mk2 phosphorylates tristetraprolin on in vivo sites including s178, a

1

MK2 phosphorylates tristetraprolin on in vivo sites including S178, a site required for 14-3-3 binding

Carol A. Chrestensen1, Melanie J. Schroeder2,*, Jeffrey Shabanowitz2, Donald F. Hunt2, Jared W.

Pelo3, Mark T. Worthington3, and Thomas W. Sturgill1,+

From the Departments of Pharmacology1, Departments of Chemistry and Pathology2, and the Digestive Health Center of Excellence3, University of Virginia Health Sciences Center,

Charlottesville, VA 22908

Running Title: MK2-induced phosphorylations of TTP +Corresponding author *Completed in partial fulfillment of the requirements for the Ph.D. degree, University of Virginia, Charlottesville, VA Address for correspondence: Thomas Sturgill M.D.-Ph.D., Box 800735, Department of Pharmacology, University of Virginia, Charlottesville, VA 22908 Telephone: 434-924-8659 Facsimile: 434-924-5207 eMail: [email protected]

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on December 19, 2003 as Manuscript M310486200 by guest on February 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

2

Summary MK2 (MAPKAP kinase 2) is required for TNF synthesis. Tristetraprolin (TTP) binds to the 3’

UTR of TNF mRNA and regulates its fate. We identified in vitro and in vivo phosphorylation

sites in TTP using nHPLC-µESI mass spectrometry and novel methods for direct digestion of

TTP bound to affinity matrices (GSH-beads or anti-myc linked to magnetic beads). MK2∆3B,

activated in E. coli by p38α, phosphorylates TTP in vitro at major sites S52 and S178 (>10 fold

in abundance) as well as at several minor sites that were detected after enriching for

phosphopeptides with IMAC. MK2 phosphorylation of TTP creates a functional 14-3-3 binding

site. In cells, TTP was phosphorylated at S52, S178, T250, S316 and at SP sites in a cluster

(S80/82/85). Anisomycin treatment of NIH 3T3 cells increased phosphorylation of S52 and

S178. Over-expression of MK2 sufficed to increase phosphorylation of S52 and S178, but not

S80/82/85 or T250. Thus, S52 and S178 are putative MK2 sites in vivo. Identified

phosphosite(s) may be biologic switches controlling mRNA stability and translation.

Key words: MAPKAP kinase 2/mRNA stability/Tristetraprolin

by guest on February 18, 2018http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

3

Introduction

MK2 mediates several p38α,β MAP kinase dependent processes (for review see (1)),

demonstrated most clearly by results from targeted disruption of the MK2 gene in mice (2).

MK2 (-,-) mice have suppressed stress responses. Cellular studies show deficits in motility,

chemotaxis, and cytokine production. Macrophages taken from MK2 (-,-) mice exhibit normal

TNF mRNA induction in response to endotoxin but do not release TNF protein. Cellular TNF

protein was markedly decreased in MK2 (-,-) macrophages, suggesting a block in production of

TNF from TNF mRNA (2). TNF expression is regulated both via mRNA stability and

translation (3,4), but is not completely understood.

The p38α,β MAP kinase pathway regulates stability of mRNAs that contain AU-rich

elements (AREs) in their 3’ untranslated regions. Examples include TNF, COX-2, IL-6, and IL-

1β (4-6). Evidence chiefly comes from mRNA stabilization caused by transfection of

mutationally activated MEK3/MEK6 or by addition of agents that activate p38 MAPK, and

conversely from destablization caused by addition of a p38α,β MAP kinase inhibitor. Since

p38α,β is required to activate MK2, these experiments do not dissect contributions from MK2.

Studies in MK2 (-,-) cells suggest MK2 regulates stability of some cytokine mRNAs (2,4). Lasa

et al. (5) first reported that expression of a mutant of MK2 with constitutive activity stabilized

COX-2 mRNA in the presence of SB2035780 and that expression of a kinase-defective MK2

blocked the stabilization induced by activated MEK6, arguing that MK2 is necessary and

sufficient to induce stabilization of at least the COX-2 mRNA (5). How MK2 regulates cytokine

production post-transcriptionally is unknown. Mahtani, K.R. et al. (7) reported that

tristetraprolin (TTP) is an in vitro substrate for MK2, motivating the detailed studies we describe.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

4

TTP (for review see (8)) destabilizes class II AREs and is the prototype for a non-zinc-

finger class of nucleic acid binding proteins. Destabilization requires integrity of TTP’s tandem

Cys3His RNA binding domains that coordinate zinc in a disk-like structure (9-11). TTP-null

mice exhibit many defects including inflammatory arthritis and systemic lupus erythematosis-

like symptoms attributed to increased production of TNF (12).

TTP is phosphorylated in cells treated with growth factors or cytokines. Phosphorylation

occurs at more than one site evident by appearance of two distinct slower migrating forms of

TTP on gels after stimulation that are reversed by phosphatase treatment (7,13). TTP undergoes

detectable gel shifts between ~1- 1.5h, and the shifts persists for several hours (7,13). The only

reported TTP phosphorylation site is S220, phosphorylated by ERK2 in vitro (14). p38α also

phosphorylates TTP in vitro, but at unresolved sites (13). S178 mutation in human TTP causes

loss of ability to bind 14-3-3, suggesting S178 is a phosphorylation site (15), but the responsible

kinase(s) have not been identified.

We studied phosphorylation of TTP by potently active, recombinant MK2∆3B produced

in E. coli as well as in situ phosphorylation of myc-TTP expressed in cells. We identified two

major (S52 and S178) and several minor MK2 sites. Description as major sites reflects

abundance of phosphopeptide and estimated stoichiometry in peptides recovered with the

methods we describe. Minor sites detected are not excluded as candidates for functionally

significant sites. We show that the major sites (S52, S178) phosphorylated by MK2 are

phosphorylated in vivo and their phosphorylation can be altered by manipulating the p38


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

5

pathway in vivo. Furthermore, phosphorylation of TTP by MK2 confers ability to bind 14-3-3

proteins. Phosphorylation(s) of TTP may provide biological switches to regulate its dynamic life

cycle and/or its functions to control mRNA stability and translation.

Experimental Procedures

Materials

Several plasmids were generous gifts from colleagues: C. Moroni provided murine TTP in

pCDNA3.1/myc-HisA (16); M. Cobb, pT7.5-mup38-huMEK4 (17); C. Marshall, pEF myc WT

and KD (D207A) huMK2 (18); M. Gaestel, pGEX-muMK2∆3B (19) and pGEX-MK2EE∆3B

(19). pKH3-hu14-3-3β is from our laboratory. Anti-TTP antibodies to the mouse whole protein

(13) and human C-terminal peptide (20) were kindly provided by J. Han and W. Rigby,

respectively. Commercially acquired reagents: horseradish peroxidase (HRP) linked anti-goat

antibodies (Santa Cruz Biotechnology); tosylactivated Dynabeads (Dynal Biotech); γ-bind

Sepharose plus, glutathione Sepharose 4B; HRP linked anti-mouse and anti-rabbit antibodies

(Amersham Biosciences); anisomycin, SB203580, and microcystin LR (Calbiochem); BHK-21

cells, NIH 3T3(ATCC).

