Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M513053200 on February 21, 2006

J. Biol. Chem., Vol. 281, Issue 16, 11115-11125, April 21, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
281/16/11115    most recent
M513053200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ostareck-Lederer, A.
Right arrow Articles by Wahle, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ostareck-Lederer, A.
Right arrow Articles by Wahle, E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Asymmetric Arginine Dimethylation of Heterogeneous Nuclear Ribonucleoprotein K by Protein-arginine Methyltransferase 1 Inhibits Its Interaction with c-Src*Formula

Antje Ostareck-Lederer{ddagger}1, Dirk H. Ostareck{ddagger}, Karl P. Rucknagel§, Angelika Schierhorn{ddagger}, Bodo Moritz{ddagger}, Stefan Huttelmaier, Nadine Flach{ddagger}, Lusy Handoko||, and Elmar Wahle{ddagger}2

From the {ddagger}Institute of Biochemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle (Saale), the §Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), ZAMED, Martin-Luther-University Halle-Wittenberg, Heinrich Damerow Strasse 1, 06120 Halle (Saale), and the ||Institute of Biochemistry, Biocenter at the University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany

Received for publication, December 7, 2005 , and in revised form, February 2, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Arginine methylation is a post-translational modification found in many RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) from HeLa cells was shown, by mass spectrometry and Edman degradation, to contain asymmetric NG,NG-dimethylarginine at five positions in its amino acid sequence (Arg256, Arg258, Arg268, Arg296, and Arg299). Whereas these five residues were quantitatively modified, Arg303 was asymmetrically dimethylated in <33% of hnRNP K and Arg287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNA-binding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Arginine dimethylation is a common post-translational modification in eukaryotes (1–5). The enzymes responsible for this modification are the protein-arginine methyltransferases (PRMTs).3 They are classified in two groups (2). Type I enzymes promote the formation of asymmetric NG,NG-dimethylated arginine. The known mammalian type I enzymes are PRMT1, PRMT2, PRMT3, PRMT4/coactivator-associated arginine methyltransferase 1, PRMT6, and the recently discovered brain-specific PRMT8 (6–11). Type II enzymes catalyze the symmetric NG,N'G-dimethylation of arginine residues. PRMT5 and PRMT7 are the mammalian type II enzymes described so far (12–14). PRMT1, which is predominantly localized to the cytoplasm (15), is thought to account for the generation of ~85% of asymmetric dimethylarginine residues (16) and is required for early post-implantation development in mice but not for the viability of embryonic stem cells (17). Arginine methylation affects several cellular processes, including histone function and transcriptional regulation (18–22), RNA processing (23–25), signal transduction (26–29), and intracellular localization of proteins (30–35).

Many proteins involved in RNA metabolism like hnRNP A1 (36), hnRNP A2 (37), Sam68 (38), and SAF-A (hnRNP U) (39) contain regions with clustered arginine residues in Arg-Gly-Gly motifs (RGG box) or RG repeats. These arginine residues are typically asymmetrically dimethylated. In addition, asymmetric arginine dimethylation has also been found in clustered RXR motifs (40) and other sequences. Therefore, the prediction of a methylated arginine is difficult. Furthermore, the enzyme responsible for the dimethylation of a particular protein is unknown in many cases, and the substrate specificities of the different methyltransferases remain poorly characterized. The exact knowledge of the methylated arginine residues and their quantitative distribution as well as the identification of the relevant methyltransferases is a prerequisite for the functional analysis of arginine methylation.

HnRNP K belongs to the family of heterogeneous nuclear RNPs that participate in the processing of pre-mRNAs and in the export of mRNAs from the nucleus. An N-terminal bipartite nuclear-localization signal and an hnRNP K-specific nuclear shuttling signal confer the capacity for bi-directional transport across the nuclear envelop (41, 42). The cytoplasmic accumulation of hnRNP K is mediated by its Erk-dependent serine phosphorylation (43). In the cytoplasm hnRNP K functions in the post-transcriptional regulation of gene expression.

For example, hnRNP K, together with hnRNP E1 and hnRNP E2, regulates human papilloma virus type 16 L2 capsid protein synthesis in the course of cellular differentiation by binding to the L2 mRNA 3'-untranslated region (44). Binding of hnRNP K, hnRNP E1, and hnRNP E2 to the internal ribosome entry site of the c-myc mRNA enhances the translation of the c-myc proto-oncogene (45). Furthermore, hnRNP K is involved in the regulation of reticulocyte 15-lipoxygenase (r15-LOX) mRNA translation. r15-LOX, a key enzyme in erythroid cell differentiation, participates in the breakdown of mitochondria in mature reticulocytes, which is a prerequisite for erythrocyte formation. Its premature expression in erythroid precursor cells is temporally restricted by translational silencing, mediated by hnRNP K and hnRNP E1 binding, individually or together, to the differentiation control element (DICE) in the r15-LOX mRNA 3'-untranslated region. The hnRNP K/E1-DICE complex blocks 80 S ribosome assembly (46, 47). The silencing mechanism can be recapitulated in vitro in rabbit reticulocyte lysate or wheat germ extract and in transfected HeLa cells (46, 47).

Interestingly, hnRNP K has been found to bind and to activate the tyrosine kinase c-Src specifically. Activated c-Src, in turn, phosphorylates hnRNP K, which leads to an inhibition of its DICE-binding activity and abolishes inhibition of mRNA translation by hnRNP K. In contrast, hnRNP E1 is neither an activator nor a substrate of c-Src (48). HnRNP K contains three proline-rich domains, allowing an interaction with the Src homology domain 3 (SH3) of c-Src (49–51). In addition, hnRNP K bears five clustered Arg-Gly-Gly (RGG) motifs within the proline-rich domains (52). HnRNP K, like other hnRNP proteins, is methylated in HeLa cells and lymphoblastoid cells (53, 54), but neither the methylation sites nor the specific methyltransferase or the functional significance of arginine methylation are known.

In this report, we have identified the methylated arginine residues in hnRNP K by mass spectrometry and Edman sequencing and quantitatively determined the methylation state. We provide biochemical and genetic evidence that PRMT1 is the only methyltransferase that methylates hnRNP K in vitro and in vivo. An hnRNP K variant in which all completely modified arginines were substituted is neither methylated by recombinant PRMT1 nor in HeLa cells. Methylation of arginine residues in hnRNP K did not influence its DICE-binding activity, its translation inhibitory function, or its cellular localization, but reduced its interaction with c-Src. This led to an inhibition of hnRNP K-dependent c-Src activation and a reduced hnRNP K phosphorylation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—pET16b-hnRNP K and pET16b-hnRNP E1 have been described in Ref. 46 and pSG5 His-hnRNP K and pSGT c-Src in Ref. 48. Arginine to glycine mutants (hnRNP K 5RG) were generated by site-directed mutagenesis (Stratagene). GFP-hnRNP K or -hnRNP K 5RG were generated using the plasmid pEGFP-C1 (Clontech). His6-PABPN1 and His6-PABPN1{Delta}C49 are described in Ref. 55.

The plasmids coding for GST-PRMT1 and pCDNA-PRMT1 (38) were a kind gift from Stéphane Richard, McGill University, Montreal, Canada. The plasmid pET28b-PRMT1 (56) was a kind gift from X. Cheng, Emory University, Atlanta, GA. Plasmids encoding GST-GAR (2) and GST-PRMT2 were a kind gift from Mark T. Bedford, University of Texas, Smithville, TX. Primers used to PCR-amplify the human PRMT3 cDNA, cloned into SalI/NotI of pGEX 5X1 (Amersham Biosciences) are listed in the supplemental materials. The full-length human cDNAs encoding PRMT4, PRMT5, and PRMT6 were PCR-amplified (primers in the supplement) and inserted into pGEX-6P-1 (Amersham Biosciences). pET28a-PRMT3 was generated by PCR and subcloned into pET28a. The leukemia inhibitory factor (LIF) was expressed as GST-LIF from the plasmid pGEX-2T-LIF (57), a kind gift from John K. Heath, University of Birmingham, UK.

