Comprehensive Analysis of in Vivo Phosphoproteome of Mouse Liver

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Comprehensive analysis of in vivo phosphoproteome of mouse liver microsomes Oh Kwang Kwon, Juhee Sim, Sun Ju Kim, Eunji Sung, Jin Young Kim, Tae Cheon Jeong, and Sangkyu Lee J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00812 • Publication Date (Web): 20 Oct 2015 Downloaded from http://pubs.acs.org on October 29, 2015

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Journal of Proteome Research

Comprehensive analysis of in vivo phosphoproteome of mouse liver microsomes

Oh Kwang Kwon †,¶, JuHee Sim †,¶, Sun Ju Kim †, Eunji Sung †, Jin Young Kim ‡, Tae Cheon Jeong §, Sangkyu Lee *,†

†

College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National

University, Daegu 41566, Republic of Korea ‡

Mass Spectrometry Research Center, Korea Basic Science Institute, Ochang, Chungbuk

28115, Republic of Korea §

College of Pharmacy, Yeungnam University, Gyeongsan 38541, Republic of Korea

¶

Those authors are equally contributed.

*Correspondence: Sangkyu Lee, Ph.D., Professor, College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu 702-701, Republic

of

Korea.

Tel.:

+82-53-950

8571;

Fax:

[email protected].

1

ACS Paragon Plus Environment

+82-53-950-8557;

E-mail:


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Abstract Protein phosphorylation at serine, threonine, and tyrosine residues are some of the most widespread reversible post-translational modifications. Microsomes are vesicle-like bodies, not ordinarily present within living cells, which form from pieces of the endoplasmic reticulum (ER), plasma membrane, mitochondria, or Golgi apparatus of broken eukaryotic cells. Here, we investigated the total phosphoproteome of mouse liver microsomes (MLM) using TiO2 enrichment of phosphopeptides coupled with in-line two-dimensional (2D)-LC-MS/MS. In total, 699 phosphorylation sites in 527 proteins were identified in MLMs. When compared with the current phosphoSitePlus database, 155 novel phosphoproteins were identified in MLM. The distributions of phosphosites were 89.4%, 8.0%, and 2.6% for phosphoserine, phosphotheronine, and phosphotyrosine, respectively. By Motif-X analysis, 8 Ser motifs and 1 Thr motif were found, and 5 acidic, 2 basophilic-, and 2 proline-directed motifs were assigned. The potential functions of phosphoproteins in MLM were assigned by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In GO annotation, phosphorylated microsomal proteins were involved in mRNA processing, mRNA metabolic processes and RNA splicing. In the KEGG pathway analysis, phosphorylated microsomal proteins were highly enriched in ribosome protein processing in ER, ribosomes, and in RNA transport. We determined that 52 and 23 phosphoproteins were potential substrates of cAMP-dependent protein kinase A and casein kinase II, respectively, many of which are 40S/60S ribosomal proteins. Overall, our results provide an overview of features of protein phosphorylation in MLMs that should be a valuable resource for the future understanding of protein synthesis or translation involving phosphorylation.

Key words Microsomes; phosphorylation; ribosomal protein; cytochrome P450; protein processing 2


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Introduction Phosphorylation of proteins at serine, threonine, and tyrosine residues is one of the most widespread reversible post-translational modifications (PTM)

1

. Generally, reversible

phosphorylation, a key mechanism of regulating protein activities, participates in diverse cellular processes including metabolism, cell communication, cell growth, and development 2. The balance between reversible phosphorylation and dephosphorylation is coordinated by the dynamic interplay between protein kinases, phosphatases, and phosphorus-binding proteins. Although more than 30% of human proteins are known to be phosphorylated, phosphorylation of regulatory proteins is a transient modification that shows low stoichiometry

3, 4

. Phosphorylation occurs only on particular proteins or at particular times,

resulting in a low steady state abundance of phosphorylated proteins. Therefore, specific and efficient phosphopeptide-enrichment methods are needed to capture and analyze the large scale of the phosphoproteome in complex samples. The enzymes found in liver microsomes normally contribute to various biological reactions, including metabolism of chemicals, the various functions of membrane bound enzymes, and lipid-protein interactions

