bond has been reported for many membraneous proteins (17,(26)(27)(28)(29).…”

Section: Methodsmentioning

confidence: 99%

“…Since the RAS proteins are present in both soluble and membrane fractions, we tested if the p36 and p40 molecules in the membrane fraction are chemically distinct from the molecules in the soluble fraction. Many membraneous proteins including p60srC and p21rlS have been reported to be modified by fatty acylation (17,(26)(27)(28)(29)(30)(31)(32)(33)(34). Therefore, we performed labeling with fatty acids.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae.

Fujiyama¹,

Tamanoi²

1986

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

100

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We demonstrate the pathway for the biosynthesis of RASI and RAS2 gene products of Saccharomyces cerevisiae leading to their localization in membranes. The primary translation products of these genes are detected in a soluble fraction. Shortly after synthesis, these precursor molecules are converted to forms that migrate slightly faster than the precursor forms on a NaDodSO4/polyacrylamide gel.These processed proteins are further modified by fatty acid acylation, which is detected by [3H]palmitic acid labeling. The acylated derivatives are found exclusively in cell membranes, indicating the translocation of the RAS proteins from cytosol to membranes during maturation process. The attached fatty acids can be released by mild alkaline hydrolysis, suggesting that the linkage between the fatty acid and the protein is an ester bond. The site of the modification by fatty acid is presumably localized to the COOH-terminal portion of the RAS proteins. Fractionation of the membranes by sucrose gradient demonstrates that a majority of the fatty-acylated RAS proteins are localized in plasma membrane.The RAS] and RAS2 genes of Saccharomyces cerevisiae were first identified as homologues of mammalian ras genes and were assigned to chromosomes XV and XIV, respectively (1-3). Amino acid sequences predicted from the DNA sequences of cloned genes have demonstrated a high level of homology with other members of the ras protein family (1, 2, 4, 5). Genetic analyses have shown that haploid cells that lack one of the RAS genes grow normally; however, mutants that are defective in both genes are incapable ofvegetative growth (3, 6). Like their mammalian counterparts, the yeast RAS proteins have been shown to bind GTP and GDP as well as to function as GTPase (7-10). Recent studies have shown that the RAS proteins are involved in the regulatory mechanisms ofadenylate cyclase activity (11,12 (12).Compared with the biochemical functions, little is known about the structural properties of the RAS proteins and how these relate to protein function. Since, as described above, the action of the RAS proteins appears to take place in membranes, it is important to elucidate subcellular localization and biosynthetic pathway of the RAS proteins. It is also of interest to investigate whether any modifications occur on the RAS proteins and which form regulates adenylate cyclase in the membranes. Many membraneous proteins are known to undergo various modifications. In the case ofp21raS protein of Harvey murine sarcoma virus, which is localized at the inner surface of the plasma membrane, detection of a soluble precursor pro-p21, conversion of pro-p21 to p21, and phosphorylation of p21 have been reported (13-15). Furthermore, the p21ras protein is modified by fatty acid acylation (13-17), which appears to be important for the transforming activity of the protein (14, 15). However, it is unclear whether the fatty acylation is responsible for the conversion of pro-p21 to p21 or whether a modification distinct from the fatty acylation is taking pla...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae.

Fujiyama¹,

Tamanoi²

1986

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

100

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“…Among these proteins, the myelin basic proteins (MBPs),' hydrophobic proteolipid proteins (PLPs), myelin-associated glycoproteins (MAGs), and 2',3'-cyclic nucleotide 3'-phosphorylase (CNP) have been identified (3)(4)(5) and their cDNAs isolated (6)(7)(8)(9). MBPs account for 30-40% of total protein in myelin sheaths and range in size from 14 to 21.5 kD (10,11). These forms are produced from a single primary transcript from a single MBP gene through alternative patterns of RNA splicing (6)(7)(8)(9)(12)(13)(14)(15).…”

Section: Introductionmentioning

confidence: 99%

Nuclear proteins in mouse brain cells bind specifically to the myelin basic protein regulatory region.