Plasmids for expression of active MK2∆3B

A bicistronic plasmid (pTYB4 His6p38α-HA1MEK6EE) was created by moving the coding

region of His6-tagged WT p38α with the ribosomal binding site engineered after the stop codon

(17) into the NcoI site of pTYB4 (New England Biolabs). HA-tagged MKK6EE (excised from

the pcDNA3 parent vector with NotI and Bgl2 followed by Klenow fill-in) was blunt-cloned into


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

6

the p38-pTYB4 construct at the NruI site. PCR was used to create an IPTG inducible MK2

expression vector with a p15A origin of replication (pAC-pET MK2∆3B), similar in overall

strategy to construction of pAC-pET RSK2CTD (21). pAC-pET MK2∆3B expresses a GST-

MK2∆3B fusion protein having an intervening 70 aa leader polypeptide containing His6 and S

tags, in addition to thrombin and enterokinase cleavage sites. All constructs were verified by

sequencing.

Protein production and purification

BL21 E. coli were transformed with pAC-pET MK2∆3B with or without pTYB4 His638α-

HA1MEK6EE using appropriate antibiotic selections. Transformed cells were grown and

induced, lysates prepared, and proteins purified as described (21). GST-TTP was expressed and

purified as in (22). Purified proteins were quantified with the Bio-Rad Protein Assay™ and with

SDS-PAGE analysis. Phosphocellulose paper assays were used to monitor kinase activity of the

purified GST or His6-tagged proteins. The kinase reactions contained (final, in 40µl) 25mM

HEPES, pH 7.4, 5mM β-glycerophosphate, 100mM NaCl, 2mM DTT, 0.25mg/ml BSA, 10mM

MgCl2, 50µM [γ-32P]-ATP (~4000cpm/pmol), 50µM peptide substrate and were incubated at

30°C for 15 min.

TTP phosporylation and in gel digestion

Reactions to phosphorylate TTP in vitro for site identification by MS/MS (see below) used the

same kinase cocktail minus [γ-32P]ATP and usually omitted BSA. GST-TTP (2µg in 20µl final

volume) was phosphorylated with 60ng of MK2∆3B for 1h (30o). A parallel reaction was always

performed that included [γ-32P]ATP. Initially, in vitro kinase reactions were stopped with


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

7

sample buffer and run on 4-20% gradient mini gels. Phosphorylation of TTP could also be

visualized by gel shift on mini gels when separation times were extended so as to run lower

molecular weight species off the gel. Details are provided in the legends.

Cell culture, transfection, and lysates

NIH 3T3 cells were grown at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium

supplemented with 10% newborn calf serum. BHK cells were grown similarly, substituting 5%

FBS for calf serum. Cells were transfected using Lipofectamine 2000 (Invitrogen) under normal

serum conditions, essentially as directed by the manufacturer using 32µg of DNA per 15cm dish

(16µg of TTP and either 16µg of MK2 or pCDNA3.1 plasmids). Post-transfection (~24hrs) the

cells were treated as described in the legends. Cell lysates were made as described (23) in 50mM

Tris, pH 8, at 4°C, 50mM NaCl, 1% Nonidet P-40, 0.5% Triton X-100, 1.5mM MgCl2, 10µg/ml

each of leupeptin and aprotinin, 1µM PefaBlock (Roche Applied Science), 200µM Na3VO4, and

1µM microcystin LR. The lysates were either processed immediately or quick frozen in liquid

N2 for later use.

Immunoprecipitation using magnetic beads

Tosylated Dynabeads (Dynal Biotech, Norway) were covalently attached to pure monoclonal

myc-antibody as directed by the manufacturer. Conjugated anti-myc IgG beads (1 x 108 of

Dynabeads) were incubated with cleared cellular lysates for 2h. After incubation, the beads

were magnetically concentrated and the lysate removed. The beads were washed 3X PBS

(Invitrogen) or for higher stringency 1X with PBS, 2X with PBS containing 0.1M NaCl, and 1X

with PBS. A portion of the IP was taken for Western blot analysis and the remainder was


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8

washed 3X with 0.1M NH4HCO3. These NH4HCO3 washed samples were then subjected to

digestion and MS/MS analysis (see below).

Sample preparation for analysis with mass spectrometry

After experimentation with gel based methods that were found to be problematic (see Results

and Discussion), we devised a new scheme to be termed ‘on beads’ digestion. Kinase reactions

were terminated by adding cold GSH-Sepharose beads (20µl of a 1:1 slurry), placing the tubes

on ice, and washing several times with cold PBS to remove ATP (necessary for IMAC). ATP

removal was monitored for sufficiency by following loss of 32P from a parallel hot reaction.

Then the beads were washed 3X with 100mM NH4HCO3 and re-suspended in 50µL. Bound

TTP was reduced with DTT (5mM final, for 1hr at 51°C) followed by iodoacetamide alkylation

(15mM final, 45mins RT, dark). Sequencing grade trypsin (Promega, Madison, WI) was then

added (1:20 ratio) with agitation for 12hrs at RT. The beads were pelleted, and the supernatant

was transferred to a new tube where acetic acid was used to lower the pH to 3.5. To generate

samples for in vivo phosphorylation, the above “on-beads” digestion procedure was repeated for

the TTP immmunoprecipitated with Dynabeads except sample reduction and alkylation was

omitted to minimize antibody digestion. Also, the digestion time was reduced to 6h for the same

reason.

Mass spectrometry

The samples were analyzed by reverse phase nanoflow HPLC micro ESI tandem mass

spectrometry (RP nHPLC µESI MS/MS) on a ThermoFinnigan LCQ Deca or XP ion trap (San

Jose, CA) operating in either data-dependent or targeted mode. Immobilized metal affinity


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

9

chromatography (IMAC) was performed according to (24) except the peptides were not reacted

to form methyl esters, and the elution was done with 7µL of 500mM ascorbic acid. A gradient

consisting of 0–60% B in 50 min, 60–100% B in 10 min (A = 100mM acetic acid (Sigma-

Aldrich) in water, B = 70% acetonitrile (Mallinkrodt), 100mM acetic acid in water) was used to

elute peptides from the reverse-phase column to the mass spectrometer through an integrated

electrospray emitter tip with a flow rate of ~60nl/min. Spectra were acquired with the instrument

operating in the data-dependent mode throughout the HPLC gradient. In this mode, every ∼10

sec, the instrument cycled through acquisition of a full-scan mass spectrum and five MS/MS

spectra (3 Da window; precursor m/z ±1.5 Da, collision energy set to 35%, dynamic exclusion

time of 1 min) recorded sequentially on the five most abundant ions present in the initial MS

scan. For the in vivo KD and WT MK2 samples, analysis was performed in the data-dependent

mode and run in duplicate. Because this sample was more complex than the in vitro sample, the

gradient was changed to 0-15%B in 7 mins, 15-25%B 20 mins, 25-60%B 35 mins and 60-

100%B 10 mins. This allowed for better resolution of the TTP peptides. In addition, these

particular in vivo samples were subjected to a time-segmented targeted analysis in order to obtain

the most data points per peptide peak of interest. Four peptides and their corresponding

phosphorylated forms were chosen for this analysis. In targeted mode, the instrument was

instructed prior to the run to repeat a cycle consisting of a full MS scan followed by a targeted

MS/MS scan of the (M+2H)++ ion for each pair of non- and phosphorylated peptide based on

their retention times (collision energy set to 35%) (see Fig. 6C).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

10

Peptide Identification

All MS/MS spectra acquired were searched against either a GST- or myc-TTP protein database

by using the SEQUEST algorithm (25). Search parameters included a differential modification

of +80 Da (presence or absence of phosphate) on serine, threonine, or tyrosine and a static

modification of 57 (alkylated Cys for the in vitro analysis only). All possible enzymatic

cleavage sites were considered. Peptide hits were manually confirmed.