Recombinant Proteins—His-hnRNP K, His-hnRNP K 5RG, and His-hnRNP E1 were prepared as described (46) except that the proteins were dialyzed against 100 mM potassium acetate, 20 mM Hepes, pH 7.4, 5% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 7 mM beta-mercaptoethanol. Expression and purification of recombinant His-PABPN1 and His-PABPN1{Delta}C49 was described in Ref. 55. His-PABPN1 was further purified by Mono-S fast protein liquid chromatography (Amersham Biosciences). His-PRMT1 and His-PRMT3 were prepared as described for His-hnRNP E1 (46). GST fusion proteins were expressed and purified as described for the pGEX system (Amersham Biosciences). GST-GAR and GST-PRMT1, His-PRMT1 and His-PRMT3 used in the in vitro methylation assay were dialyzed against 150 mM potassium acetate, 20 mM Hepes, pH 7.4, 5% glycerol, 1 mM EDTA, 1 mM dithiothreitol. Protein concentrations were determined by the Bradford assay, SDS-PAGE, and Coomassie staining using bovine serum albumin as a standard. GST-LIF was expressed as described in a previous study (57). SWISSPROT accession numbers for the expressed PRMTs are: PRMT1: Q63009 [GenBank] ; PRMT2: P55345 [GenBank] ; PRMT3: O60678 [GenBank] ; PRMT4: Q9NR22; and PRMT6: Q96LA8.

Antibodies—Anti-PRMT1 antibody was a kind gift from Stéphane Richard, McGill University, Montreal, Canada. PRMT2, PRMT4, and PRMT6 antibodies were raised against GST-tagged proteins. PRMT3 and PRMT5 antibodies were generated against GST-tagged proteins and affinity-purified using the respective His-tagged proteins immobilized on CnBr-activated Sepharose beads (Amersham Biosciences).

Immunopurification and Identification of hnRNP K—HnRNP K was precipitated from 1.7 mg of cytosolic HeLa cell extract prepared according to a previous study (58) (a kind gift from Reinhard Lührmann, Max Planck Institute, Göttingen, Germany) with 50 µg of a polyclonal hnRNP K antibody (Matritech R20475 [GenBank] ) using 200 µl of protein-A Sepharose CL-4B (Amersham Biosciences). Precipitated protein was eluted from affinity beads with a small volume of formic acid at 4 °C. The solution was diluted to 5% formic acid, and hnRNP K was purified by HPLC on a Nucleosil 500-5 C3-PPN column (150 x 2 mm, Macherey-Nagel) equilibrated with 0.09% trifluoroacetic acid and eluted by a 30–60% solvent B (0.08% TFA in acetonitrile) gradient over 28 min with a flow rate of 0.2 ml/min at 40 °C. HnRNP K was identified in column fractions by SDS-PAGE and Western blot analysis as well as peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) following digestion with proteinase Lys-C. An aliquot was submitted to N-terminal Edman degradation.

Purification of hnRNP K from HeLa Cell Extract—Cytosolic HeLa cell extract (58) was centrifuged for 30 min at 20,000 x g, and the supernatant was loaded onto a DEAE-Sepharose column (10 mg of protein per ml column volume) equilibrated in 50 mM Tris, pH 7.4, 50 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol (buffer A). Proteins were eluted by a gradient of 50 mM to 500 mM KCl in buffer A (10 column volumes). Fractions were assayed for PRMT activity as described below with 500 ng of recombinant hnRNP K as a substrate. Fractions containing hnRNP K were pooled, dialyzed against buffer B (50 mM Hepes, pH 7.4, 50 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol), and applied to a Macroprep-S column (7 mg of protein per ml column volume). The column was washed with one volume of buffer B, and proteins were eluted by a gradient of 50 mM to 500 mM KCl in buffer B (10 column volumes). Fractions containing hnRNP K were pooled, dialyzed against buffer B, and applied to a Heparin-Sepharose column (2 mg of protein per ml column volume). The column was washed with 2.5 volumes of buffer B, and proteins were eluted by a gradient of 50 mM to 1 M KCl in buffer B (10 column volumes). Fractions containing hnRNP K were further purified by reversed-phase HPLC as above. The protein in HPLC fractions was dried under a nitrogen stream, alkylated with vinylpyridine as described (59), and desalted by HPLC. An aliquot was used for MALDI-TOF MS and electrospray ionization (ESI) mass spectrometry.

In Vitro Methylation Assay—Reaction mixture (25µl volume): 1 nmol of [S-14C]adenosylmethionine (60 mCi/mmol, Amersham Biosciences), 50 mM Hepes, pH 8.0, 40 mM potassium acetate, 0.2 mg/ml bovine serum albumin, 0.01% Nonidet P-40, 10% glycerol, 1 mM EDTA, 0.5 mM dithiothreitol, and protein as indicated, incubated at 30 °C for 2 h. After incubation the reaction was split: 11 µl were added to 500 µl of bovine serum albumin (50 µg/ml), followed by the addition of 500 µl of 20% trichloroacetic acid. The mixture was incubated for 30 min on ice and filtered through Glass Microfibre Filters GF/C (Whatman). Filters were washed with 4 ml of ice-cold trichloroacetic acid (10%) and 4 ml of ice-cold ethanol (96%), air dried, and added to 3 ml of scintillation mix (Lumasafe Plus). Precipitated radioactivity was measured in a Liquid Scintillation Analyzer (Tri-Carb 2100PR, Packard). The remaining volume was subjected to SDS-PAGE and autoradiography.

In Vitro Transcription and Northwestern Analysis—In vitro transcription and Northwestern blot assays were carried out as described previously (48).

ES Cell Culture—ES(+/+) and ES(-/-) cells (17) obtained from Earl H. Ruley, Vanderbilt University, Nashville, TN, were cultured on 0.2% gelatinized cell culture plates in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, L-glutamine, nonessential amino acids, penicillin/streptomycin (all Invitrogen), and 0.5 ng/ml GST-LIF. For the in vitro methylation assay, 1 x 106 cells were lysed in 0.5 ml of lysis buffer (300 mM NaCl, 1% Triton, 50 mM Tris, pH 7.4, 10 µg/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride) and centrifuged for 5 min at 20,800 x g to obtain a cytoplasmic extract. Metabolic labeling of ES(+/+) and ES(-/-) cells was performed in methionine-free Dulbecco's modified Eagle's medium supplemented with 10 µCi L-[methyl-3H]methionine (70–85 Ci/mmol, Amersham Biosciences) per ml for 3 h in the presence of cycloheximide and chloramphenicol as described previously (53). Cells were lysed as above. For immunoprecipitation, anti-hnRNP K antibody (Matritech R20475 [GenBank] ) was used. Precipitated 3H-labeled proteins were subjected to SDS-PAGE and fluorography.

HeLa Cell Transfection and Analysis—HeLa cells were transiently transfected by the calcium phosphate method (60). For the experiment shown in Fig. 7B, 5 µg of GFP-hnRNP K or GFP-hnRNP K 5RG was cotransfected with 5 µg of pcDNA or PRMT1. For the experiments shown in Fig. 8, 5 µg of cDNAs coding for pSG5 His-hnRNP K or His-hnRNP K 5RG was cotransfected with 5 µg of pSGT-cSrc or pcDNA PRMT1 or empty vector (pSGT or pcDNA, respectively) as indicated. HeLa cell lysate preparation and immunoprecipitation was performed as described (48) with an anti-His antibody (H5, Qiagen). Antibodies against His and p-Tyr (Santa Cruz Biotechnology), c-Src (Abcam), and PRMT1 were used (Fig. 8).

Immunofluorescence—For single or double immunofluorescence, cells were essentially processed as described (61). An E600 (Nikon) microscope equipped with a digital camera (Hamamatsu) was used for conventional fluorescence microscopy. Images were acquired by Lucia software (Nikon) and compiled with Adobe Photoshop.

Enzymatic Digestion of hnRNP K—Sequencing grade proteases were obtained from Roche Applied Science. Lys-C digestion of alkylated hnRNP K was performed in 50 µl of 25 mM Tris-HCl, pH 8.5, with a Lys-C to hnRNP K ratio of 1:200 (w/w) overnight at 37 °C. The digest was separated by HPLC as above with a 0–40% solvent B gradient over 60 min followed by a 40–60% solvent B gradient over 20 min and a flow rate of 0.2 ml/min at 40 °C. Fractions were dried under a nitrogen stream and analyzed by MALDI-TOF MS or ESI MS. Fragments are labeled Lys-C1–23 in the order of theoretical cleavage sites (Table 1). Secondary tryptic digestion was carried out on Lys-C17 in 25 mM NH4HCO3, pH 8.5, with a trypsin to peptide ratio of 1:20 (w/w) overnight at 37 °C. An aliquot of the complete digest was analyzed directly by MALDI-TOF MS. The remaining digest was separated by HPLC like the Lys-C digest above. Early eluting fractions (retention time, <16 min) were combined, dried under a stream of nitrogen, and rechromatographed on a Nucleosil 500-5 C18-PPN column (150 x 2 mm, Macherey-Nagel) using parameters as above. Peptide-containing fractions were analyzed by MALDI or ESI MS. Tryptic fragments are labeled Tryp1–21 in the order of theoretical cleavage sites in the unmodified sequence of Lys-C17 (Table 1). Missed cleavages are assigned by a `/' symbol between numbers. Carboxypeptidase B type I diisopropyl fluorophosphate was purchased as a frozen solution from Sigma. 1 µl of sample was incubated with 0.4 µl of a 1:1000 dilution (50 mM NH4HCO3 buffer) of the carboxypeptidase B stock solution for 1 h at room temperature.