Lashgari

Devine-Beach²,

Haas³

et al. 1990

J. Clin. Invest.

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Expression of myelin basic protein (MBP) in mice is regulated in a cell-and stage-specific manner during brain development. The MBP control region contains multiple cis-acting elements, shown by in vivo and in vitro assays, which are responsible for its unique pattern of transcription. Using synthetic DNA fragments spanning the MBP control region, we have analyzed nuclear proteins obtained from newborn (2-3 d),. young adult (18-30 d), and adult (60 d) animals; these nuclear proteins form DNA-protein complexes with the MBP regulatory region. Brain extracts from young adult and adult mice showed enhanced binding activities with the sequences supporting transcriptional activation in glial cells.

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“…Protein fatty-acid acylation in eukaryotes is a well-established modification of specific cellular and viral proteins (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). However, the function of protein acylation is only beginning to be understood.…”

mentioning

confidence: 99%

Protein fatty acid acylation: enzymatic synthesis of an N-myristoylglycyl peptide.

Towler¹,

Glaser²

1986

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

117

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(26)(27)(28)(29). In all cases, the enzymatic activity is membraneassociated, and little specificity for fatty-acid chain length has been demonstrated. In contrast, the enzymatic activity(s) responsible for myristoylation of proteins at NH2-terminal glycine residues has not been identified. We report here that Saccharomyces cerevisiae contains proteins with NH2-terminal myristoylglycine residues. We describe an enzymatic activity that specifically transfers myristic acid to the aminoterminal glycine residue of the synthetic peptide Gly-AsnAla-Ala-Ala-Ala-Arg-Arg. GBq) of 3H-labeled fatty acid, cooled for 5 min on ice, and cells were pelleted at 4°C by centrifugation at 7600 x g for 10 min. Cell pellets were washed, centrifuged with 1 ml of 10 mM NaN3 in 140 mM NaCl/10 mM phosphate, pH 7.2, then suspended in 100 ,ul of 5 mM Tris-HCl, pH 7.4/3 mM dithiothreitol/1% NaDodSO4/1 mM phenylmethylsulfonyl fluoride. Cells were broken with one cell volume equivalent of 0.5-mm glass beads by six 30-sec spurts of vigorous mixing in a Vortex, cooling on ice between each mixing. Debris was removed by centrifugation for 30 sec at 8000 x g in a tabletop Eppendorf centrifuge. The supernatant was then alkylated with 125 ,ul of 20 mM iodoacetamide in 8 mM Tris HCl (pH 8.0) for 1 hr at room temperature. Twenty-microliter aliquots were analyzed by NaDodSO4/polyacrylamide gel electrophoresis and fluorography (9). MATERIALS AND METHODSAnalysis of the Linkage of 3H-Labeled Fatty Acids to Proteins. Twenty microliters of reduced and alkylated 3H-labeled fatty acid-labeled yeast protein was treated for 4 hr at 23°C with 7 Azl offreshly prepared 4 M hydroxylamine/20 mM glycine, pH 10, followed by electrophoresis and fluorography.To determine the hydroxylamine-stable linkage of[3H]myristic acid to the 20-kDa acyl protein in JR153, the cultures were labeled as described above except that the cells were treated for 15 min prior to addition of fatty acid with cerulenin at 2 ug/ml, a known inhibitor of yeast fatty-acid synthesis (31) that enhances the labeling of the specific acyl proteins in JR153 severalfold (data not shown). After separation of protein by NaDodSO4/12% polyacrylamide gel electrophoresis, gel slices were cut from the undried gel sample lanes through the 20-kDa region, rinsed once rapidly with 0.5 ml of 10% methanol in water, then individually *To whom reprint requests should be addressed. 2812The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Phosphorylation in vivo of four basic proteins of rat brain myelin

Cited by 54 publications

References 33 publications

Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae.

Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae.

Nuclear proteins in mouse brain cells bind specifically to the myelin basic protein regulatory region.

Protein fatty acid acylation: enzymatic synthesis of an N-myristoylglycyl peptide.

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