Calculations

Estimated stoichiometries were calculated by dividing the sum of the area under the curve from

the phosphopeptide in all of its charge states and modified forms by the total peptide signal. In a

single sample, this is an approximation because phosphopeptides and their nonphosphorylated

forms generally do not ionize with the same efficiency. In addition, we estimated changes in

stoichiometry of site phosphorylation by ratios of the estimated stoichiometries. Provided that

this comparison is confined to the same phospho- and dephosphopeptide in the two conditions,

we can make direct comparisons of the relative change in stoichiometry between two conditions

with greater confidence.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

11

Results

Selective activation of GST-MK2∆3B by p38α in E. coli

To activate MK2∆3B, we created a GST-MK2∆3B expression plasmid with a p15A origin of

replication (Methods) that would be compatible with the bicistronic vector expressing both

constitutively active MEK1 and WT ERK2 (17). This co-expression system was used previously

in our lab to activate GST-RSK CTD (21) and ERK2 has been reported to activate MK2 (26).

For this reason, we co-expressed MK2∆3B with active ERK2 but this resulted in no activation of

MK2∆3B. The two proteins also showed no detectable interaction, demonstrated by lack of

ERK2 retention on the affinity column (Figure 1A, panels 1-4).

We designed a new bicistronic plasmid expressing constitutively active HA-MEK6 and

WT HIS-tagged p38α. When MK2∆3B is expressed in E. coli with active p38α produced from

this bicistronic plasmid, the kinase cascade was reconstituted. MK2∆3B was phosphorylated,

demonstrated by reduced mobility of the protein in SDS-PAGE analysis (Fig. 1A & B) and

increased peptide kinase activity (Fig. 1C). In contrast to ERK2, p38α was retained on the

affinity column and binding to GST-MK2∆3B was disrupted by washes with 2M LiCl, consistent

with an electrostatic interaction (Fig. 1A, panels 5-6). Phosphorylated MK2∆3B is dramatically

more active than the mutant MK2EE∆3B (Fig. 1C).

To identify sites in MK2∆3B whose phosphorylation was induced by p38α in E. coli, a

tryptic digest of the activated, purified GST-MK2∆3B was analyzed with LC MS/MS.

Phosphorylation of two of three S/T-Pro sites, at T222, S272 and T334 in human MK2, are


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

12

required for activation (26). T222 is within the activation loop, S272 is located in the C-lobe of

the kinase domain (27), and T334 is adjacent to an autoinhibitory helix. Our analysis showed

that the equivalent three sites are phosphorylated on MK2∆3B activated by p38α in E. coli (data

not shown). Phosphorylation at the equivalent of T338, a presumed autophosphorylation site in

human MK2 (26), was also detected in the purified active enzyme. These same sites were not

detectably phosphorylated in MK2∆3B co-expressed with active ERK2 (data not shown).

MK2∆3B phosphorylates recombinant GST-TTP

GST-TTP protein was phosphorylated by MK2∆3B in a time-dependent manner similarly to the

known in vivo substrate HSP27 while GST alone was either minimally or not at all

phosphorylated (Fig. 2A; data not shown). MK2 phosphorylation sites in TTP were initially

analyzed using peptides recovered from silver-stained SDS-PAGE bands of phosphorylated TTP

(28). An example of a gel run for this analysis is shown in Fig. 2B. Phosphorylation of TTP

causes a mobility shift of TTP that can be resolved with increased electrophoresis. Analyses of

TTP from gels were problematic due to multiple phosphorylated bands, co-migration of the most

gel-shifted band with BSA, and poor recovery that precluded IMAC to identify minor sites.

‘On beads’ digestion of TTP increases both coverage and recovery

To resolve these problems, we devised ‘on beads’ digestion methodologies of general utility for

future MS/MS analyses. We purified the GST-TTP from the kinase reaction with GSH-

Sepharose beads. While still bound, TTP was reduced, alkylated and digested with trypsin. TTP

peptides in the supernatant were analyzed by reverse phase and IMAC MS/MS (Fig. 3). Sample

from ‘on beads’ digestion was found to have increased protein coverage (70%) compared to the


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

13

in-gel digested sample (49%). ‘On beads’ digestion allows for better protein recovery,

eliminates the possibility of selection bias in excision of gel bands, and provides more material

for repeated analyses from a given sample.

S52 and S178 are major in vitro sites for MK2

Reverse phase MS/MS (Fig. 3) allowed us to compare m/z intensities for both the

unphosphorylated and phosphorylated peptides and to calculate an estimate of the percentage of

TTP phosphorylated at a particular residue of interest (Table I). The ability to enrich for

phosphopeptides on an Fe3+ POROS 20 IMAC column (Fig. 3) significantly increased our ability

to detect phosphopeptides, allowing us to identify four additional in vitro phosphorylation sites.

The calculated percent phosphorylation from the reverse phase analysis corresponds well with

peptide intensity signals from the IMAC analysis. Because these calculations of stoichiometry

are semi-quantitative, we will describe the TTP residues we have defined as sites of MK2∆3B

phosphorylation as “major” and “minor” phosphosites.

In Table I, the sites identified in muTTP (accession P22893) include serines 52, 58, 105,

176, 178, and 316. Of note, phospho-S52 and phospho-S178 peptides were more than an order

of magnitude more abundant than the other phosphopeptides identified, and are therefore

referred to throughout as major sites. Two additional singly phosphorylated peptides were also

identified as minor sites (T249 or T250 and S264 or S266, respectively), based on the available b

and y ions. T250 or S264 if assigned are in S/T-P motifs that could alternatively be

phosphorylated by small amounts of p38α present in the preparations of GST-MK2 (Fig. 1A).

The other, more prevalent, sites are phosphorylated by MK2∆3B.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

14

Minor sites S105 and S266 are in motifs that exactly match the current consensus

sequence for MK2 phosphorylation (ΦxRxxS) where Φ is a bulky hydrophobic residue (29).

The major MK2 sites on TTP have similarity to the current consensus but are not exact matches.

S178 has L at p-5; S52 has R at the p-3 position. Their preferential use may indicate

unrecognized determinants, possibly the p+1 F for S178, or secondary and tertiary factors. Other

MK2 substrates, including p16-Arc and vimentin, have non-consensus sites (30,31).