View this table:
[in this window]
[in a new window]
  		TABLE 1
Origin, assignment, and mass of hnRNP K proteolytic digestion fragments If not otherwise stated, masses were determined by MALDI-MS. Lys-C17: The numbers proximate to the observed mass [M+H]+ indicate the mass ratio of the peptides.

 
Mass Spectrometry—Peptide and protein masses were determined with a Q-TOF 2 mass spectrometer (Waters) with an electrospray source or an Ultraflex-II TOF-TOF mass spectrometer (Bruker Daltonic) with MALDI source. The Q-TOF 2 was equipped with a modified nano-ESI source to hold a pico-tip (New Objective). Samples were injected with a syringe pump (Harvard Apparatus) with a flow rate of 300 nl/min. The collision energy for MS/MS experiments varied between 25 and 35 eV. The MaxEntTM1 or MaxEntTM3 algorithms were used for deconvoluting the data for single charge state and de-isotoping. For MALDI-TOF MS, 0.5µl of a saturated solution of{alpha}-cyano-4-hydroxycinnamic acid in acetone was deposited onto the target. 1 µl of the peptide sample was mixed with 1 µlof {alpha}-cyano-4-hydroxycinnamic acid (saturated solution in acetonitrile/0.1% trifluoroacetic acid (2:1 v/v)) and spotted onto the matrix surface. Protein analysis was carried out using a saturated solution of sinapinic acid (30:20:50 acetonitrile:methanol:0.1% trifluoroacetic acid, v/v/v) as a matrix. The software Flex Control 2.4, Flex Analysis 2.4, and Biotools 3.0 were used to operate the mass spectrometer and analyze the data. For external calibration a peptide calibration mixture (Bruker Daltonik) was used. The nomenclature of Roepstorff and Biemann was used for the fragment ion notation (62, 63).

Amino Acid Sequencing—Edman degradation was performed with an Applied Biosystems Procise cLC sequencer. Monomethylated and symmetrically or asymmetrically dimethylated arginine were applied directly to the sequencing filter, and after one cycle of sequencing the phenylthiohydantoin derivatives served as standards in the HPLC detection system.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Location of Modified Arginine Residues in hnRNP K Purified from HeLa Cell Extract—Arginine methylation of hnRNP K has been reported earlier (53, 54). To map the methylated arginines in hnRNP K, we first used hnRNP K immunopurified from HeLa cells for an initial analysis to verify the protein identity by mass spectrometry and to establish HPLC purification conditions. Next, we purified the protein from cytoplasmic HeLa cell extract by DEAE-Sepharose, Macroprep-S, and heparin-Sepharose chromatographies and reversed-phase HPLC. The protein was then alkylated with vinylpyridine and digested with proteinase Lys-C. The peptides generated were separated by reversed-phase HPLC and subsequently analyzed by sequencing and MALDI-TOF MS or ESI MS. Lys-C fragments that could be assigned to the hnRNP K sequence were labeled Lys-C1–23 in the order of theoretical cleavage sites. The masses of the isolated peptides were compared with the masses predicted from the cDNA sequence. All peptides with a predicted mass above 750 Da could be identified (Table 1). Of the 23 Lys-C fragments only Lys-C17 contained methylated arginine residues. The masses of all other peptides, whether they contained arginine residues or not, corresponded closely to those predicted for unmodified peptides (Table 1 and supplementary information). The only exception was the N-terminal fragment Lys-C1, which does not contain an arginine. The difference of 42 mass units between observed and predicted mass of Lys-C1 resulted from N-terminal acetylation of Met1. This was confirmed by MS/MS experiments.


Figure 1
View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 1.
Amino acid sequence of human hnRNP K (SWISSPROT accession number: P61978 [GenBank] ). Completely asymmetrically dimethylated arginine residues 256, 258, 268, 296, and 299 (bold, gray background) in hnRNP K, purified from cytoplasmic HeLa cell extract, were identified by MS/MS and Edman sequencing. Partially asymmetrically dimethylated Arg303 (33%) (bold) and partially monomethylated Arg287 (10%) (bold) were found only in a fraction of peptides analyzed. Proline-rich domains P1 (265–278), P2 (285–297), and P3 (303–318) are underlined.

 


Figure 2
View larger version (62K):
[in this window]
[in a new window]
  		FIGURE 2.
Specific arginine methylation of hnRNP K in vitro. A, Coomassie stain of recombinant proteins used in the methylation assay resolved on 12% SDS-PAGE. B, 500 ng of each polypeptide shown in A was methylated in vitro using [14C]SAM and cytoplasmic HeLa cell extract, followed by 12% SDS-PAGE and autoradiography. Methylated PABPN1 (lane 1) and hnRNP K (lane 3) are indicated.

 
Lys-C17 contained 23 of the 36 arginine residues in hnRNP K. This peptide was digested with trypsin. The digestion products were separated by reversed-phase HPLC and subsequently analyzed by sequencing and MALDI-TOF MS or ESI MS. Tryptic fragments were labeled Tryp1–21 in the order of theoretical cleavage sites within the fragment Lys-C17 (Table 1; for details see supplementary information). Only peptides Tryp1 (Gly220-Arg221) and Tryp9 (Arg278) were not detected. Because the flanking fragments Tryp2 and Tryp10 were found and trypsin in our hands only cleaves unmodified Arg-X bonds, we concluded that Arg221 and Arg278 were not modified. This was consistent with our data using an hnRNP K arginine substitution variant (see below). The arginine residues at position 256, 258, 268, 296, and 299 were quantitatively asymmetrically dimethylated. In addition, Arg303 was asymmetrically dimethylated in less than 33% of hnRNP K, and Arg287 was monomethylated in <10% of the protein (for details see supplementary information). All other arginine residues were unmethylated. In summary, the methylations demonstrated in the tryptic digest of Lys-C17 almost perfectly explained the observed mass of this fragment. Of all the arginine residues present in hnRNP K only Arg409 escaped our analysis. However, the lack of arginine methylation of an hnRNP K variant in which the identified five quantitatively asymmetrically dimethylated arginine residues (arginines 256, 258, 268, 296, and 299) were substituted by glycine (hnRNP K 5RG) (see data below), makes Arg409 an unlikely target of arginine methyltransferases. ESI MS measurements of the complete alkylated hnRNP K were obscured by the tendency of the protein to form adducts, as also observed with the fragment Lys-C17. However, the smallest detectable mass was 51682.0 Da. This is in good agreement with the calculated value of 51683.9 Da for hnRNP K containing five dimethylarginine residues, as detected in the peptide analysis (Fig. 1).

HnRNP K Can Be Methylated in Vitro—To identify the relevant PRMT(s), we developed an in vitro methylation system: cytoplasmic HeLa cell extract was incubated with recombinant hnRNP K, which is not methylated in Escherichia coli (Fig. 2A, lane 3). S-[14C]Adenosylmethionine was used as the methyl donor, and PABPN1, which carries thirteen asymmetrically dimethylated arginines in the C terminus, as a positive control (40) (Fig. 2A, lane 1). A deletion mutant lacking the C-terminal 49 amino acids (PABPN1{Delta}C49), the hnRNP K substitution variant hnRNP K 5RG and hnRNP E1, which does not contain RGG boxes, were used as negative controls (Fig. 2A, lanes 2, 4, and 5). One part of the reaction was used for trichloroacetic acid precipitation and scintillation counting; the second part was analyzed by SDS-PAGE (Fig. 2B). Both PABPN1 and hnRNP K were methylated in HeLa cell extract (Fig. 2B, lanes 1 and 3), whereas PABPN1{Delta}C49, hnRNP K 5RG, and hnRNP E1 were not methylated (Fig. 2B, lanes 2, 4, and 5). Methylated bands appearing at 40 kDa when hnRNP K served as a substrate were identified by mass spectrometry as C-terminally truncated hnRNP K fragments, still containing the methylation sites. hnRNP K was also methylated in rabbit reticulocyte lysate, whereas hnRNP K 5RG was not (data not shown).