5, 6

. Microsomes are spontaneously formed vesicle-

like bodies, not ordinarily present in living cells, derived from pieces of the endoplasmic reticulum (ER), plasma membrane, mitochondria, or Golgi apparatus of broken eukaryotic cells 7. Typically, microsomes are separated by differential centrifugation of homogenized cells or tissues. Using methods based on enrichment of ER fractions, prepared microsomes usually show a high level of metabolic activity due to large amounts of metabolic enzymes, such as cytochrome P450 (CYP) 8. CYPs are important components of xenobioticmetabolizing

monooxygenase,

which

accounts

biotransformation of clinically relevant drugs

for

approximately

9

75%

of

the

. Hepatic CYPs are ER-anchored

3



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hemoproteins involved in the metabolism of numerous endo- and xenobiotics, including drugs, steroids, and carcinogens 10. During the past decades, global phosphoproteomic analyses have been conducted in H. sapiens and M. musculus using MS-based proteomic and phosphopeptide enrichment methods 1, 11, 12. Nevertheless, only proteomic analyses have been applied to microsomes, and the extent of phosphorylation of microsomal proteins remains undefined

6, 13, 14

. A better

understanding of protein phosphorylation in microsomes is needed to improve our knowledge of important microsomal processes. Here we investigated the global microsomal phosphoproteome using TiO2 enrichment of phosphopeptides coupled with in-line twodimensional (2D)-LC-MS/MS.

4


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Experimental procedures Preparation of mouse liver microsomes Specific pathogen-free male ICR mice (28 to 30 g) were obtained from the Orient Co. (Seoul, Korea). The animals purchased at 5 weeks of age were acclimated for at least 2 weeks. They were randomly housed four per cage and strictly maintained at 23 ± 3 °C and 40–60% relative humidity. A 12 h light/12 h dark cycle was used with an intensity of 150–300 lx. After animals were subjected to necropsy, the livers were removed and homogenized with four volumes of ice-cold 0.1 M potassium phosphate buffer, pH 7.4. The liver homogenates were centrifuged at 9,000 ×g for 20 min at 4 °C in order to obtain an S9 fraction. The S9 fraction was centrifuged at 105,000 ×g for 1 h at 4 °C using an ultracentrifuge, and the supernatants were discarded. The liver microsomal pellets were washed by pipetting using ice-cold 0.1 M potassium phosphate buffer, pH 7.4, and were centrifuged once again under the same conditions. The microsomal pellets were ground on ice in 250 mM sucrose in 0.1 M potassium phosphate buffer by using a Dounce tissue grinder. The prepared micosome were confirmed by Western blotting (Supplemental Figure S1). The liver microsomal pellets isolated by differential centrifugation were stored at -80 °C until use

15, 16

. The concentration

of microsomal protein was determined using Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). All animal procedures complied with the guidelines issued by the Society of Toxicology (USA; 1989). This study was approved by the Institutional Review Board of the Kyungpook National University (IRB-KNU 2014-0082-1).

Tryptic digestion of microsomal proteins To analyze the proteins, the microsome fractions were lysed using lysis buffer at 4 °C for 5



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30 min and centrifuged at 4 °C for 10 min. Lysis buffer contained RIPA buffer (25 mM TrisHCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, Thermo Scientific, Rockford, IL) with protease inhibitor cocktail (1 mM AEBSF, 0.8 µM aprotinin, 50 µM bestatin, 15 µM E-64, 20 µM leupeptin, and 10 µM pepstatin A; Calbiochem, Merck Millipore, Billerica, MA) and phosphatase inhibitor (10 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM β-glycerophosphate disodium salt hydrate, 2 mM sodium pyrophosphate decahydrate, Sigma Aldrich, St. Louis, MO). The supernatants were transferred to new tubes and precipitated with 10% trichloroacetic acid (TCA) at 4 °C for 4 h. The precipitated protein was centrifuged at 13,000 ×g, 4 °C for 10 min, and washed twice with cold acetone. Two milligrams of protein were reduced using 15 mM dithiothreitol in 25 mM ammonium bicarbonate at 56 °C for 30 min, and then alkylated using 60 mM iodoacetamide in 25 mM ammonium bicarbonate for 30 min at RT in the dark. Protein samples were then added to 75 mM cysteine in 25 mM ammonium bicarbonate at 25 °C for 30 min in the dark. After adding trypsin to the protein (at a ratio of 1:50 w/w), peptide samples were digested at 37 °C overnight. Digested peptides were dried using a centrifugal vacuum concentrator, and then additional trypsin was added at 1:50 (w/w). Finally, the mixed peptides were dried using a centrifugal vacuum concentrator.