MK2 phosphorylation of TTP creates a 14-3-3 binding site

If S178 is a phosphorylation site for MK2, MK2 phosphorylation should cause a gain of function

for 14-3-3 binding. To test this, GST-TTP was phosphorylated in vitro by either MK2∆3B or

p38α and then tested for altered 14-3-3 binding compared to unmodified GST-TTP. As a source

we used BHK cell lysates that either were transfected with 14-3-3β (Fig. 4A) or were not

transfected (Fig. 4B). After incubation of phosphorylated TTP with lysate, GSH-Sepharose

beads were added to the mixture. After further incubation the beads were recovered and washed

extensively before elution of TTP for analysis by Western blotting with anti-TTP and pan anti-

14-3-3 antibodies. MK2 phosphorylation caused TTP to gel shift. GST-TTP phosphorylated by

MK2 bound 14-3-3 proteins from the lysates, including endogenous 14-3-3, while in contrast the

unphosphorylated and p38α phosphorylated proteins did so only slightly, and only when 14-3-3β

was over-expressed (Fig. 4A).

TTP phosphorylated by MK2 in vitro binds RNA

TTP phosphorylated by MK2∆3B bound a TNF ARE-probe (22) to a similar extent as untreated

TTP (data not shown). This suggests that phosphorylation of the MK2 sites does not inhibit


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

15

RNA binding. Similarly, phosphorylation of TTP by ERK, p38 or JNK did not alter binding of

TTP to a different ARE probe (32).

TTP is predominantly phosphorylated in growing BHK cells

A significant portion of the expressed myc-TTP in BHK cells is phosphorylated at the 24h time

point (Fig. 5; data not shown). Expressed myc-TTP was detected in Western blots using two

different antibodies, a polyclonal antisera to recombinant human TTP (left panel) (13) and

monoclonal antibody to the myc tag (right panel). Endogenous TTP in untransfected BHK cells

was not detectable. In lysates from these cells and others, we usually observe three TTP bands in

different proportions by Western blotting. In BHK cells, the majority of TTP was detected in the

band having the slowest mobility (Fig. 5).

We treated equivalent plates of BHK cells for 1h with or without 10µM SB203580, a

p38α,β inhibitor. SB203580 caused a small but significant downshift, evident by an increase in

the fraction of TTP migrating as the middle band (Fig. 5 and data not shown). This result

suggests that activation of pathways other than p38α,β are sufficient to induce shifted

phosphorylated states of TTP, an expected result since mitogens induce TTP and TTP

phosphorylation (14). Mitogen and cytokine stimuli may induce phosphorylation of the same or

overlapping physiologic sites. Since TTP was predominantly gel shifted and phosphorylated in

BHK cells, we expressed myc-TTP to generate mass spectrometry samples for identification of

in vivo phosphosites on TTP.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

16

‘On beads’ digestion of TTP bound to anti-tag Ab enables direct in vivo MS/MS analyses

Our success with ‘on beads’ digestion in the in vitro studies led us to devise an ‘on beads’

approach of general utility for studying post translational modifications of proteins in cells.

Myc-antibody was covalently bound to magnetic Dynabeads, chosen because the

physicochemical properties of the beads decrease non-specific interactions. The antibody-linked

beads were used to immunoprecipitate myc-tagged TTP from transfected BHK cells. Then, after

extensive washing with PBS and 100 mM NH4HCO3, the antibody-bound protein was subjected

to trypsin digestion without reduction and alkylation in order to minimize digestion of the myc-

antibody.

Identification of TTP phosphosites in BHK cells

To identify the sites phosphorylated, we analyzed peptides obtained by ‘on beads’ digests of

myc-TTP from proliferating BHK cells by MS/MS. The resultant peptides enabled us to clearly

detect the major MK2 phosphorylation sites (S52 and S178), two minor sites (S316 and

T249/250) that could resolve to an S/T-P site (T250), and an additional S/T-P site, S82 (likely

S80/82/85), which was not phosphorylated in our in vitro experiments (data not shown). S220

was in an unfavorably sized tryptic peptide and was not detectable in vivo in either phospho- or

dephosphoform.

To determine whether the p38 pathway was contributing to phosphorylation of the major

MK2 phosphosites in BHK cells, we treated transfected cells in parallel with the p38α,β

inhibitor. SB203580 caused a small decrease in phosphorylation of S178 in myc-TTP (Fig. 5)

(the S52 site was not well resolved in these analyses, data not shown) consistent with the small


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

17

decrease in the fraction gel shifted. These results necessitated finding a suitable cell line in

which expressed TTP was less extensively phosphorylated so that phosphorylation of TTP might

be induced by experimental manipulation of the p38 pathways.

TTP was less extensively phosphorylated in both RAW 264.7 macrophages and NIH 3T3

cells (data not shown). 3T3 cells transfected well enough to provide sufficient myc-TTP and

RAW cells did not. BHK cells were transfected and maintained in complete medium containing

fetal bovine serum (FBS). Twenty-four h post transfection, 3T3 as well as BHK cells were

normal in appearance without evidence of apoptosis. Apoptosis occurs to a variable but

significant extent when some cell lines, such as 3T3 cells (BHK cells were not studied), are

transfected with TTP constructs using different conditions that included removal of serum for 6 h

(33,34). Continued maintenance in media with FBS may be important in avoiding apoptosis, but

was not studied here.

S52, S178, T250, S316 and SP sites within a cluster (S80/82/85) are phosphosites in 3T3

cells

We analyzed ‘on beads’ digests of myc-TTP in duplicate in the data-dependent mode. These

results confirmed that, as in BHK cells, S52 and S178 were the major phosphosites in 3T3 cells

(data not shown). We also pursued peptides of interest more aggressively, by operating the ion

trap in targeted mode (Fig. 6C). In this way, we obtained the most data points per peptide of

interest as well as better quality spectra. This helped to clarify the minor phosphorylation site as

T250 rather than T249 and revealed that the peptide PGPELSPSPTSPTATPTTSSR was actually

a co-eluting mixture of three singly phosphorylated S*P species: (Ser80, 82, and 85).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

18

Both anisomycin treatment and MK2 expression increases S52 and S178 phosphorylation

As a test of p38 pathway responsiveness, we treated cells with or without anisomycin, which is

known to activate p38α and MK2 (35). In addition, 3T3 cells were co-transfected with TTP and

kinase defective (KD) or wild type (WT) MK2 plasmids. Treatment with anisomycin resulted in

increased phosphorylation at S52 and S178 (Fig. 6A). Co-expression of WT MK2 with TTP

resulted in a clear increase of S178 and S52 phosphorylation, but not S80/82/85 or T250, which

were possible MAP kinase sites. No increase in phosphorylation was observed with transfection

of the KD construct (Fig. 6B). The data obtained by averaging all three analyses as compared to

the targeted analysis alone produced very similar results. The KD construct did not act as a

dominant negative, as observed in other systems (36). We conclude that MK2 does

phosphorylate S52 and S178 in vivo, but our results do not rule out the possibility that these sites

may also be substrates for other kinases.