Identification of the Relevant PRMTs—Cytoplasmic HeLa cell extract was fractionated by DEAE-Sepharose. Each fraction was tested for methylation activity by the addition of recombinant hnRNP K as a substrate and S-[14C]adenosylmethionine as a methyl donor. The distribution of type I PRMTs was analyzed in Western blot assays using antibodies against PRMT1, -2, -3, -4, and -6 (Fig. 3). Brain-specific PRMT8 (11) was not tested. The purification revealed a major hnRNP K methylation activity peak (Fig. 3A, fractions 10–16), which corresponded to PRMT1, but also contained PRMT3. The trailing end of methylation activity (fractions 20–23) coincided with a second peak of PRMT1 (Fig. 3B). PRMT2 and -4 were detected in the flow-through, which did not show hnRNP K methylation activity (data not shown). PRMT6 was eluted in fractions 15–19 (Fig. 3B). The experiment suggested that PRMT1, PRMT3, and PRMT6 might contribute to the methylation of hnRNP K and that PRMT2 and PRMT4 do not.


Figure 3
View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 3.
In vitro methylation of hnRNP K and distribution of PRMTs in DEAE-Sepharose-fractionated cytoplasmic HeLa cell extract. A, in vitro methylation ([14C]SAM incorporation) of recombinant hnRNP K in cytoplasmic HeLa cell extract (input) and eluted DEAE-Sepharose fractions (1–35). The position of methylated hnRNP K is indicated (left lane). Methylated proteins were visualized by autoradiography. B, distribution of PRMT1, -2, -3, -4, and -6 in DEAE fractions 1–35. The PRMTs were detected by specific antibodies. For each individual PRMT the position is indicated in cytoplasmic HeLa cell extract (left lane).

 
PRMT1 and PRMT3 were expressed as His tag fusion proteins and analyzed for methylation activity in vitro (Fig. 4). PABPN1, which is methylated by PRMT1 and PRMT3 in vitro served as a positive control (40). HnRNP K was methylated by PRMT1 (Fig. 4A, lanes 4–8) but not by PRMT3 (below 2% of the signal for hnRNP K methylated by PRMT1, lanes 10–14), whereas both enzymes showed PABPN1 methylation activity (Fig. 4B, lanes 4–8 and 10–14; methylation efficiency of PRMT3 was 25% of that of PRMT1). In addition, GST-PRMT6 was tested for methylation of hnRNP K and the artificial substrate GST-GAR (10). GST-GAR, but not hnRNP K was methylated by PRMT6 (data not shown). These data strongly indicate that the recombinant enzymes display substrate specificity. We conclude that hnRNP K is specifically methylated by PRMT1 in vitro.

Characterization of PRMT1 in Vitro—As estimated by Western blot assay, 44 µg of cytoplasmic HeLa cell protein contained ~100 ng (=2.3 pmol) of PRMT1 (data not shown) and had a methylation activity of 16.25 pmol/2 h under standard reaction conditions. With 2.3 pmol of His-tagged recombinant PRMT1, a methyl incorporation of 42 pmol/2 h was detected (Fig. 4C). Because the methylation activity of His-PRMT1 was in excess compared with the incorporation observed by the equivalent amount of HeLa cell extract, it is unlikely that PRMT1 activity depends on additional cellular factors.

Interestingly, when we compared the methylation activity of His-PRMT1 and GST-PRMT1 in titration experiments in the presence of 9 pmol of hnRNP K, we found a 35-fold higher activity at low concentrations of His-PRMT1 compared with GST-PRMT1, indicating that the GST tag disturbs the methyltransferase activity (Fig. 4C).

Titration of the substrate protein hnRNP K in the presence of 40 µM SAM revealed a Km of 0.25 µM and a kcat of 1/min. The dependence of the reaction rate on the concentration of SAM, measured in the presence of 0.72 µM hnRNP K, was complex and did not correspond to Michaelis-Menten kinetics (data not shown).

PRMT1 Methylates hnRNP K in Vivo—To provide a definitive proof for the specific methylation of hnRNP K by PRMT1, ES cells carrying a homozygous prmt1 gene null mutation (ES(-/-) cells) were used (17). In Western blot assays no PRMT1 was detected in ES(-/-) cells, whereas hnRNP K was present in both ES(+/+) and ES(-/-) cells (Fig. 5A, lanes 1 and 2). Recombinant hnRNP K was methylated in HeLa and ES(+/+) cell extract but not in ES(-/-) cell extract (Fig. 5B, lanes 1–6). Complementation of ES(-/-) cell extract with increasing amounts of His-PRMT1 resulted in the methylation of both added recombinant hnRNP K and endogenous hnRNP K, which was identified by immunoprecipitation, and other cellular proteins (Fig. 5B, lanes 7–12 and supplementary information Fig. S1). Efficient methylation of endogenous hnRNP K in ES(-/-) cell extracts after the addition of PRMT1 suggests that it was unmethylated and, therefore, that PRMT1 is responsible for its methylation in vivo. PRMT3 could not complement extracts from ES(-/-) cells (Fig. 5B, lanes 13–18), again demonstrating substrate specificity.

The role of PRMT1 in hnRNP K methylation in vivo was analyzed by metabolic labeling of ES(+/+) and ES(-/-) cells using L-[methyl-3H]methionine in the presence of the translation inhibitors cycloheximide and chloramphenicol (Fig. 5C). Labeling was followed by cell lysis and immunoprecipitation of endogenous hnRNP K (Fig. 5C, lanes 2 and 5). Endogenous hnRNP K was immunoprecipitated from both ES(+/+) and ES(-/-) cells (Fig. 5C, lanes 2 and 5), but it was methylated only in ES(+/+) and not in (-/-) cells (Fig. 5C, lanes 2 and 5). Coprecipitation of endogenous PRMT1 was detected in ES(+/+), but not (-/-) cells, whereas PRMT3, present in both ES(+/+) and ES(-/-) cells, was not copurified with hnRNP K (Fig. 5C, lanes 2 and 5). Other methyltransferases are still present in ES(-/-) cells: methylation activity was detectable (Fig. 5C, lane 4) and PRMT3 (Fig. 5C, lane 4) and PRMT6 (data not shown) were detected by Western blot assays. The background of the immunoprecipitation was reproducibly increased in the ES(-/-) cell lysate in a nonspecific, antibody-independent manner (see "Discussion"). Nevertheless, there was no discernible band of hnRNP K in the immunoprecipitation and no significant difference between the hnRNP K antiserum and the pre-immune serum (Fig. 5C, lanes 5 and 6). Taken together, the results show that PRMT1 is the only methyltransferase acting on hnRNP K.


Figure 4
View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 4.
HnRNP K is specifically methylated by PRMT1 in vitro. Arginine methylation of hnRNP K and PABPN1 by His-PRMT1 and His-PRMT3 was analyzed by incorporation of [14C]SAM in the in vitro methylation assay. Recombinant hnRNP K (9 pmol) (A) or PABPN1 (15 pmol) (B) was incubated with cytoplasmic HeLa cell extract (lane 2) or increasing amounts (1.4–23 pmol) of His-PRMT1 (lanes 4–8) or His-PRMT3 (lanes 10–14), respectively. No hnRNP K (A) or PABPN1 (B) were added in lanes 1, 3, and 9. Reaction products were resolved on a 12% SDS-PAGE followed by autoradiography. C, 9 pmol of recombinant hnRNP K was incubated with increasing amounts (0.27 fmol to 42 pmol) of GST- or His-PRMT1 and 1 nmol of [14C]SAM under standard conditions. The reaction was analyzed by trichloroacetic acid precipitation and scintillation measurement.

 
Methylation of hnRNP K Does Not Influence Its DICE-binding Activity—To examine whether PRMT1-dependent arginine methylation of hnRNP K affects its DICE-binding activity, recombinant hnRNP K or hnRNP K 5RG were incubated with His-PRMT1 or buffer. Complete methylation was detected for hnRNP K (90 pmol of methyl-14C incorporation in 9 pmol of hnRNP K), whereas hnRNP K 5RG was not methylated (<2.4 pmol of methyl-14C incorporation in 9 pmol of hnRNP K 5RG) (Fig. 6A, upper panel, lanes 1–4). Importantly, in a Northwestern blot with radioactively labeled DICE, non-methylated or completely methylated hnRNP K showed no difference in DICE binding activity, and the same DICE-binding activity was observed with hnRNP K 5RG (Fig. 6A, lower panel, lanes 1–4). To test whether endogenous hnRNP K behaves the same, hnRNP K was immunoprecipitated from ES(+/+) and ES(-/-) cells with an anti-hnRNP K antibody (Fig. 6B, upper panel, lanes 1–6). Again, the methylated and the non-methylated endogenous hnRNP K showed similar DICE-binding activity (Fig. 6B, lower panel, lanes 1–6). From these experiments we conclude that arginine methylation of hnRNP K does not influence its DICE-binding activity. This is consistent with the result of our previous experiments showing that equal amounts of recombinant hnRNP K and protein from methylation active reticulocyte lysate, both purified by RNA affinity chromatography, had a similar translation inhibitory activity (46).