Enrichment of phosphopeptides The phosphopeptides were captured using a phosphopeptide enrichment titanium dioxide (TiO2) kit (TitansphereTM, Phos-TiO kit, GL Sciences Inc., Japan). The TiO2 tips were activated with 200 µl of 100% ACN and equilibrated with 200 µl of solution B (1 M glycolic acid in 80% ACN with 2% TFA) using centrifugation at 3,000 ×g for 2 min. For adsorption 6


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of peptides, peptides were mixed with 1000 µl of solution B at RT, rinsed using solution B and then with solution A (80% ACN with 2% TFA), each at 1,000 ×g for 5 min. Phosphopeptides were eluted with 200 µl of 0.5% ammonium hydroxide solution, and with a 200 µl volume of 30% ACN for an additional elution. Enriched phosphopeptides were dried using a centrifugal vacuum concentrator. The samples were desalted using C18 Zip-tip (Thermo Pierce, Rockford, IL) according to manufacturer’s instructions and dried using a centrifugal vacuum concentrator. The sample was analyzed by 2D-LC-MS/MS performed on a nano ACQUITY Ultra Performance LC System (Waters; Milford, MA) and an LTQOrbitrap velos pro (Thermo Scientific; Rockford, IL) fitted with a nano-electrospray source. The details of the LC-MS/MS method are described in Supporting Information.

Western blotting The population of phosphoproteins in microsomes was screened by Western blotting. The microsomes (30 µg) were separated by 12% SDS-PAGE and transferred to a PVDF membrane (Roche, Germany) by wet transfer at 300 mA for 1 h on ice. After membranes were blocked with TBST (20 mM Tris, 137 mM sodium chloride, 0.1% Tween-20) containing 5% bovine serum albumin (BSA) for 3 h at RT, membranes were incubated with each primary

antibody;

anti-phosphoserine

(Abcam,

UK),

anti-phosphothreonine,

anti-

phosphotyrosine (Cell Signaling, Danvers, MA) by rotation at 4 °C overnight. Membranes were washed with TBST, exposed to horseradish peroxidase (HRP)-linked secondary antibody (Cell Signaling, Danvers, MA) with rotation for 2 h at RT, and washed with TBST. The membranes were developed using ECL reagent (GE Healthcare, UK) and detected using Image Quant LAS 4000 mini (GE Healthcare, UK).

7



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PKA and CKII enzyme reactions For reaction with cAMP-dependent protein kinase A (PKA) using a PKA assay kit (Promega, Madison, WI), 500 µg of pooled CD-1 male mouse liver microsome (MLM, XenoTech, LLC., Lenexa, USA) were diluted in 5× PKA reaction buffer (100 mM Tris-HCl, pH 7.4, 50 mM MgCl2, 5 mM ATP), plus PKA activator 5× solution (5 µM cAMP in water), and preincubated at 30 °C for 1 min. To the MLM reaction samples were added PKA solution (2 µg/ml PKA in 350 mM K3PO4, 0.1 mM DTT) or buffer only without PKA solution (negative control, only added 350 mM K3PO4, 0.1 mM DTT), and incubation was continued at 30 °C for 2.5 h. For reaction with casein kinase II (CKII) using recombinant CKII (New England BioLabs Inc., Ipswich, MA), 500 µg of pooled MLM were diluted in CKII reaction buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2) with 0.2 mM ATP solution in water and preincubated at 30 °C for 1 min. CKII (2500 U/5 µL) was added and incubation continued at 30 °C for 2.5 h. Both kinase reactions were incubated in the presence of phosphatase inhibitor cocktail (10 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM β-glycerophosphate disodium salt hydrate, 2 mM sodium pyrophosphate decahydrate). For immunoblotting, approximately 10 µg of the reaction samples were transferred to new tubes and 5× SDS sample buffer was added. Reaction samples (400 µg) were treated with lysis buffer to obtain trypsin-digested peptides.

Identification and validation of phosphorylation Phosphopeptides and proteins were identified using MaxQuant (version 1.4.1.2)

17

. The

resulting MS/MS spectral data were used to search a database containing 45,269 protein sequences

of

Mus

musculus

downloaded

from

UniProtKB

(http://www.uniprot.org/proteomes/UP000000589). Search parameters were as follows: 8


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trypsin digestion with up to 2 missed cleavages; fixed modification of carbamidomethylation (Cys); variable modification of oxidation (Met) and phosphorylation (Ser/Thr/Tyr). Maximum mass tolerance allowance was 5 ppm (monoisotopic) for MS/MS tolerance and 0.02 Da (monoisotopic) for fragment ions. MaxQuant search results were filtered with a MaxQuant score ≥40 and occupation of phosphorylation site probability ≥ 0.5, with reverse and contamination peptides removed from the results. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD003043 18.