S52A/S178A TTP exhibits similar electrophoretic mobility to TTP

To determine the requirement for S52 and S178 for generation of phosphorylated forms with

reduced electrophoretic mobility, we made the double mutant. In cycling BHK cells,

S52A/S178A TTP has reduced electrophoretic mobility similar to wild type TTP (Fig. 6E). This

suggests that S52/S178 are not required for the changes in mobility. The reduced mobility of

S52A/S178A TTP is due to phosphorylation on other sites, evident by increased mobility after

treatment with calf intestinal phosphatase.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

19

Discussion

TTP is downstream of MK2 Previous work showed that TTP was a substrate for MK2 without defining the sites of

phosphorylation (7). We confirm phosphorylation of TTP by MK2 and identify specific

phosphorylation sites at S52, S105, S58, S176, S178, and S316. Two other sites (T250 and

S264) we identified in vitro may be phosphorylated by p38α, since it is a minor contaminant in

our preparations of active MK2. All of these sites are novel excepting S178 previously identified

through mutational analysis as a necessary residue for 14-3-3 binding (15).

We showed that phosphorylation of TTP by MK2 creates a binding site for 14-3-3

proteins. (In a different pathway, MK2 phosphorylation of the tuberous sclerosis 2 gene product

also creates a 14-3-3 binding site and serves as a precedent for our finding (37)). Recently, a

further indication of a regulatory network involving MK2 and 14-3-3 proteins was obtained in a

functional proteomics screen to identify MK2 substrates (38). MK2 interacts with 14-3-3ζ and

phosphorylates the monomer preferentially on a site in the dimer interface, S58 in subsequence

GARRSS58, causing decreased dimerization. Thus, both TTP and MK2, but not p38α, are 14-3-

3 interactors.

All of the potential MK2 phosphorylation sites identified as in vitro sites are conserved as

equivalent sites in mouse, rat, bovine, and human TTP. Of these, only the S178 and S316 sites

are present in the closely related human proteins Tis11b/ERF-1 or Tis11d/ERF2, although


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

20

sequences around these sites are not completely conserved. TTP, but not Tis11b or Tis11d,

shows synergy with TNF to cause apoptosis (15).

In our in vivo studies, we observed TTP phosphorylation at S52 and S178, two minor

sites S316 and T250, and the cluster of singly phosphorylated S80/82/85 sites we did not observe

in vitro. These sites were found in both BHK and 3T3 cells. The other minor sites were not

observed in vivo. This lack of detection does not indicate that these sites are not present in vivo,

as factors may have prevented their observation.

TTP phosphorylation at the putative MK2 sites was manipulated in vivo both by

anisomycin treatment that activates p38α and MK2 and by over-expression of MK2.

Importantly, increasing cellular WT MK2 was sufficient to induce phosphorylation. KD MK2

failed to induce phosphorylation, demonstrating that the effect of WT MK2 required its kinase

activity. KD MK2 did not lower basal phosphorylation, similar to previous results where co-

transfection of a similar KD MK2 construct had no detrimental effect on LPS-stimulation of

TNF production in WT macrophages (36). Although it is difficult to prove that a

phosphorylation in vivo is direct for any kinase/substrate pair these correlations strongly support

our conclusion that MK2 directly phosphorylates TTP in vivo.

MK2 could phosphorylate TTP in either the nuclear or the cytoplasmic compartment or

both. MAP kinases p38α,β are activated outside and then enter the nucleus. MK2 is activated in

the nucleus, and once activated may phosphorylate nuclear targets (see (1)) before it is exported


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

21

to the cytoplasm. Export of active MK2 in stressed fibroblasts is rapid, and complete by 20 min

(39), prior to maximal TTP expression.

MK2 may not be the only kinase phosphorylating TTP at the putative MK2 sites we

identify. MAPKAP kinase 3 and MAPKAP kinase 5/Prak are closely related to MK2, and thus

candidates. MK2 itself has two forms in mammals, revealed by in gel kinase assays as p55 and

p45 bands (35), which both become absent in MK2 (-,-) macrophages (2). The origin of these

MK2 forms is still unclear because evidence for cDNAs in EST databases that could correspond

to the shorter form identified biochemically is rare (36). In addition, kinases phosphorylating

MK2 sites in vivo could include kinases in the ERK pathways.

Novel procedure for obtaining Mass Spectrometry samples

So far as we are aware, we are the first to utilize direct digestion of protein samples bound to

affinity purification matrices, either GSH-Sepharose beads or myc-antibody linked to magnetic

Dynabead, to generate peptides for LC MS/MS analysis. In both cases, digestion directly from

the beads was an efficient way of producing sample, with a considerable increase in recovery

that provided sufficient peptides for multiple analyses (data-dependent or targeted) and with

different types of chromatography (RP or IMAC) from a small amount of protein. We

phosphorylated 2µg of TTP, and could identify all of the sites from a single ‘on beads’ digest

using only 10% of the material. In addition, the ability to analyze a protein purified from a crude

lysate after ‘on beads’ digestion should find immediate applications. Many signaling proteins of

importance can be expressed in a functional form as GST-fusions, allowing for direct application

of ‘on beads’ digestion after reduction and alkylation of the recovered protein. These methods


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

22

are applicable to other affinity reagents such as Ni2+ NTA beads for His-tagged proteins

(unpublished results), maltose bound beads for MBP-tagged proteins, and others (40).

It is important to note that the approach we used to minimize antibody contamination of

the in vivo sample (eliminating both reduction and alkylation of the sample) can produce altered

cleavage patterns of a protein and is a potential cause for reduced protein coverage. This is a

potential limitation, depending on the targeted protein. Protein interactors if still present could

also reduce the extent of cleavage. This is offset by the improved recovery of peptides by

avoiding gels, and by the practicality for studying in vivo phosphorylations by minor alteration of

generally available immunoprecipitation protocols. Bosworth, H.B. et al. (41) also used the

resistance of native IgG to trypsin to identify antigen epitopes by MALDI/MS.

For myc-TTP, cleavage ‘on beads’ reduced the extent of digestion in comparison to in

vitro digests somewhat, but we still recovered peptides to define in vivo phosphosites and

estimate stoichiometries. We cannot exclude that we have missed sites. For example, S105 was

a minor in vitro phosphosite, and S105 peptides were not abundant enough to detect in the in

vivo sample. S105 phosphopeptide recovery may be affected by proximity to the Cys3His repeat

and its physical properties. The S105 peptide elutes very early in the gradient and, therefore,

may not retain well on C18. This may also partly explain the observation that the S105

phosphopeptide was not recovered after IMAC, which depends on a final separation by C18.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

23

Points for TTP regulation by phosphorylation

In quiescent serum-starved fibroblasts or resting macrophages there is little TTP mRNA or

protein. Induction of TTP mRNA requires p38α,β (7) or ERK1,2 dependent pathways (42),

depending on the stimulus. The same pathways also induce mRNAs containing AU-rich

elements that are targets for TTP. In particular, LPS causes p38-dependent induction of TNF

and TTP mRNAs (43), but with differential regulation. TTP mRNA levels increase more

rapidly, peaking at ~1h and declining more rapidly thereafter, whereas levels of TNF mRNA

peak at ~2h and decline more gradually over the next 2-10h (13,43,44). Once translated, TTP

protein may localize to the nucleus (15,45,46), the cytoplasm (47,48) or both (48,49).