Figure 5
View larger version (50K):
[in this window]
[in a new window]
  		FIGURE 5.
HnRNP K is methylated in ES(+/+) but not ES(-/-) cells. A, extracts from ES(+/+) and ES(-/-) cells were resolved on a 12% SDS-PAGE and analyzed in a Western blot assay using antibodies against hnRNP K and PRMT1 (lanes 1 and 2). B, in vitro methylation assay using cytoplasmic HeLa cell extract (lanes 1 and 2), ES(+/+) cell extract (lanes 3 and 4), or ES(-/-) cell extract (lanes 5–18) in the presence of [14C]SAM. Recombinant hnRNP K (9 pmol) was added as indicated. Increasing amounts (1.4–23 pmol) of His-PRMT1 (lanes 7–12) or His-PRMT3 (lanes 13–18), respectively, were added to the reactions in ES(-/-) cell extract (lanes 7–18). Proteins were resolved on a 12% SDS-PAGE, and methylation was detected by autoradiography. C, in vivo methylation reactions in ES(+/+) or (-/-) cells were performed for 3 h in the presence of L-[methyl-3H]methionine, cycloheximide, and chloramphenicol. Following cell lysis, endogenous hnRNP K was immunoprecipitated using a specific antibody (lanes 2 and 5) or preimmune serum (lanes 3 and 6). Cytoplasmic extract (lanes 1 and 4) or the precipitation products from ES(+/+)(lanes 2 and 3) or ES(-/-) cells (lanes 5 and 6) were resolved on a 12% SDS-PAGE and analyzed by Western blotting using antibodies against hnRNP K, PRMT1, and PRMT3 or fluorography.

 
Methylation of hnRNP K Does Not Influence Its Cellular Localization— Next we examined whether arginine methylation affects the cellular distribution of hnRNP K in ES cells. To determine the localization of hnRNP K in ES(+/+) and ES(-/-) cells, immunofluorescence with an anti-hnRNP K antibody was employed (Fig. 7A). In both, ES(+/+) and ES(-/-) cells, hnRNP K was clearly localized to the nucleus. In HeLa cells cotransfected with plasmids coding for GFP-hnRNP K or GFP-hnRNP K 5RG, with or without PRMT1, hnRNP K and hnRNP K 5RG were also localized to the nucleus independently of the presence of PRMT1 (Fig. 7B). This indicates that arginine methylation has no effect on the steady-state localization of hnRNP K.

Methylation of hnRNP K Reduces Its Ability to Activate c-Src in HeLa Cells—The following experiment was carried out to determine if arginine methylation of hnRNP K affects its interaction with c-Src. HeLa cells were cotransfected with plasmids coding for His-hnRNP K or His-hnRNP K 5RG, c-Src, and PRMT1. Protein expression was monitored by Western blot assays of total lysate using antibodies against the His-tag (His), PRMT1, c-Src, and glyceraldehyde-3-phosphate dehydrogenase as a loading control. Phosphorylation was detected with a phosphotyrosine-specific antibody (p-Tyr) (Fig. 8, A and B). As shown before (48), when His-hnRNP K was cotransfected with c-Src, the phosphorylation of cellular proteins was significantly increased, indicating that hnRNP K activates c-Src (Fig. 8 A, lanes 1 and 3). When His-hnRNP K was cotransfected with c-Src and PRMT1, tyrosine phosphorylation of cellular proteins was strongly reduced compared with His-hnRNP K cotransfected with c-Src in the absence of PRMT1 (Fig. 8A, lanes 3 and 5). These data indicate that hnRNP K-dependent activation of c-Src was impaired significantly in the presence of PRMT1. In contrast, when His-hnRNP K 5RG was cotransfected with either c-Src alone or c-Src and PRMT1, no difference in the phosphorylation of cellular proteins could be detected; i.e. c-Src was equally active (Fig. 8B, lanes 3 and 5). Because hnRNP K 5RG cannot be methylated (see Figs. 2 and 6A), the inhibitory activity of PRMT1 on hnRNP K-dependent c-Src activation is due to hnRNP K methylation. To test whether the interaction between hnRNP K and c-Src is affected in the presence of PRMT1, His-hnRNP K, or His-hnRNP K 5RG were immunoprecipitated with an anti His-antibody, followed by Western blot assays (Fig. 8, A and B). When His-hnRNP K and c-Src were cotransfected, hnRNP K interacted with c-Src and became tyrosine-phosphorylated (Fig. 8A, lane 2). Cotransfection of hnRNP K with c-Src and PRMT1 resulted in a reduced interaction of hnRNP K with c-Src, and tyrosine phosphorylation of hnRNP K was strongly diminished (Fig. 8A, lane 4). No differences could be detected when His-hnRNP K 5RG was cotransfected with either c-Src alone or c-Src and PRMT1 (Fig. 8B, lanes 2 and 4). This suggests that arginine methylation of hnRNP K diminishes the activation of c-Src by hnRNP K, which, in turn, leads to a reduced tyrosine phosphorylation of hnRNP K.


Figure 6
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 6.
Methylation of hnRNP K does not affect its DICE-binding activity. A, recombinant hnRNP K or hnRNP K 5RG (9 pmol) were incubated with [14C]SAM in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 2.3 pmol of His-PRMT1, followed by a 12% SDS-PAGE and autoradiography (upper panel). DICE-binding activity was examined with a 32P-labeled DICE probe in a Northwestern blot assay (lower panel). B, hnRNP K was immunoprecipitated from ES(+/+) (lane 2) or (-/-) (lane 5) cell extract by an hnRNP K-specific antibody. Preimmune serum was used in lanes 3 and 6. Cell extract (lanes 1 and 4) or the precipitation products from ES(+/+) (lanes 2 and 3) or (-/-) cells (lanes 5 and 6) were resolved on a 12% SDS-PAGE and analyzed by Western blotting (upper panel) or in a Northwestern blot assay with 32P-labeled DICE (lower panel).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In hnRNP K purified from cytoplasmic HeLa cell extract 5 of the 36 arginine residues were identified as 100% asymmetrically dimethylated (Arg256, Arg258, Arg268, Arg296, and Arg299). In addition, Arg303 was partially (33%) asymmetrically dimethylated and Arg287 was monomethylated to a low extent (10%). Dimethylated Arg296 and Arg299 have been detected earlier; however, the asymmetric state and the quantitative distribution of methylation were not described (64). An earlier comparison of 20 mapped sites of asymmetric dimethyl arginines revealed a preferred amino acid consensus: (G/F)GGRGG(G/F) with the C-terminal-flanking glycine considered obligatory (36). All completely dimethylated arginines identified here have either one (Arg256) or two (Arg258, Arg268, Arg296, or Arg299) C-terminal-flanking glycine residues. The partially monomethylated Arg287 is followed by a C-terminal flanking glycine and the partially dimethylated Arg303 by an alanine. It is conceivable that partial methylation occurs because these residues are in close proximity to a cluster of preferred substrate sites. Of all the 36 arginine residues present in hnRNP K, only Arg409 could not be analyzed. However, it is unlikely that this arginine is methylated, because the substitution variant hnRNP K 5RG was not methylated.

We identified PRMT1 as the only enzyme responsible for hnRNP K methylation in vitro using fractionation of HeLa cell extract, recombinant PRMTs, and extracts of ES cells with a homozygous deletion of the prmt1 gene (ES(-/-) cells). Metabolic labeling of ES(+/+) and ES(-/-) cells confirmed that specifically PRMT1 acts on hnRNP K in vivo. PRMT1 is the major methyltransferase catalyzing asymmetric arginine dimethylation in mammalian cells (16). The crystal structure of the PRMT1 core revealed a homodimeric structure. Dimerization is required for SAM binding and thus for catalytic activity (56). Dimers associate to larger oligomers (6, 56). We found that the activity of GST-PRMT1 is significantly reduced compared with His-PRMT1, possibly due to an interference of the GST tag with dimer or oligomer formation.


Figure 7
View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 7.
Arginine methylation of hnRNP K does not influence its cellular localization. A, nuclear localization of hnRNP K in ES(+/+) and ES(-/-) cells was determined by immunofluorescence using an hnRNP K antibody and a Cy3-coupled secondary antibody. 4',6-Diamidino-2-phenylindole (DAPI) and {alpha}-tubulin (fluorescein isothiocyanate) staining were used to determine the nuclear and cytoplasmic compartment. B, HeLa cells were cotransfected with GFP-hnRNP K or GFP-hnRNP K 5RG in the absence or presence of PRMT1. HnRNP K and hnRNP K 5RG were determined by GFP fluorescence (pseudocolored) and PRMT1 using a PRMT1 antibody and a Cy5-coupled secondary antibody. 4',6-Diamidino-2-phenylindole and a-tubulin (Cy3) staining were used to determine the nuclear and cytoplasmic compartment.