Bioinformatic analysis To obtain information about the identified phosphoproteins, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and the annotated protein KEGG database were searched. For GO annotation, phosphoproteins were mapped to GO ID. If the identified phosphorylation substrates were not annotated in the UniProt or GOA databases, InterProScan software was used to annotate a protein’s GO function based on a protein sequence alignment method 19. The annotated phosphoproteins were further classified by GO annotation based on three categories: biological process, cellular component, and molecular function. The pathways associated with phosphoproteins in microsomes were annotated with KEGG online service tool KAAS. The annotation results were mapped onto the KEGG pathway database using the KEGG online service tool KEGG mapper

20

. WoLF

PSORT was used for subcellular localization prediction 21. For functional enrichment analysis, Fisher’s exact test was used to test for enrichment or depletion (right-tailed test) of specific annotation terms among members of the resulting 9



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protein clusters. Derived p-values were further adjusted to address multiple hypothesis testing by the method proposed by Benjamini and Hochberg

22

. Terms having adjusted p-values

below 0.05 in any of the clusters were treated as significant. For motif analysis, Software motif-x was used to analyze the models of sequences composed of amino acids in specific positions within phospho-13-mers (6 amino acids upstream and downstream of the phosphorylation site) in all protein sequences 23. Further, all the database protein sequences were used as the background database parameter, with all other parameters set at default.

10


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Results Global phosphoproteome in mouse liver microsomes The liver microsomal lysates were prepared from ICR male mice, with 3 individual livers pooled prior to sample digestion. The separated microsomes had been confirmed by Western blotting using CYP2B and cytochrome c oxidase subunit IV (COX IV) indicating specific microsomal and mitochondrial proteins, respectively (Supplemental Figure S1). All samples had strong blots for CYP2B, on the other hand COX IV was not showed in prepared samples. To confirm the distribution of microsomal phosphorylation, microsomal proteins were separated by 1D SDS-PAGE (Figure 1). The Coomassie blue-stained gel showed a closely migrating set of proteins in the range of 43–55 kDa, typical of the migration of CYP proteins 24

(Figure 1A). The distribution of phosphorylations in prepared microsomes was detected by

Western blot using anti-phospho-Ser, phospho-Thr, and phospho-Tyr antibodies (Figure 1B). Analogous patterns of immunoblots with well-characterized patterns were obtained from three individual microsome preparations. To identify the global phosphoproteome in mouse liver microsomes, microsomal lysates were prepared as solution tryptic digests (Figure 2A). After trypsin digestion, the peptide mixture was enriched for phosphopeptides using TiO2 chromatography. Enriched phosphopeptides were analyzed by nano-LC-MS/MS on Orbitrap velos pro. Phosphoproteins were identified by MaxQuant with the use of the current UniProt database (containing 45,269 protein sequences). To ensure the quality of the analysis, annotated spectra of all detected phosphopeptides were examined manually, and the mass tolerance for precursor ions was less than 5 ppm (Supplemental Figure S2A). Using orthogonal filtering criteria, we filtered the search results sequentially and established a phosphopeptide data set with a MaxQuant score ≥ 40, modified sites probability ≥ 0.5, and removal of reverse and contaminating peptides (Supplemental 11



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Figure S2B). In total, 699 phosphorylation sites in 527 phosphorylated proteins were identified in mouse liver microsomal fractions (Supplemental Table S1). 372 phosphoproteins were identified in the phosphoSitePlus database

25

, and 155 (29%) novel phosphoproteins

were identified for the first time in mouse liver microsomes (Figure 2B). Detailed information of the novel phosphorylated peptides and proteins are summarized in Supplemental Table S2.

Characterization of phosphorylation in mouse liver microsomes The detected distributions of phosphosites were 89.4%, 8.0%, and 2.6% for phosphoserine, phosphothreonine, and phosphotyrosine, respectively (Figure 2C). The high ratio of phosphoserine was close to that observed in a large-scale phosphoproteomics study of a mouse liver cell line 12, 26.

In this study, the Motif-X algorithm was used to extract specific

microsomal phosphorylation motifs from the data (Figure 3). The kinase classification for these motifs was confirmed by reference to a previously reported motif database

27

. As a

result, 8 Ser motifs and 1 Thr motif were found, and 5 acidic (n = 178), 2 basophilic (n = 108) and 2 proline-directed motifs (n = 190) were assigned. The acidic motifs containing E or D at positions +1, +2, or +3 were dominant, consistent with reactions by CKII 4. We identified 2 basophilic motifs (RxxS), which are specific target sites of PKA or protein kinase

28

, and 2

proline-directed motifs (SP and TP) found as substrates of mitogen-activated protein kinase (MAPK) 4, 29. To understand potential functional implications of phosphorylation in the microsomal fractions, we analyzed enrichments of GO annotation and KEGG pathway analysis (Figure 4 and Supplemental Figure S3). GO analysis showed that microsomal phosphorylations were enriched for proteins that are involved in mRNA processing, mRNA metabolic processes, and 12


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RNA splicing in biological processes (Figure 4A). With respect to cellular compartment, the phosphorylations were mainly detected in membrane proteins associated with the ER. Phosphorylation

of

microsomal

proteins

is

therefore

functionally

important

for

oxidoreductase activity and RNA binding. In the KEGG pathway analysis, the most highly enriched categories for microsomal phosphorylation were ribosome protein processing in the ER, ribosomes, and RNA transport (Figure 4B). The greatest enrichment of microsomal phosphorylation was in proteins involved in processing in the ER and translation of mRNA (Supplemental Figures S4 and S5). A total of 27 phosphosites in 18 phosphoproteins related to protein processing was identified, and three phosphorylation sites in ribosome-binding protein 1 were revealed as novel phosphorylations in microsomal proteins (Table 1). Phosphorylations of CYP isoforms were previously identified in humans 24. Specifically, the phosphorylation at S478 of hCYP3A4 is well-known to enhance the ubiquitinationmediated degradation of CYP

30

. Previous results from studies that focused on the

identification of phosphorylation of human and murine CYP were poorly analyzed. In this study, we identified a total of 10 phosphorylation sites in 8 murine CYP proteins (Table 2). Four phosphorylation sites, S128, T275, S134, and S138 in CYP 2B10, 2D10, 3A11, and 3A13, respectively, had not been reported previously, and the S129 phosphorylation of CYP2E1 had been previously identified as a phosphorylation site in humans 24.

Identification of potential PKA and CKII kinase substrates by motif analysis Based on identified motifs, we hypothesized that PKA and CKII might be the main kinases regulating the microsomal phosphorylation. To investigate the substrates of PKA and CKII, mouse microsomal proteins were incubated with or without PKA or CKII kinase. We confirmed an increased phosphorylation of microsomal proteins using immunoblotting, 13



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except Tyr-phosphorylation (Figure 5). The 52 and 23 phosphoproteins as potential substrates of PKA and CKII, respectively, were identified and are listed in Supplemental Tables S3 and S4. The 17 and 4 phosphopeptides in 40S/60S ribosomal proteins that were identified as substrates of PKA and CKII kinase in mouse liver microsomes are listed in Table 3. When compared with the PhosphoSite database, 8 phosphorylation sites were revealed as novel phosphorylation sites at positions S272, T18, T60, S9, S82, S31, S53, and S32 in 60S ribosomal proteins L5, S16, S20, L23, S25, S29, L35, and L36a, respectively.

14


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Discussion From a global phosphoproteome analysis of mouse liver microsomes, an enrichment of phosphorylation in ribosomal proteins and proteins involved in processing was revealed. Specifically, we identified phosphorylations at 27 sites in 18 proteins involved in protein processing in microsomal proteins. Among these, calnexin is a molecular chaperone of ER with folding-promoting functions and an abundant integral membrane phosphoprotein of the ER of eukaryotic cells

31, 32

. We verified the 3 phosphorylation sites on calnexin that were

previously identified as phosphorylated sites at Ser553, 563, and 582 33. Several kinases have been shown to have roles in the phosphorylation of calnexin. The phosphorylation at the Ser563 in calnexin by CK2 increases its association with ribosomes

34

. ERK1, MAPK was

also shown to phosphorylate Ser563 both in vitro and in vivo 35. In this study, the previously known 3 phosphorylation sites in calnexin were confirmed, indicating the reliability of the characterized phosphorylation sites in microsomal proteins we have identified. Here we also identified 3 phosphorylation sites at Ser154, 155, and 167 in ribosomebinding protein 1 (RRBP1). RRBP1 is a membrane-associated and ribosome-binding protein located on the rough ER that has essential roles in the transportation of intracellular proteins in mammalian cells

36

. The abnormal expression of RRBP1 is related to tumorigenesis and

progression of colorectal and lung cancers 37, 38. In breast cancer, RRBP1 is upregulated at the mRNA level and overexpressed in 84% (177/219) of the breast carcinoma cases tested 39. In addition, RRBP1 overexpression highly affects overall survival in patients with early-stage (I and II) tumors, indicating that RRBP1 is a potentially important marker for disease prognosis 40

. In general, the phosphorylated forms of proteins are typically associated with biologically

activated forms, as compared with non-phosphorylated proteins 2. Therefore, our findings suggest that the phosphorylation of RRBP1 is an interesting molecule that may be involved in 15



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Page 16 of 34

progression of cancer, and the potential role of phosphorylation in RRBP1 can be identified in further studies to serve as a cancer biomarker or prognostic factor. CYPs are categorized into 1A, 2A, 2B, 2C, 2D, 2E, and 3A subfamilies, which have important roles in drug metabolism

41

. There are 57 human and 102 murine putatively

functional genes 42. In 2003, PKA was identified as a major contributor of phosphorylation of CYP

43

. The phosphorylation of CYP leads to a marked decrease in the monooxygenase

activity associated with UBC7/gp78–mediated CYP3A4 and CYP2E1 ubiquitination

30, 44, 45

.

Its cell surface receptor, gp78/AMFR, is also localized to the endoplasmic reticulum, where it functions as an E3 ubiquitin ligase

46

. The turnover of CYP3A4 involves endoplasmic

reticulum-associated degradation via the ubiquitin (Ub)-dependent 26S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase complexes, as well as PKA and PKC

47

. It is therefore important to identify the phosphorylation sites of CYPs

since the phosphorylation of CYP is involved in the regulation of multiple metabolic activities. Here, we identified the 10 phosphorylation sites of murine cytochrome P450s using a global phosphoproteome approach in mouse liver microsomes. As shown in Table 2, the phosphorylation of CYP was highly similar in humans and mice. We identified a consensus phosphorylated sequence [RFS(ph)XXXX] in humans and mice for CYP2A12, 2B10, 2D26, and 2E1 24. The phosphorylation at S134 on CYP3A11 also showed the same result as seen in hCYP3A4 30. The phosphorylation in CYPs is significantly involved in regulation of catalytic activities, and the variation of CYP activities may seriously affect results of pharmacokinetic studies during drug development processes. In this manuscript, a high similarity of phosphorylation patterns between human and mouse CYP isoforms was revealed, suggesting that it will be helpful to search for correlations between results in human and mouse 16


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pharmacokinetic studies. In mouse liver microsomes, PKA mediated a total of 66 phosphorylations, and 17 phosphorylation sites found in 40S/60S ribosomal proteins that comprise the ribosome complex were mediated by PKA’s catalytic activity (Table 3). PKA is the archetypical phosphokinase, sharing a catalytic core with the entire protein kinase superfamily

48

.

Moreover, PKA contributes to peptide-chain elongation in mammalian cells by phosphorylating the eukaryotic translation elongation factor 2 (eEF2) kinase

49

. The

phosphorylation of eEF2 kinase causes phosphorylation and inactivation of eEF2, resulting in a decrease in the elongation of peptide chains

50

. Although PKA contributes to diverse

cellular biological functions, such as cellular signaling, cell cycle, proliferation, and metabolism

51, 52

, its interaction with ribosomal proteins was not well-known. Moreover, the

function of phosphorylation of ribosomal proteins in the ribosome complex is also not clear. Here we showed that the subunit proteins of ribosomes were widely phosphorylated by PKA, and the modified sites in phosphoproteins were detected in mouse liver microsomes. The modulation of PKA activity might be an essential regulating factor of proteins in ribosomes or ER membrane. The biological functions of the phosphorylations of ribosome subunits should be the subjects of further study. In present study, we identified global levels of phosphorylation in mouse liver microsomes that were focused on ribosomal proteins and CYPs. 155 phosphoproteins were newly identified, and 10 phosphorylation sites in CYPs and a large population of phosphorylation sites in ribosomal proteins were also investigated. Use of the mouse as a model mammal, and as a surrogate for human biology, assumes reasonable similarity between the two. It is therefore of interest to compare the similarities and differences of protein phosphorylations, and to clarify the limits of extrapolation from mouse to human. Therefore, 17



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Page 18 of 34

it will be very important to expand on in vivo experiments that generate information about phosphorylation in murine models, as was presented here.

Acknowledgements This study was supported by the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No: A112026) and by the National Research Foundation

(NRF)

grant

funded

by

the

Korean

government

2012R1A4A1028835).

18


(MSIP)

(NRF-

Page 19 of 34

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Table 1. Selected list of phosphopeptides from proteins in processing pathways in endoplasmic reticulum from mouse liver microsomes Protein accession A2AVJ7

Protein names

Ribosome-binding protein 1

Gene names RRBP1

KEGG name P180

Position

Modified sequence

S167

_S(ph)AILEATPK_

S154

_VEPAVS(ph)SIVNSIQVLASK_

S155

_VEPAVSS(ph)IVNSIQVLASK_

O08795

Glucosidase 2 subunit beta

GLU2B

GIcII

S168

_S(ph)LEDQVETLR_

P07901

Heat shock protein (HSP) 90-alpha

HSP90A

Hap90

S263

_ESDDKPEIEDVGS(ph)DEEEEEKK_

P08113

Endoplasmin (Fragment)

F7C312

GRP94

S447

_GVVDSDDLPLNVS(ph)R_

P11499

Heat shock protein (HSP) 90-beta (Fragment)

E9Q3D6

Hap90

S255

_IEDVGS(ph)DEEDDSGKDKK_

P20029

78 kDa glucose-regulated protein

GRP78

BiP

T649

_LYGSGGPPPTGEEDT(ph)SEKDEL_

T474

_VYEGERPLT(ph)K_

S582

_AEEDEILNRS(ph)PR_

S553

_QKS(ph)DAEEDGVTGSQDEEDSKPK_

S563

_QKSDAEEDGVTGS(ph)QDEEDSKPK_

P35564 Q01853 Q3TDQ1

Calnexin Transitional endoplasmic reticulum ATPase Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3B

CALX

CNX

TERA

P97

S197

_EDEEES(ph)LNEVGYDDIGGCR_

STT3B

OSTs

S495

_ENPPVEDS(ph)SDEDDKRNPGNLYDK_

T375

_EELEQQT(ph)DGDCDEEDDDKDGEVPK_

S356

_SQHS(ph)SGNGNDFEMITK_ _GTENGVNGTVTSNGADS(ph)PR_

Q8BU14

Translocation protein SEC62

SEC62

Sce62/63

Q91V04

Translocating chain-associated membrane protein 1

TRAM1

TRAM

S365

Q922R8

Protein disulfide-isomerase A6

Q3TML0

PDIs

S428

_EPWDGKDGELPVEDDIDLS(ph)DVELDDLEKDEL_

S13

_PGPTPSGTNVGS(ph)SGR_

S17

_PGPTPSGTNVGSSGRS(ph)PSK_

S49

_TTS(ph)AGTGGMWR_

S268

_VEM(ox)GTSSQNDVDMSWIPQETLNQINKAS(ph)PR_

Q9CQS8 Q9CY50

Protein transport protein Sec61 subunit beta Translocon-associated protein subunit alpha

SC61B SSRA

Sec61 TRAP 24


Page 25 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


Q9DCF9

Translocon-associated protein subunit gamma

Q9JKR6

Hypoxia upregulated protein 1

Q9R049

E3 ubiquitin-protein ligase AMFR

Q9Z2B5

Eukaryotic translation initiation factor 2-alpha kinase 3

SSRG

TRAP

HYOU1

NEF

AMFR

gp78

E9QQ30

PERK

S11

_QQS(ph)EEDLLLQDFSR_

S567

_VESVFETLVEDS(ph)PEEESTLTK_

S552

_LEETLDFS(ph)EVELEPIEVEDFEAR_

S542

_VPLDLS(ph)PR_

S711

_EQIEVIAPS(ph)PER_

2

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

3

Page 26 of 34

Table 2. Selected list of phosphopeptides of murine cytochrome P450s in mouse liver microsomes

Human#

Mouse

4

Protein accession

Protein names

Position

Modified sequence

Score

Sequence

Protein

P56593

Cytochrome P450 2A12

S130

_RFS(ph)IATLR_

136.38

_RFS(ph)SIATLR_

CYP2A6

Q9WUD0

Cytochrome P450 2B10

S128*

_RFS(ph)LATMR_

53.567

_RFS(ph)VTTMR_

CYP2B6

P24456

Cytochrome P450 2D10

T275*

_NLT(ph)DAFLAEIEK_

211.26

-

-

Q8CIM7

Cytochrome P450 2D26

S138

_FS(ph)VSTLR_

152.23

-RFS(ph)VSTLR_

CYP2D6

Q05421

Cytochrome P450 2E1

S129

_RFS(ph)LSILR_

142.97

_RFS(ph)LTTLR_

CYP2E1

P33267

Cytochrome P450 2F2

S128

_RFS(ph)VQILR_

136.53

-

-

Q64464


S134*

_ALLS(ph)PTFTSGR_

164.68

_SLLS(ph)PTFTSGK_

CYP3A4

Q64464


S501

_VVSRDETVS(ph)DE_

79.089

-

-

Q64459


T138*

_ALLSPTFT(ph)SGK_

162.48

-

-

Q64459


S139

_ALLSPTFTS(ph)GK_

169.58

_RLRSLLSPTFTS(ph)GK_

CYP3A4

#

*, novel phosphorylation sites in mouse liver microsomes. , Described in literature as phosphorylation

5

26


24, 30

.

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6


Table 3. The phosphorylation of ribosomal proteins by PKA and CK2 in mouse liver microsomes Kinase

PKA

CK2

7

Protein accession

Protein names

Score

Modified sequence

P27659

60S ribosomal protein L3

155.3

_HGS(ph)LGFLPR_

S13

Q9D8E6


55.8

_RNT(ph)ILRQAR_

T339

P47962


54.7

_MS(ph)LAQKKDR_

S272*

P47911


109.2

_AGS(ph)DAAASRPR_

S21

P14131

40S ribosomal protein S16

67.1

_T(ph)ATAVAHCK_

T18*

P62717

60S ribosomal protein L18a

86.9

_AHS(ph)IQIM(ox)K_

S123

P60867


62.5

_MPTKT(ph)LR_

T60*

P62830


88.1

_GGS(ph)SGAKFR_

S9*

Q8BP67


100.5

_AITGAS(ph)LADIMAK_

S86

P62852


117.5

_IRGS(ph)LAR_

S82*

P47915


57.4

_SQRYES(ph)LK_

S31*

50.2

_AVS(ph)ARAEAIK_

S66

P41105


122.96

_RAS(ph)AILR_

S115

Q9D1R9


99.5

_RLSYNT(ph)ASNK_

T15

62.861

_RLS(ph)YNTASNKTR_

S12

Q6ZWV7


72.1

_VVRKS(ph)IAR_

S53*

P83882

60S ribosomal protein L36a

69.5

_GKDS(ph)LYAQGK_

S32*

Q9D8E6


184.2

_ILKS(ph)PEIQR_

S295

P47963


155.6

_KGDS(ph)S(ph)AEELK_

S139

155.6

_KGDS(ph)S(ph)AEELK_

S140

P62270


173

_RAGELT(ph)EDEVER_

T60

*, novel phosphorylation site in ribosomal proteins. 27


Position


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

8

Figure legends

9

Figure 1. Detection of phosphoproteome in mouse liver microsomes. Electrophoretic

10

separation of microsomal proteins, as revealed by Coomassie staining (A) and

11

screening of phosphoproteome by Western blot (B). The individually prepared three

12

microsomal fractions (lanes 1–3, 30 µg of protein per lane) were separated on a 12%

13

Tris-glycine gel, followed by immunoblotting using anti-phospho-Ser, phospho-Thr,

14

and phospho-Tyr antibodies, respectively.

15

Figure 2. Scheme of the analytical workflow to identify phosphorylation sites in mouse

16

liver microsomes. Experimental workflow (A), area-proportional Venn diagram for

17

overlapping identified unique phosphoproteins (B), and frequency of phosphoserine,

18

phosphothreonine, and phosphotyrosine (C). The microsomes had been prepared from

19

three independent liver microsome preparations.

20

Figure 3. Motif analysis of Ser and Thr phosphorylation sites in mouse liver microsomes.

21

Acidic (A), basophilic (B) and proline-directed (D) motifs. Sites that were within six

22

residues of protein termini were not used in the motif logo. Ser and Thr

23

phosphorylation sites were identified in mouse liver microsomes by Motif X using the

24

13-mer sequences.

25

Figure 4. Characterization of mouse liver microsomal phosphoproteome. Phosphorylated

26

protein functional enrichment by biological processes, cellular component, and

27

molecular function (A) and phosphorylated protein functional enrichment by KEGG

28

pathway (B).

29

Figure 5. Immunoblots to detect phosphorylation in mouse liver microsomes after

30

reaction with PKA and CKII. Lane 1; reaction without PKA, lane 2: reaction with 28


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31


PKA, lane 3: reaction without CKII; lane 4: reaction with CKII.

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

32

Figure 1.

33

30


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34


Figure 2

35 36

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

37

Figure 3

38 39

32


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40


Figure 4

(A)

mRNA processing

4.84

Organic substance transport

3.91

Translation

Biological Process

3.74

Establishment of localization in cell

3.58

mRNA metabolic process

3.31

RNA processing

2.17

Cellular localization

2.12

RNA splicing

2.08

Ribosome

Cellular Component

5.98

Spliceosomal complex

4.32

Organelle membrane

4.18

Ribonucleoprotein complex

4.16

Endoplasmic reticulum part

3.85

Endoplasmic reticulum membrane

3.69 2.98

Endoplasmic reticulum Nuclear outer membrane-endoplasmic reticulum membrane network Endomembrane system

Molecular Function

2.77 2.07

Oxidoreductase activity

10.8

RNA binding

3.75

0

2

4

6

8

10

Fold enrichment

(B) (mmu03010) Ribosome

4,40

(mmu04141) Protein processing in endoplasmic reticulum

4.29

(mmu00140) Steroid hormone biosynthesis

3.87

(mmu03013) RNA transport

3.79

(mmu03040) Spliceosome

3.45

(mmu04910) Insulin signaling pathway

2.27

0

1

2

3

4

Fold enrichment

41 42

33


5

12


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

43

Figure 5

44

34


Page 34 of 34

Comprehensive Analysis of in Vivo Phosphoproteome of Mouse Liver

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