Leptomycin causes TTP to accumulate in the nucleus, proving that TTP shuttles. Induced TTP

protein levels are down regulated within 8h in human monocytes. The profiles vary with other

cell types and stimuli (20), suggesting that down regulation is regulated separately from

induction.

The established effector function of TTP is to negatively regulate mRNA stability. TTP

null mice have increased TNF mRNA and plasma TNF, and TNF mRNA is more stable in TTP

(-,-) cells than in WT cells. TTP additions to a cell-free system destabilize TNF mRNA by

stimulating deadenylation (50). Interestingly, endogenous TTP sediments with polysomes in

sucrose gradients (47), suggesting an additional unsuspected role for TTP in translation, and

possibly a positive one since polysomes usually contain the pool of actively translating mRNA.

Confocal microscopy data of endogenous TTP stained with a C-terminal antibody (47) suggest to

us a cytoplasmic localization of TTP into macromolecular assemblies. Endogenous TTP is

concentrated within unidentified, punctate substructures with more distinct borders in resting


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

24

cells and within more diffuse and more numerous substructures in stimulated cells. These

substructures are reminiscent of stress granules (51).

TTP phosphorylation could regulate its life cycle as well as its effector functions in

nuclear or cytoplasmic compartments. The sites are localized schematically in Fig. 7. When

TTP is nuclear in fibroblasts, its export to the cytosol can be stimulated by mitogens (15,46),

suggesting involvement of kinase cascades activated by mitogens.

TTP has an active nuclear export sequence (NES) within the first 14 amino acids at the

N-terminus (48,49). A sequence required for nuclear localization was mapped to sequence

surrounding the first Cys3His RNA binding domain, residues 95-130 in muTTP (48,49). Minor

in vitro phosphosite Ser105 lies within the NLS, and could silence NLS function. Major site

Ser52 and minor site Ser58 are located between the NES and NLS in the N-terminus as are

S80/82/85, making them candidates to regulate cellular localization and/or effector functions.

The N-terminus along with tandem Cys3His RNA binding domains, but not the C-

terminus, are required for induction of apoptosis caused by expression of TTP (34). The N-

terminus is not required to bind mRNA or to destabilize mRNA either in cells or in cell free

assays (50). Thus the requirement for the N-terminal domain could be confined to regulation of

import/export, but the N-terminus’s role might also include modulation of effector functions.

These may be regulated by N-terminal phosphorylations we describe. The N-terminus (aa 1-100

in muTTP) fused to GAL4 DNA has apparent transactivating activity in PC12 cells, which is

inhibitable by phorbol ester (42). Observed transactivating functions of TTP in macrophages are


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

25

also altered directly or indirectly by p38 kinase cascades. Expression of TTP robustly down

regulates basal luciferase activity from reporters of cytokine genes TNF and IL8, and it decreases

relative induction of the same reporters that, in the absence of TTP expression, are markedly

activated by LPS (13). Stimulation of p38α,β with LPS blocks suppression, and suppression is

blocked by p38α,β inhibition.

Major phosphosite S178 is in the C-terminal domain. Phosphorylation of S178 by MK2

may increase TTP levels in the cytoplasmic compartment following activation of the p38

pathway. S178 is not required for mitogen-stimulated export of TTP from the nucleus but is

required for 14-3-3 binding. Co-expression of TTP with 14-3-3β increased the level of

cytoplasmic TTP (15). Ser178 is not required for TTP effector function, assayed by ability of

S178A TTP to induce apoptosis.

Currently we do not know whether phosphorylation impacts the effector activity of TTP

as opposed to its life cycle, but phosphorylation could change protein/protein interactions that

facilitate deadenylation activity. Multienzyme complexes coordinate what happens at the 5’ and

3’ ends of mRNA (52). MK2 has also been shown to phosphorylate poly(A) binding protein 1

(53) and hnRNP AO (54). If TTP has a different function that favors, or is permissive, for

translation of cytokine, this would have to have a biologic switch. The major block in TNF

production in MK2 (-,-) cells is translation not stability (4). In TTP (-,-) mice, TNF is

overproduced. Thus as a hypothesis, phosphorylation of TTP at an N-terminal site (S52, S58) or

Ser105 in the nucleus could silence the NLS and/or enhance the NES activity, causing export of

TTP. Phosphorylation of S178 could increase its concentration or dwell time or bring another


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

26

protein to it via the other pocket of bound bifunctional 14-3-3, provided its other pocket does not

bind internally to another phosphosite in TTP. Loss of TTP does not block translation. If MK2

regulates translation in part by TTP phosphorylation, TTP should be a repressor of translation

when dephosphorylated and an activator (or neutral to) translation when phosphorylated.

Acknowledgements

We thank Mary Harp for technical assistance and many colleagues (named herein) who have

generously provided reagents. This work was supported by the United States Public Health

Service, National Institutes of Health Grants GM62890 (to T.W.S.), GM37537 (to D.F.H.), and

and DK/GM60720 (to M.T.W.). Support for C.A.C. was provided by Training Grant T32-

DK07320 (to C.A.C.)

References 1. Shi, Y., and Gaestel, M. (2002) Biological Chemistry 383(10), 1519-36 2. Kotlyarov, A., Neininger, A., Schubert, C., Eckert, R., Birchmeier, C., Volk, H. D., and

Gaestel, M. (1999) Nat Cell Biol 1(2), 94-7. 3. Han, J., Brown, T., and Beutler, B. (1990) J Exp Med 171(2), 465-75 4. Neininger, A., Kontoyiannis, D., Kotlyarov, A., Winzen, R., Eckert, R., Volk, H. D.,

Holtmann, H., Kollias, G., and Gaestel, M. (2002) J Biol Chem 277(5), 3065-8. 5. Lasa, M., Mahtani, K. R., Finch, A., Brewer, G., Saklatvala, J., and Clark, A. R. (2000)

Molecular & Cellular Biology 20(12), 4265-74 6. Kotlyarov, A., and Gaestel, M. (2002) Biochem Soc Trans 30(Pt 6), 959-63. 7. Mahtani, K. R., Brook, M., Dean, J. L., Sully, G., Saklatvala, J., and Clark, A. R. (2001)

Mol Cell Biol 21(19), 6461-9. 8. Blackshear, P. J. (2002) Biochemical Society Transactions 30(Pt 6), 945-52 9. Worthington, M. T., Amann, B. T., Nathans, D., and Berg, J. M. (1996) Proc Natl Acad

Sci U S A 93(24), 13754-9. 10. Amann, B. T., Worthington, M. T., and Berg, J. M. (2003) Biochemistry 42(1), 217-21. 11. Lai, W. S., Carballo, E., Thorn, J. M., Kennington, E. A., and Blackshear, P. J. (2000) J

Biol Chem 275(23), 17827-37. 12. Taylor, G. A., Carballo, E., Lee, D. M., Lai, W. S., Thompson, M. J., Patel, D. D.,

Schenkman, D. I., Gilkeson, G. S., Broxmeyer, H. E., Haynes, B. F., and Blackshear, P. J. (1996) Immunity 4(5), 445-54.

13. Zhu, W., Brauchle, M. A., Di Padova, F., Gram, H., New, L., Ono, K., Downey, J. S., and Han, J. (2001) Am J Physiol Lung Cell Mol Physiol 281(2), L499-508.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

27

14. Taylor, G. A., Thompson, M. J., Lai, W. S., and Blackshear, P. J. (1995) J Biol Chem 270(22), 13341-7.

15. Johnson, B. A., Stehn, J. R., Yaffe, M. B., and Blackwell, T. K. (2002) J Biol Chem 277(20), 18029-36.

16. Stoecklin, G., Ming, X. F., Looser, R., and Moroni, C. (2000) Mol Cell Biol 20(11), 3753-63.

17. Khokhlatchev, A., Xu, S., English, J., Wu, P., Schaefer, E., and Cobb, M. H. (1997) J Biol Chem 272(17), 11057-62.

18. Ben-Levy, R., Hooper, S., Wilson, R., Paterson, H. F., and Marshall, C. J. (1998) Curr Biol 8(19), 1049-57.

19. Engel, K., Schultz, H., Martin, F., Kotlyarov, A., Plath, K., Hahn, M., Heinemann, U., and Gaestel, M. (1995) J Biol Chem 270(45), 27213-21.

20. Fairhurst, A.-M., Connolly, J., Hintz, K., Goulding, N., Rassias, A., Yeager, M., Rigby, W., and Wallace, P. (2003) Arthritis Res Ther 5(4), R214 - R225

21. Chrestensen, C. A., and Sturgill, T. W. (2002) J Biol Chem 277(31), 27733-41. 22. Worthington, M. T., Pelo, J. W., Sachedina, M. A., Applegate, J. L., Arseneau, K. O., and

Pizarro, T. T. (2002) J Biol Chem 277(50), 48558-64. 23. Poteet-Smith, C. E., Smith, J. A., Lannigan, D. A., Freed, T. A., and Sturgill, T. W.

(1999) J Biol Chem 274(32), 22135-8 24. Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M.,

Shabanowitz, J., Hunt, D. F., and White, F. M. (2002) Nature Biotechnology 20(3), 301-5 25. Eng, J., McCormack, A.L., and Yates, J.R. (1994) J Am. Soc. Mass Spectrom. 5, 976-989 26. Ben-Levy, R., Leighton, I. A., Doza, Y. N., Attwood, P., Morrice, N., Marshall, C. J., and

Cohen, P. (1995) Embo J 14(23), 5920-30. 27. Meng, W., Swenson, L. L., Fitzgibbon, M. J., Hayakawa, K., Ter Haar, E., Behrens, A.

E., Fulghum, J. R., and Lippke, J. A. (2002) J Biol Chem 277(40), 37401-5. 28. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Analytical Chemistry 68(5),

850-8 29. Stokoe, D., Caudwell, B., Cohen, P. T., and Cohen, P. (1993) Biochem J 296(Pt 3), 843-

9. 30. Singh, S., Powell, D. W., Rane, M. J., Millard, T. H., Trent, J. O., Pierce, W. M., Klein, J.

B., Machesky, L. M., and McLeish, K. R. (2003) J Biol Chem. 278(38), 36410-7 31. Cheng, T. J., Tseng, Y. F., Chang, W. M., Chang, M. D., and Lai, Y. K. (2003) J Cell

Biochem 89(3), 589-602. 32. Cao, H., Dzineku, F., and Blackshear, P. J. (2003) Arch Biochem Biophys 412(1), 106-

120. 33. Johnson, B. A., Geha, M., and Blackwell, T. K. (2000) Oncogene 19(13), 1657-64. 34. Johnson, B. A., and Blackwell, T. K. (2002) Oncogene 21(27), 4237-46. 35. Cano, E., Doza, Y. N., Ben-Levy, R., Cohen, P., and Mahadevan, L. C. (1996) Oncogene

12(4), 805-12. 36. Kotlyarov, A., Yannoni, Y., Fritz, S., Laass, K., Telliez, J. B., Pitman, D., Lin, L. L., and

Gaestel, M. (2002) Mol Cell Biol 22(13), 4827-35. 37. Li, Y., Inoki, K., Vacratsis, P., and Guan, K. L. (2003) J Biol Chem 278(16), 13663-71. 38. Powell, D. W., Rane, M. J., Joughin, B. A., Kalmukova, R., Hong, J.-H., Tidor, B., Dean,

W. L., Pierce, W. M., Klein, J. B., Yaffe, M. B., and McLeish, K. R. (2003) Mol. Cell. Biol. 23(15), 5376-5387


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

28

39. Engel, K., Kotlyarov, A., and Gaestel, M. (1998) Embo J 17(12), 3363-71. 40. Terp, K. (2003) Applied Microbiology and Biotechnology 60(5), 523-533 41. Bosworth, H. B., Clipp, E. C., McNeilly, M., Christakis, N. A., Parker, J., Tulsky, J. A.,

and Parker, C. E. (2002) Journal of Palliative Medicine 5(6), 829-41 42. Murata, T., Hikita, K., and Kaneda, N. (2000) Biochem Biophys Res Commun 274(2),

526-32. 43. Carballo, E., Lai, W. S., and Blackshear, P. J. (1998) Science 281(5379), 1001-5. 44. Baseggio, L., Charlot, C., Bienvenu, J., Felman, P., and Salles, G. (2002) Eur Cytokine

Netw 13(1), 92-8. 45. DuBois, R. N., McLane, M. W., Ryder, K., Lau, L. F., and Nathans, D. (1990) J Biol

Chem 265(31), 19185-91. 46. Taylor, G. A., Thompson, M. J., Lai, W. S., and Blackshear, P. J. (1996) Mol Endocrinol

10(2), 140-6. 47. Brooks, S. A., Connolly, J. E., Diegel, R. J., Fava, R. A., and Rigby, W. F. (2002)

Arthritis Rheum 46(5), 1362-70. 48. Phillips, R. S., Ramos, S. B., and Blackshear, P. J. (2002) J Biol Chem 277(13), 11606-

13. 49. Murata, T., Yoshino, Y., Morita, N., and Kaneda, N. (2002) Biochem Biophys Res

Commun 293(4), 1242-7. 50. Lai, W. S., Kennington, E. A., and Blackshear, P. J. (2003) Molecular & Cellular

Biology 23(11), 3798-812 51. Kedersha, N., and Anderson, P. (2002) Biochemical Society Transactions 30(Pt 6), 963-9 52. Wilkie, G. S., Dickson, K. S., and Gray, N. K. (2003) Trends Biochem Sci 28(4), 182-8 53. Bollig, F., Winzen, R., Gaestel, M., Kostka, S., Resch, K., and Holtmann, H. (2003)

Biochem Biophys Res Commun 301(3), 665-70. 54. Rousseau, S., Morrice, N., Peggie, M., Campbell, D. G., Gaestel, M., and Cohen, P.

(2002) Embo J 21(23), 6505-14


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

29

Figure Legends

Fig. 1. Activation of MK2 in bacteria requires co-expression with active p38α. A)

Purification of GST-MK2∆3B after co-expression with activated ERK2 or p38 shows that only

p38 interacts with and activates MK2. Panel 1 - SDS-PAGE analysis of GST-MK2∆3B

expressed alone. Panel 2- SDS-PAGE analysis of GST-MK2∆3B co-expressed with active

ERK2. Panel 3- Western analysis of GST-MK2∆3B co-expressed with active ERK2 probed for

anti-ERK2. Panel 4- Panel 3 probed for anti-active ERK. Panel 5-SDS-PAGE analysis of GST-

MK2∆3B expressed with active p38α. Panel 6- Western analysis of GST-MK2∆3B co-

expressed with active p38 probed for anti-p38. An electrostatic interaction, disrupted by 2 M

LiCl, is observed between MK2 and p38 but not between MK2 and ERK2. B) SDS-PAGE

analysis of GST-MK2∆3B (GST-MK2EE∆3B) purified either alone or with activated ERK2 or

p38, only MK2 expressed with p38 is gel shifted. C) Kinase activity of purified GST-MK2∆3B

proteins using the p81 paper assay with a peptide substrate (KKLNRTLSVA). MK2 activated

by p38 has a much greater specific activity in comparison to the mutational activated protein

MK2EE∆3B. Results are representative of at least 2 independent experiments.

Fig. 2. Activated MK2 phosphorylates TTP in vitro. A) Kinase assays were analyzed by SDS-

PAGE, and incorporation of 32P into the substrates was visualized using a phosphoimager.

Active GST-MK2∆3B phosphorylates TTP and HSP27. The lower panel is the Coomassie stain

of these gels. B) SDS-PAGE analysis (4-20% gradient) of concentrated GST-TTP (6µg)

phosphorylated with activated GST-MK2∆3B (0.05µg), silver stain (left panel) and

PhosphoImage (right panel). Results are indicative of more than 3 independent experiments.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

30

Fig. 3. MS/MS analyses of MK2 phosphorylated TTP. A) Reverse phase and IMAC MS/MS

analysis of TTP peptides. TTP was treated as described in Fig. 2 and then the protein was re-

purified with GSH-Sepharose and digested with trypsin (1% of the digest analyzed for reverse

phase, 10% for IMAC).

Fig. 4. GST-TTP phosphorylated in vitro by MK2 interacts with endogenous 14-3-3. A)

GST-TTP (5µg) was incubated in various kinase reactions with or without activated GST-

MK2∆3B, or p38 (0.05µg) and then the reaction was combined with BHK lysates transfected

with 14-3-3. After incubation, the GST-TTP was bound to GSH-Sepharose beads, which were

washed and then analyzed by SDS-PAGE for interacting proteins. B) GST-TTP was treated as

in A but pre-treated with or without SB203580 (10µM), and the BHK cells used to make the

lysates were not transfected with 14-3-3, so only endogenous protein is detected. Results are

representative of 4 independent experiments

Fig. 5. Myc-TTP transfected into BHK cells is predominantly phosphorylated. BHK cells

transfected with myc-TTP were treated with or without SB203580 (10µM) 60 min. prior to

harvest. Anti-TTP, and Anti-myc antibodies were used on duplicate Western blots to gauge the

effect of p38 pathway inhibition on protein mobility. Results are representative of at least 4

independent experiments.

Fig. 6. In vivo analysis of TTP phosphorylation sites and modulation by MK2. A) Change in

relative TTP phosphorylation by treatment with anisomycin; 3T3 cells were transiently

transfected with expression vectors containing TTP and then treated with 5 µg/ml anisomycin for


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

31

20 minutes prior to harvest. Dynabead-purified proteins were digested into tryptic peptides,

which were analyzed in data-dependant mode (4% of digest). The results are presented as % of

peptide phosphorylated in treated and untreated sample. Results are the average ± range of two

MS/MS analyses. B) Lysates from cells co-transfected with TTP and either WT or KD MK2

were treated as in A, results of three independent runs averaged ± S.D. *- in targeted analysis on

samples from B (see part C), this was determined to be a co-eluting peak of three singly

phosphorylated species (S80/82/85) C) Elution order and abundance of eight TTP peptides

analyzed using targeted analysis of sample from B. D) Western blot analysis of cellular lysates

and an IP from 3T3 cells. E) Western blot analysis (anti-myc) of lysates from WT and

S52A/S178A mutant transfected BHK cells treated with or without calf intestine alkaline

phosphatase. The S80/82/85 peptide is ***PGPELSPSPTSPTATPTTSSR.

Fig. 7. Identified Sites in muTTP. NES (Red) is present in the first 14 residues. A segment

required for NLS activity includes a loop of the first Cys3His domain (Blue). MK2 sites S52 and

S178 were sensitive to anisomycin and MK2 expression. S80/82/85 were anisomycin sensitive

and MK2 expression insensitive, and are putative p38 sites.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

32

Table I - Summary of in vitro MK2 phosphorylation sites on TTP.

Phosphopeptide

Signal for Nonphos C18a

Signal for Phospho C18a

Percent Phosphorylatedb

Signal IMAC a

175QSIS*FSGLPSGR186

175QS*IS*FSGLPSGR186 1.3x108 1.3x108

1.4x108 53 1.2x108 3.3x106

50STS*LVEGR57 1.1x108 2.2x108 67 3.6x108 103TYS*ESGR109 4.6x107 8.2x106 15 Not

detected 308RLPIFNRIS*VSE319 9.0x106 3.5x106 28 2.9x107

58S*CGWVPPPPGFAPLAPR74 1.8x108 3.6x106 248ST?T?PSTIVGPLGGLAR263 1.5x108 2.3x106

264S?PS?AHSLGSDPDDYASSGSSL.. .GGSDSPVFEAGVFGPPQTPAPPR307

2.8x107 1.4x107

a) The signals for the non- and phosphorylated peptides are the sum of peak intensities for all detected charge states.

b) The percent phosphorylation is an estimate based on the ratio of the phosphorylated signal to the total signal.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

0%

10%

20%

30%

40%

50%

60%

Ser 52 Ser 178 Ser 80,82,85* Thr 250

Peptide

Perc

ent p

hosp

hory

latio

n

UntreatedAnisomycin

Fig. 6, Chrestensen et al.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

Ser 52 Ser 178 Ser 80,82,85* Thr 250

Peptide

Perc

ent p

hosp

hory

latio

n

Wild typeKinase dead

B

A


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Fig. 7, Chrestensen et al.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

W. Pelo, Mark T. Worthington and Thomas W. SturgillCarol A. Chrestensen, Melanie J. Schroeder, Jeffrey Shabanowitz, Donald F. Hunt, Jared

14-3-3 bindingMK2 phosphorylates tristetraprolin on in vivo sites including S178, a site required for

published online December 19, 2003J. Biol. Chem.

10.1074/jbc.M310486200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M310486200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M310486200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/12/19/jbc.M310486200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2003/12/19/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/12/19/jbc.M310486200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

mk2 phosphorylates tristetraprolin on in vivo sites including s178, a

Documents