 
Type I PRMTs have not been purified to homogeneity so far, and it remains unknown whether the catalytic polypeptides are associated with additional subunits, as it has been found for the type II enzyme PRMT5 (24). However, the finding that hnRNP K was methylated with similar efficiencies in HeLa cell extract and by a comparable amount of His-PRMT1 strongly indicates that no additional cofactor or activator of PRMT1 is required for methylation of hnRNP K. We determined a Km of 0.25 µM for the methylation of hnRNP K by His-PRMT1. Previously, with a partially purified methyltransferase from rat liver, which probably contained mainly PRMT1, a Km of 0.54 µM was found for the methylation of hnRNP A1 (65).


Figure 8
View larger version (56K):
[in this window]
[in a new window]
  		FIGURE 8.
Arginine methylation of hnRNP K strongly reduces its interaction with c-Src. HeLa cells were transiently transfected with His-tagged hnRNP K (A) or His-tagged hnRNP K 5RG (B) and PRMT1, c-Src, or empty vector (pSGT or pcDNA, respectively) as indicated. HeLa cell total lysates (A and B, upper panel) were resolved on a 12% SDS-PAGE and analyzed by Western blot assays with either anti-His, anti-PRMT1, anti-Src, anti-phosphotyrosine (p-Tyr) or anti-glyceraldehyde-3-phosphate dehydrogenase antibodies. His-tagged hnRNP K or hnRNP K 5RG was immunoprecipitated with an anti-His antibody (A and B, lower panel), resolved on a 12% SDS-PAGE and analyzed by Western blot assays with antibodies against His, to assess the amounts of precipitated hnRNP K or hnRNP K 5RG, c-Src, and PRMT1 to visualize coprecipitation and p-Tyr to analyze the tyrosine phosphorylation status of hnRNP K or hnRNP K 5RG.

 
Arginine methylation of the yeast RNA-binding protein Npl3 has been shown to reduce its self-association (66). Interestingly, immunoprecipitation from the ES(-/-) cells showed a strong tendency of proteins to aggregate and/or nonspecifically associate with protein-A beads in a nonspecific, antibody-independent manner. Thus, arginine methylation may generally promote correct protein folding or increase solubility.

In contrast to protein-protein interaction, the interaction of RNA-binding proteins like mammalian PABPN1 and yeast Hrp1p with RNA is not affected by arginine methylation (55, 67). This is consistent with our results showing that arginine methylation of hnRNP K does not influence its DICE binding activity. Equal amounts of RNA affinity-purified hnRNP K from reticulocyte lysate, which contains PRMT1 and shows methylation activity (data not shown, this study), or expressed in E. coli had a similar translation inhibitory activity (46). The mammalian RNA-binding proteins hnRNP A2 and Sam68, both targets of PRMT1, accumulate in the cytoplasm in the presence of methylation inhibitors (37, 38). In ES(-/-) cells we could not observe an effect on hnRNP K localization. In contrast, serine phosphorylation by Erk drives the cytoplasmic accumulation of hnRNP K (43).

HnRNP K contains three proline-rich domains, which interact with the SH3 domain of c-Src (49–51), leading to c-Src activation and phosphorylation of hnRNP K (48). The five methylated arginines are located adjacent to the three proline-rich regions. As a result of hnRNP K arginine dimethylation, its association with c-Src is diminished, which leads to a reduced activation of c-Src and results in a reduced tyrosine phosphorylation of hnRNP K. The mechanism by which arginine dimethylation interferes with the binding of Pro-Arg-rich polypeptides to SH3 domains is unknown. In the case of the RNA-binding protein Sam68, arginine methylation of RG repeats within proline-rich domains selectively modulates the interaction with the SH3 domain of the tyrosine kinase Src-family tyrosine kinase, but not with the WW domain of forming binding protein 30 in vitro (68).

According to our current model, synthesis of r15-LOX, the enzyme which participates in mitochondria degradation, is repressed in early stages of erythroid differentiation. It becomes derepressed in mature reticulocytes upon activation of c-Src by hnRNP K and subsequent hnRNP K phosphorylation. Tyrosine phosphorylation of hnRNP K results in a loss of DICE-binding activity and translation activation (48, 69). The results of our study imply that arginine methylation of hnRNP K could prevent premature activation of c-Src and, thereby, may restrain tyrosine phosphorylation of hnRNP K until r15-LOX mRNA translation is required.

To date, there is no red blood cell line that can be induced to differentiate to the state where nuclear exclusion and degradation of mitochondria can be detected quantitatively, and thus translation control by hnRNP K cannot be recapitulated in an appropriate cell system at present. However, from the analysis of cytoplasmic extracts of rabbit erythroid cells, we know that hnRNP K, PRMT1, and c-Src are co-expressed at different maturation stages.4 Therefore, arginine methylation of hnRNP K by PRMT1 is likely to function as a modulator for the transition of c-Src from its kinase-inactive to the active state catalyzing tyrosine phosphorylation of hnRNP K.

At least three scenarios regarding the modulation of the hnRNP K-c-Src interaction can be envisaged as follows: Arginine methylation of hnRNP K can either be reversible, assuming that a demethylation activity exists (70–72). Alternatively, the pool of arginine-methylated hnRNP K could be exchanged by nonmethylated protein as shown for histone H3.3 (73, 74). Finally, it is possible that methylation of hnRNP K is constitutive in vivo and that it masks the protein until c-Src reaches the regulatory expression level during erythroid differentiation. Future experiments will further address the question how hnRNP K arginine methylation by PRMT1 and phosphorylation by c-Src are regulated.


   FOOTNOTES
 
* This work was supported in part by a Heisenberg fellowship of the Deutsche Forschungsgemeinschaft (DFG) (to A. O.-L.) and by the DFG (Grants Os 290/2-1 to A. O.-L., Os 135/2-1,2 to D. H. O. and Grant SFB 610 to E. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Ref. 1, and Fig. S1. Back

2 Supported by the Fonds der Chemischen Industrie. Back

1 To whom correspondence should be addressed. Tel.: 49-(0)345-552-4949; Fax: 49-(0)345-552-7014; E-mail: aostareck{at}biochemtech.uni-halle.de.

3 The abbreviations used are: PRMT, protein-arginine methyltransferase; hnRNP K, heterogeneous ribonucleoprotein K; ES, embryonic stem; Hrp1, nuclear polyadenylated RNA-binding protein 4 (Nap4p); Sam68, Src associated in mitosis 68-kDa protein; PABPN1, mammalian nuclear poly(A)-binding protein 1; r15-LOX, reticulocyte-15-lipoxygenase; DICE, differentiation control element; L2, protein L2 of human papillomavirus type 16; GST, glutathione S-transferase; LIF, leukemia inhibitory factor; GST-GAR, GST fusion protein containing the glycine- and arginine-rich N-terminal region of fibrillarin; HPLC, high pressure liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry; ESI MS, electrospray ionization-mass spectrometry; SAM, S-adenosylmethionine; SH3-domain, Src homology-3 domain; Src, tyrosine kinase of the Rous sarcoma virus; WW, denotes two conserved tryptophan residues within this domain; Erk, extracellular signal-regulated kinase; GFP, green fluorescent protein; MS/MS, tandem mass spectrometry. Back

4 A. Ostareck-Lederer and D. H. Ostareck, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank A. Haase for generating hnRNP K 5RG and M. T. Bedford, X. Cheng, J. K. Heath, R. Lührmann, S. Richard, and E. A. Ruley for providing reagents. We are grateful to U. Fischer and S. Richard for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bedford, M. T., and Richard, S. (2005) Mol. Cell 18, 263-272[CrossRef][Medline] [Order article via Infotrieve]
  2. Gary, J. D., and Clarke, S. (1998) Prog. Nucleic Acids Res. 61, 65-133[Medline] [Order article via Infotrieve]
  3. Jenuwein, T., and Allis, C. D. (2001) Science 293, 1074-1080[Abstract/Free Full Text]
  4. Lee, D. Y., Teyssier, C., Strahl, B. D., and Stallcup, M. R. (2004) Endocr. Rev. 2, 147-170
  5. McBride, A. E., and Silver, P. (2001) Cell 106, 5-8[CrossRef][Medline] [Order article via Infotrieve]
  6. Lin, W. J., Gary, J. D., Yang, M. C., Clarke, S., and Hershman, H. R. (1996) J. Biol. Chem. 271, 15034-15044[Abstract/Free Full Text]
  7. Qui, C., Chang, J., Zhu, Y., Yeldani, A. V., Rao, S. M., and Zhu, Y. J. (2002) J. Biol. Chem. 277, 28624-28630[Abstract/Free Full Text]
  8. Tang, J., Gary, J. D., Clarke, S., and Hershman, H. R. (1998) J. Biol. Chem. 273, 16935-16945[Abstract/Free Full Text]
  9. Schurter, B. T., Koh, S. S., Chen, D., Bunick, G. J., Harp, J. M., Hanson, B. L., Henschen-Edman, A., Mackay, D. R., Stallcup, M. R., and Aswad, D. W. (2001) Biochemistry 40, 5747-5756[CrossRef][Medline] [Order article via Infotrieve]
  10. Frankel, A., Yadav, N., Lee, J., Branscombe, T. L., Clarke, S., and Bedford, M. T. (2002) J. Biol. Chem. 277, 3537-3543[Abstract/Free Full Text]
  11. Lee, J., Sayegh, J., Daniel, J., Clarke, S., and Bedford, M. T. (2005) J. Biol. Chem. 280, 32890-32896[Abstract/Free Full Text]
  12. Branscombe, T. L., Frankel, A., Lee, J. H., Cook, J. R., Yang, Z. Z., Pestka, S., and Clarke, S. (2001) J. Biol. Chem. 276, 32971-32976[Abstract/Free Full Text]
  13. Miranda, T. B., Miranda, M., Frankel, A., and Clarke, S. (2004) J. Biol. Chem. 279, 22902-22907[Abstract/Free Full Text]
  14. Lee, J. H., Cook, J. R., Yang, Z. H., Mirochnitchenko, O., Gunderson, S. I., Felix, A. M., Herth, N., Hoffmann, R., and Pestka, S. (2005) J. Biol. Chem. 280, 3656-3664[Abstract/Free Full Text]
  15. Herrmann, F., Lee, J., Bedford, M. T., and Fackelmayer, F. O. (2005) J. Biol. Chem. 280, 38005-38010[Abstract/Free Full Text]
  16. Tang, J., Kao, P. N., and Hershman, H. R. (2000) J. Biol. Chem. 275, 19866-19876[Abstract/Free Full Text]
  17. Pawlak, M. R., Scherer, C. A., Chen, J., Roshon, M. J., and Ruley, H. E. (2000) Mol. Cell. Biol. 20, 4859-4869[Abstract/Free Full Text]
  18. An, W., Kim, J., and Roeder, R. G. (2004) Cell 117, 735-748[CrossRef][Medline] [Order article via Infotrieve]
  19. Covic, M., Hassa, P. O., Saccani, S., Buerki, C., Meier, N. I., Lombardi, C., Imhof, R., Bedford, M. T., Natoli, G., and Hottiger, M. O. (2004) EMBO J. 24, 85-96[CrossRef][Medline] [Order article via Infotrieve]
  20. Huang, S., Litt, M., and Felsenfeld, G. (2005) Genes Dev. 19, 1885-1893[Abstract/Free Full Text]
  21. Rezai-Zadeh, N., Zhang, X., Namour, F., Fejer, G., Wen, Y. D., Yao, Y. L., Gyory, I., Wright, K., and Seto, E. (2003) Genes Dev. 17, 1019-1029[Abstract/Free Full Text]
  22. Xu, W., Chen, H., Du, K., Asahara, H., Tini, M., Emerson, B. M., Montminy, M., and Evans, R. M. (2001) Science 294, 2507-2511[Abstract/Free Full Text]
  23. Brahms, H., Meheus, L., de Brabandere, V., Fischer, U., and Lührmann, R. (2001) RNA 7, 1531-1542[Abstract]
  24. Meister, G., and Fischer, U. (2002) EMBO J. 21, 5853-5863[CrossRef][Medline] [Order article via Infotrieve]
  25. Friesen, W. J., Paushkin, S., Wyce, A., Massenet, S., Pesiridis, G. S., Van Duyne, G., Mann, M., and Dreyfuss, G. (2001) Mol. Cell. Biol. 21, 8289-8300[Abstract/Free Full Text]
  26. Abramovich, C., Yakobson, B., Chebath, J., and Revel, M. (1997) EMBO J. 16, 260-266[CrossRef][Medline] [Order article via Infotrieve]
  27. Chen, W., Daines, M. O., and Hershey, G. K. (2004) J. Immunol. 172, 6744-6750[Abstract/Free Full Text]
  28. Hsu, I. W., Hsu, M., Li, C., Chuang, T. W., Lin, R. I., and Tarn, W. Y. (2005) J. Biol. Chem. 280, 34507-34512[Abstract/Free Full Text]
  29. Mowen, K. A., Tang, J., Zhu, W., Schurter, B. T., Shuai, K., Herschman, H. R., and David, M. (2001) Cell 104, 731-741[CrossRef][Medline] [Order article via Infotrieve]
  30. Shen, E. C., Henry, M. F., Weiss, V. H., Valentinin, S. R., Silver, P. A., and Lee, M. S. (1998) Genes Dev. 12, 679-691[Abstract/Free Full Text]
  31. McBride, A. E., Weiss, V. H., Kim, H. K., Hogle, J. M., and Silver, P. A. (2000) J. Biol. Chem. 275, 3128-3136[Abstract/Free Full Text]
  32. McBride, A. E., Cook, J. T., Stemmler, E. A., Rutledge, K. L., McGrath, K. A., and Rubens, J. A. (2005) J. Biol. Chem. 280, 30888-30898[Abstract/Free Full Text]
  33. Green, D. M., Marfatia, K. A., Crafton, E. B., Zhang, X., Cheng, X., and Corbett, A. H. (2002) J. Biol. Chem. 277, 7752-7760[Abstract/Free Full Text]
  34. Xu, C., and Henry, M. F. (2004) Mol. Cell. Biol. 24, 10742-10756[Abstract/Free Full Text]
  35. Yun, C. Y., and Fu, X. D. (2000) J. Cell Biol. 150, 707-717[Abstract/Free Full Text]
  36. Kim, S., Merrill, B. M., Rajpurohit, R., Kumar, A., Stone, K. L., Papov, V. V., Schneiders, J. M., Szer, W., Wilson, S. H., Paik, W. K., and Williams, K. R. (1997) Biochemistry 36, 5185-5192[CrossRef][Medline] [Order article via Infotrieve]
  37. Nichols, R. C., Wang, X. W., Tang, J., Hamilton, B. J., High, F. A., Herschman, H. R., and Rigby, W. F. C. (2000) Exp. Cell Res. 256, 522-532[CrossRef][Medline] [Order article via Infotrieve]
  38. Côté, J., Boisvert, F. M., Boulanger, M. C., Bedford, M. T., and Richard, S. (2003) Mol. Biol. Cell 14, 274-287[Abstract/Free Full Text]
  39. Herrmann, F., Bossert, M., Schwander, A., Akgun, E., and Fackelmayer, F. O. (2004) J. Biol. Chem. 279, 48774-48779[Abstract/Free Full Text]
  40. Smith, J. J., Rücknagel, K. P., Schierhorn, A., Tang, J., Nemeth, A., Linder, M., Herschman, H. R., and Wahle, E. (1999) J. Biol. Chem. 274, 13229-13234[Abstract/Free Full Text]
  41. Michael, W. M., Choi, M., and Dreyfuss, G. (1995) Cell 83, 415-422[CrossRef][Medline] [Order article via Infotrieve]
  42. Michael, W. M., Eder, P. S., and Dreyfuss, G. (1997) EMBO J. 16, 3587-3598[CrossRef][Medline] [Order article via Infotrieve]
  43. Habelhah, H., Shah, K., Huang, L., Ostareck-Lederer, A., Burlingame, A. L., Shokat, K. M., Hentze, M. W., and Ronai, Z. (2001) Nat. Cell Biol. 3, 325-330[CrossRef][Medline] [Order article via Infotrieve]
  44. Collier, B., Goobar-Larsson, L., Sokolowski, M., and Schwartz, S. (1998) J. Biol. Chem. 273, 22648-22656[Abstract/Free Full Text]
  45. Evans, J. R., Mitchell, S. A., Spriggs, K. A., Ostrowski, J., Bomsztyk, K., Ostareck, D. H., and Willis, A. E. (2003) Oncogene 22, 8012-8020[CrossRef][Medline] [Order article via Infotrieve]
  46. Ostareck, D. H., Ostareck-Lederer, A., Wilm, M., Thiele, B. J., Mann, M., and Hentze, M. W. (1997) Cell 89, 597-606[CrossRef][Medline] [Order article via Infotrieve]
  47. Ostareck, D. H., Ostareck-Lederer, A., Shatsky, I. N., and Hentze, M. W. (2001) Cell 104, 281-290[CrossRef][Medline] [Order article via Infotrieve]
  48. Ostareck-Lederer, A., Ostareck, D. H., Cans, C., Neubauer, G., Bomsztyk, K., Superti-Furga, G., and Hentze, M. W. (2002) Mol. Cell. Biol. 22, 4535-4543[Abstract/Free Full Text]
  49. Taylor, S. J., and Shalloway, D. (1994) Nature 368, 867-871[CrossRef][Medline] [Order article via Infotrieve]
  50. Van Seuningen, I., Ostrowski, J., Bustelo, X. R., Sleath, P. R., and Bomsztyk, K. (1995) J. Biol. Chem. 270, 26976-26985[Abstract/Free Full Text]
  51. Weng, Z., Thomas, S. M., Rickles, J. J., Taylor, J. A., Brauer, A. W., Seidel-Dugan, C., Michael, W. M., Dreyfuss, G., and Brugge, J. S. (1994) Mol. Cell. Biol. 14, 4509-4521[Abstract/Free Full Text]
  52. Matunis, M. J., Michael, W. M., and Dreyfuss, G. (1992) Mol. Cell. Biol. 12, 164-171[Abstract/Free Full Text]
  53. Liu, Q., and Dreyfuss, G. (1995) Mol. Cell. Biol. 15, 2800-2808[Abstract]
  54. Huang, H. M., Tam, M. F., Tam, T. C. S., Chen, D. H., Hsieh, M., and Li, C. (2002) J. Biochem. 132, 813-818[Abstract/Free Full Text]
  55. Kühn, U., Nemeth, A., Meyer, S., and Wahle, E. (2003) J. Biol. Chem. 278, 16916-16925[Abstract/Free Full Text]
  56. Zhang, X., and Cheng, X. (2003) Structure 11, 509-520[Medline] [Order article via Infotrieve]
  57. Mereau, A., Grey, L., Piquet-Pellorce, C., and Heath, J. K. (1993) J. Cell Biol. 122, 713-719[Abstract/Free Full Text]
  58. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489[Abstract/Free Full Text]
  59. Lee, T. D., and Shively, J. E. (1990) Methods Enzymol. 193, 361-374[Medline] [Order article via Infotrieve]
  60. Graham, F. L., and Van der Eb, A. J. (1973) Virology 52, 456-464[CrossRef][Medline] [Order article via Infotrieve]
  61. Huettelmaier, S., Illenberger, S., Grosheva, I., Rüdiger, M., Singer, R. H., and Jockusch, B. M. (2001) J. Cell Biol. 155, 775-786[Abstract/Free Full Text]
  62. Roepstorff, P., and Fohlman, J. (1984) Biomed. Mass Spectrom. 11, 601[CrossRef][Medline] [Order article via Infotrieve]
  63. Biemann, K. (1990) Methods Enzymol. 193, 886-887[Medline] [Order article via Infotrieve]
  64. Ong, S. E., Mittler, G., and Mann, M. (2004) Nat. Methods 1, 1-8
  65. Rawal, N., Rajpurohit, R., Paik, W. K., and Kim, S. (1994) Biochem. J. 300, 483-489[Medline] [Order article via Infotrieve]
  66. Yu, M. C., Bachand, F., McBride, A. E., Komili, S., Casolari, J. M., and Silver, P. A. (2004) Genes Dev. 18, 2024-2035[Abstract/Free Full Text]
  67. Valentini, S. R., Weiss, V. H., and Silver, P. A. (1999) RNA 5, 272-280[Abstract]
  68. Bedford, M. T., Frankel, A., Yaffe, M. B., Clarke, S., Leder, P., and Richard, S. (2000) J. Biol. Chem. 275, 16030-16036[Abstract/Free Full Text]
  69. Ostareck-Lederer, A., and Ostareck, D. H. (2004) Biol. Cell 96, 407-411[CrossRef][Medline] [Order article via Infotrieve]
  70. Shi, Y., Lan, F., Matson, C., Mulligan, P., Whetstine, J. R., Cole, P. A., Casero, R. A., and Shi, Y. (2004) Cell 119, 941-953[CrossRef][Medline] [Order article via Infotrieve]
  71. Cuthbert, G. L., Daujat, S., Snowden, A. W., Erdjument-Bromage, H., Hagiwara, T., Yamandy, M., Schneider, R., Gregory, P. D., Tempst, P., Bannister, A. J., and Kouzarides, T. (2004) Cell 118, 545-553[CrossRef][Medline] [Order article via Infotrieve]
  72. Wang, Y., Wysocka, J., Sayegh, J., Lee, Y. H., Perlin, J. R., Leonelli, L., Sonbuchner, L. S., McDonald, C. H., Cook, R. G., Dou, Y., Roeder, R. G., Clarke, S., Stallcup, M. R., Allis, C. D., and Coonrod, S. A. (2004) Science 306, 279-283[Abstract/Free Full Text]
  73. Ahmad, K., and Henikoff, S. (2002) Mol. Cell 9, 1191-1200[CrossRef][Medline] [Order article via Infotrieve]
  74. Janicki, S. M., Tsukamoto, T., Salghetti, S. E., Tansey, W. P., Sachidanandam, R., Prasanth, K. V., Ried, T., Shav-Tal, Y., Bertrand, E., Singer, R. H., and Spector, D. L. (2004) Cell 116, 683-698[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
Z. Yu, T. Chen, J. Hebert, E. Li, and S. Richard
A Mouse PRMT1 Null Allele Defines an Essential Role for Arginine Methylation in Genome Maintenance and Cell Proliferation
Mol. Cell. Biol., June 1, 2009; 29(11): 2982 - 2996.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
O. O. Koyuncu and T. Dobner
Arginine Methylation of Human Adenovirus Type 5 L4 100-Kilodalton Protein Is Required for Efficient Virus Production
J. Virol., May 15, 2009; 83(10): 4778 - 4790.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Kolbel, C. Ihling, K. Bellmann-Sickert, I. Neundorf, A. G. Beck-Sickinger, A. Sinz, U. Kuhn, and E. Wahle
Type I Arginine Methyltransferases PRMT1 and PRMT-3 Act Distributively
J. Biol. Chem., March 27, 2009; 284(13): 8274 - 8282.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
T. Fukuda, T. Naiki, M. Saito, and K. Irie
hnRNP K interacts with RNA binding motif protein 42 and functions in the maintenance of cellular ATP level during stress conditions.
Genes Cells, February 1, 2009; 14(2): 113 - 128.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Fronz, S. Otto, K. Kolbel, U. Kuhn, H. Friedrich, A. Schierhorn, A. G. Beck-Sickinger, A. Ostareck-Lederer, and E. Wahle
Promiscuous Modification of the Nuclear Poly(A)-binding Protein by Multiple Protein-arginine Methyltransferases Does Not Affect the Aggregation Behavior
J. Biol. Chem., July 18, 2008; 283(29): 20408 - 20420.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
I. S. Naarmann, C. Harnisch, N. Flach, E. Kremmer, H. Kuhn, D. H. Ostareck, and A. Ostareck-Lederer
mRNA Silencing in Human Erythroid Cell Maturation: HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN K CONTROLS THE EXPRESSION OF ITS REGULATOR c-Src
J. Biol. Chem., June 27, 2008; 283(26): 18461 - 18472.
[Abstract] [Full Text] [PDF]

Home page
 RNAHome page
I. Abaza and F. Gebauer
Trading translation with RNA-binding proteins
RNA, March 1, 2008; 14(3): 404 - 409.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
I. Goulet, G. Gauvin, S. Boisvenue, and J. Cote
Alternative Splicing Yields Protein Arginine Methyltransferase 1 Isoforms with Distinct Activity, Substrate Specificity, and Subcellular Localization
J. Biol. Chem., November 9, 2007; 282(45): 33009 - 33021.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
D. Adolph, N. Flach, K. Mueller, D. H. Ostareck, and A. Ostareck-Lederer
Deciphering the Cross Talk between hnRNP K and c-Src: the c-Src Activation Domain in hnRNP K Is Distinct from a Second Interaction Site
Mol. Cell. Biol., March 1, 2007; 27(5): 1758 - 1770.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
281/16/11115    most recent
M513053200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ostareck-Lederer, A.
Right arrow Articles by Wahle, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ostareck-Lederer, A.
Right arrow Articles by Wahle, E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement