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[Cell Cycle 6:14, e1-e10, EPUB Ahead of Print:; 15 July 2007]; ©2007 Landes Bioscience


Autophosphorylation Properties of Inactive and Active JNK2

Genaro Pimienta 1 Scott B. Ficarro 3

Gustavo J. Gutierrez 2

Anindita Bhoumik 2

Eric C. Peters 3

Ze’ev Ronai 2

Jaime Pascual 1, *

1 Inflammation and Infectious Diseases Center; 2 Cancer Center; Burnham Institute

for Medical Research; La Jolla, California USA

3 Genomics Institute of the Novartis Research Foundation; San Diego, California


*Correspondence to: Jaime Pascual; Burnham Institute for Medical Research;

10901 North Torrey Pines Road; La Jolla, California 92037 USA; Tel.:

858.646.3100; Fax: 858.646.3195; Email:

Original manuscript submitted: 04/23/07

Manuscript accepted: 05/10/07

This manuscript has been published online, prior to printing for Cell Cycle, Volume

6, Issue 14. Definitive page numbers have not been assigned. The current citation is:

Cell Cycle 2007; 6(14):

Once the issue is complete and page numbers have been assigned, the citation

will change accordingly.

KEy woRdS

JNK2/tandem mass spectrometry/autophos-

phorylation/time-dependent phosphorylation



This work was partially funded by grant

P01CA102583 to J.P.


Supplementary material can be found at:





The c‑Jun N‑terminal kinases (JNKs) are ubiquitous proteins that phosphorylate their

substrates, such as transcription factors, in response to physical stress, cytokines or UV

radiation. This leads to changes in gene expression, ensuing either cell cycle progression

or apoptosis. Active phospho JNK1 is the main in vivo kinase component of the JNK

cascade, whereas JNK2 is presumed not to participate as a kinase during JNK signal‑

ling. However, there is evidence that JNK isoforms interact functionally in vivo. Also, a

recent chemical genetics investigation has confirmed that JNK transient activation leads

to cellular proliferation, whereas a sustained one is pro‑apoptotic. Here we investigate

the phosphorylation pattern of JNK2, with protein biochemistry tools and tandem mass

spectrometry. We choose to focus on JNK2 because of its reported constitutive activity in

glioma cells. Our results indicate that purified JNK2 from transfected nonstressed 293T

cells is a mixture of the mono‑sites pThr183 and pTyr185 of its activation loop and of

pThr386 along its unique C‑terminal region. Upon UV stimulation, its phosphorylation

stoichiometry is upregulated on the activation loop, generating a mixture of mono‑pTyr185

and the expected dual‑pThr183/pTyr185 species, with the pThr386 specie present but

unaltered respect to the basal conditions.


The c-Jun N-terminal kinases (JNKs) are a subgroup of the mitogen activated protein

kinases (MAPKs), cloned initially from cDNA libraries of human liver 1 and rat brain. 2

These protein homologues were shown to phosphorylate the transcription factor c-Jun on

its N-terminal region, upon activation by TNFa (ref. 1 and ultraviolet (UV) radiation. 2-4

JNKs relay cellular signals conveyed by the MAPK cascade, when stimulated by physical

stress, inflammatory cytokines, such as IL-1 and TNF, or DNA-damaging agents in the

form of UV or ionizing radiation. 5 The JNK signalling cascade relies on protein scaffolds

to achieve specificity and spatial localization of the transferred signal. 6 It also functions

within the context of other signalling systems, such as the NFB cascade 7-9 and those

orchestrated by the PKC. 10,11 A consequence is that JNK signalling can initiate events of

cell cycle progression or apoptosis, depending on the cell-type and the nature of a given

stimuli. 12 Current models suggest that a transient stimulation of JNK generally leads to

cell proliferation, whereas a persistent activation is conducive to apoptosis. 12,13

Three genes are found in humans and mice that code for three JNK protein homo-

logues giving rise to 10 differentially spliced isoforms. 14 JNK1 and JNK2 are ubiquitous,

whereas JNK3 is tissue-specific and found mainly in the brain. 14,15 Like other MAPKs,

JNKs have a conserved protein kinase fold and a typical activation loop. This loop has the

sequence signature of Thr (T) -x- Tyr (Y) motif, where x is a Pro (P) in the case of JNKs. 16

A hallmark in JNK activation is its induction by the dual phosphorylation (on T and Y) of

the aforementioned motif. 17,18 This event is orchestrated by two upstream dual-specificity

(S/T and Y) MAPK kinases, the MKK4 19,20 and MKK7, 21,22 which in turn are activated

by a large number of upstream S/T MKK kinases (MAP3K). 17,18 JNKs are fully active

only when MKK4 and MKK7 act synergistically, to achieve the dual phosphorylation of its activation loop. 23-25 The bulk of endogenous JNK is represented by two JNK homologues (p46 and p55) that derive from the genes jnk1 and jnk2 respectively. Their amino acid sequences are about 80% identical, differing mainly by a C-terminal extension that makes JNK2 (p55) longer than JNK1 (p46). 14 Despite having similar gene expression patterns, their apparent redundancy is controversial and their individual biological relevance still not deciphered.

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Autophosphorylation Properties of Inactive and Active JNK2

In particular, genetic studies with mice embryonic fibroblasts (MEFs) suggest that active JNK1 is the main in vivo kinase, relaying the JNK cascade signal. 26,27 This has lead to the model that JNK1 and JNK2 have distinct, rather opposite functional roles. 28 This concept has recently been challenged by a chemical genetics report, which shows that JNK1 and JNK2 display a functional in vivo cross talk and rather have compensatory functional roles. 29 A possible explanation is that the differences among JNKs are dictated at the post-transla-

tional level, in the form of a differential phosphorylation pattern. If true, this may underpin their in vivo functional cross-talk. Here we explore this hypothesis by investigating with biochemical tools and tandem mass spectrometry (MS), the phosphorylation

pattern of JNK2 when overexpressed in E. coli and 293T cells. We

choose to work with JNK2 (p55), because we find in the literature

an emerging notion that JNK2 has autocatalytic properties. 30-32

Our results show that JNK2 but not JNK1 auto-activates in vitro

because it has dual-specificity autophosphorylation properties. It

phosphorylates itself on the T and Y residues of the activation loop.

In addition we identify a previously unknown phosphorylation

site, T386 that is an amino acid unique to JNK2 located along its

C-terminal extension.


Protein expression constructs. The DNA expression constructs

encoding GST-c-Jun N-terminal fragment (a.a. 1-89), Flag-JNK2,

His-JNK1 and His-JNK2 are those described previously. 10 Mutations

on His-JNK2 were introduced by using the Quick Change

Site-Directed Mutagenesis Kit (Stratagene) and confirmed by DNA

sequencing. Four single point mutants (K55R, T183D, Y185E and

T243A) were used in this study, one double phosphomimic point

mutant (T183D/Y185E) and a truncated form (a.a. 1-387) of JNK2

that overlaps with JNK1.

Protein expression in E. coli. The GST and 5xHis fusion

proteins were produced growing bacteria in Luria-Bertani Broth

(LB) supplemented with ampicillin at 37˚C. Typically, a fresh

colony transformed with a particular DNA construct, was picked

up from a solid LB plate and used to inoculate 1–2 mL of liquid

LB and incubated overnight. Next, this preculture was diluted to

1 L of LB in a 2 L flask and cultured until the optical density of the

media measured at 600 nm reached a value of 0.6–0.9. Recombinant

protein over-expression was induced with 0.25 mM of IPTG at this

point. Protein expression experiments lasted either 4 or 12 hours and

were performed at 15˚C or 37˚C, depending on the experiment. See

the results sections for details.

Protein purification from E. coli. All recombinant proteins were

prepared in a similar way. Only the affinity column used to enrich

them was different, as specified below. For the liquid chromatog-

raphy (LC) protocols described here, we have used a ƒKTA-prime

fast performance liquid chromatography (FPLC) system, with chro-

matographic columns (XK 16), affinity resins and other LC utilities,

from GE-Amersham Biosciences. E. coli expression milieu was

harvested at 4˚C by centrifugation (4500 rpm) during 30 minutes,

using a Sorvall SLA30 rotor. Cells were resuspended in ice-cold lysis buffer (25 mM Tris-HCl, pH 8.0, 500 mM NaCl and 1 tablet/L of an EDTA-free protease inhibitors cocktail from Roche) and lysed by sonication. Next, the lysate was centrifuged with a Sorvall SS34 rotator at 12,000 rpm/4˚C, to remove the insoluble cell debris. The resulting supernatant was loaded onto a 50 mL super-loop and injected on a preequilibrated affinity column, manually packed

with 20 mL of either Nickel-NTA or Glutathione Sepharose High Performance resin, for His or GST fusion proteins, respectively. After extensive washing (5 column volumes) with equilibration buffer (25 mM Tris-HCl, pH 8.0, 500 mM NaCl, supplemented with 2.5 mM imidazole in the case of Nickel column), the bound proteins were eluted with a linear gradient of either 2.5–200 mM imidazole for His fusion proteins or 0–50 mM reduced glutathione in the case of GST fusion polypeptides. The elution peak was collected and dialyzed over-night against 2 L of 25 mM HEPES, pH 7.6,

50 mM NaCl and 10 mM DTT. Proteins were purified further by

anion exchange, with a manually packed column containing 20 mL

of Mono Q Sepharose resin, using a linear NaCl gradient 0-1M,

with the same buffer conditions used to dialyze overnight. The pure

proteins were concentrated by centrifugation with an Amicon Ultra

concentrator (10,000 Da molecular weight cut off, MILLIPORE),

to about 1 mg/mL and used immediately or flash frozen with liquid

nitrogen and stored at ~80˚C.

SDS‑PAGE and Western blotting protocols. SDS-PAGE and

Western blot (WB) transfers were performed on preassembled Bio-Rad

systems, using standard protocols, with precast 4–20% Tris-Glycine

minigels and polyvinylidene difluoride (PVDF) membranes, respec-

tively (Invitrogen). The samples were loaded with reducing buffer.

SDS-PAGE gels were stained and imaged with Pro-Q Diamond and

Sypro Ruby as indicated by the manufacturer (Invitrogen). PVDF

membranes were probed sequentially with primary rabbit anti-JNK

total, mouse anti-pTP motif and mouse anti-pY monoclonal anti-

bodies (Cell Signaling). For imaging, Alexa Fluor700-conjugated

anti-mouse/rabbit secondary antibodies were used (Cell Signaling).

An ODYSSEY infrared imaging system (LI-COR) was utilized for

blot documentation and analysis.

In vitro kinase assays. Kinase assays were performed at 30˚C for

an hour. Pure recombinant His-JNK and its substrate GST-c-Jun

N-terminal were mixed at various ratios in reaction buffer (25 mM

HEPES, pH 7.4, 1 mM DTT, 25 mM MgCl2, 150 mM NaCl

and 50 (M ATP). Kinase reactions were separated by SDS-PAGE

and documented with ProQ-Diamond/Sypro Ruby staining or by

exposure of the dried SDS-PAGE on an X-ray film (Fuji). When

radioactivity was used, 5 (M of (32P)(-ATP was added per reaction.

Cell culture and transient transfection. Human embryonic

kidney (HEK) 293T cells were maintained at 37˚C and 5% CO 2 , in

Dulbeccoís modified Eagle’s medium (DMEM), supplemented with

bovine serum (10%) and penicillin/streptomycin 1% (v/v). Cells were

transfected at 30–35% confluent with a Flag-JNK2 DNA construct.

This was done by calcium phosphate or with the Lipofectamine

Plus Reagent (Invitrogen), following the manufacturerís protocol. At

hours post-transfection, the medium was aspirated and replaced

with fresh one.

UV‑stimulation of cells and time course analysis. At near 100%

confluent, cells were either exposed to UV (45J) and harvested or

harvested nonstimulated. The medium from each plate was aspirated

and the cells washed twice with ice-cold phosphate buffered saline

(PBS). The open plates where exposed to 45J of UV and immediately

incubated with fresh medium for a fixed amount of time (0.5/1/4/8

hours). Each UV-stimulation time point was harvested and lysed, as indicated below. A control time point (time zero) of cells not treated with UV was also prepared. For each time point, 5–10 plates (100 mL) were cultured and transfected to obtain sufficient Flag-JNK2

for proteomic analysis. Purification of flag‑tagged JNK2 from 293T cells. Confluent ± UV treated cells were washed twice with ice-cold PBS. The cells


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Autophosphorylation Properties of Inactive and Active JNK2

Autophosphorylation Properties of Inactive and Active JNK2 Figure 1. JNK2 is a phosphoprotein when expressed in

Figure 1. JNK2 is a phosphoprotein when expressed in E. coli. The kinase

activity of JNK2 was monitored with SDS‑PAGE and stained with Sypro

Ruby (upper) and ProQ‑Diamond (lower). The reaction was set at various

enzyme/substrate ratios, with values of 1/1; 1/3; 3/3 and 3/1 in mg/ml,

as indicated on lanes 1–4 respectively. Lane 5 is the substrate as a negative

control. Lanes 6 and 7 are recombinant JNK2 before and after treatment

with l‑phosphatase.

were gently dislodged from the culture plate with a disposable cell

scraper. This was done with the cells immersed in 5 mL of ice-cold

PBS, supplemented with phosphatase and protease inhibitors (phos-

phatase cocktail inhibitors 1 and 2 from Sigma, and protease

inhibitors cocktail from Roche). The extract was transferred to a

50 mL Falcon tube (Corning) and harvested by centrifugation at

4000 rpm/4˚C. The cell pellet was either flash frozen in liquid

nitrogen and stored at ñ80˚C or resuspended immediately in lysis

buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA,

10% v/v glycerol, 1% v/v Triton X-100, phosphatase cocktail inhibi-

tors 1 and 2, and protease cocktail inhibitors) and incubated for one

hour, with smooth rocking at 4˚C. Next, the lysate was centrifuged

at 12,000 rpm/4˚C to remove the insoluble cell debris. The resulting

supernatant was incubated for 1.5 hours with anti-Flag antibody M2

Agarose beads (Roche), at 4˚C with gentle rotation. The Flag-JNK2/

Agarose-beads complex was harvested by centrifugation at 12,000

rpm/4˚C and washed 5 times with lysis buffer. Finally, Flag-JNK2

was eluted by pH disruption, with 100 mM Glycine pH 3.0. The

eluted protein was immediately neutralized with 1M Tris-HCl,

pH 8.0 and concentrated to a minimum volume by centrifugation

with an Amicon Ultra concentrator (10,000 Da molecular weight

cut off, Millipore). The samples were either flash frozen with liquid

nitrogen and stored at ~80˚C or immediately analyzed by mass

spectrometry (MS).

Mass spectrometry experiments. Recombinant JNK2 protein was

purified as described above from either E. coli or mammalian 293T

cells. Equal amounts of the pure protein were subject to SDS-PAGE

electrophoresis and digested in-gel with trypsin, chymotrypsin,

or Asp-N using a previously described method. 33 Peptides were

analyzed by automated nano-LC/MS using a vented column strategy. Briefly, digests were loaded onto precolumns (360 m x 100 mm I.D. fused silica packed with 4 cm 5 mm Monitor C18 from Column Engineering, Ontario, CA) at a flow rate of 4 mL/min for 10 minutes using an autosampler and HPLC pump (Agilent, Palo Alto, CA). After sample loading, the vent was closed putting the precolumn in-line with the analytical column (360 mm x 75 mm I.D. fused silica

the analytical column (360 m m x 75 m m I.D. fused silica Figure 2. JNK2

Figure 2. JNK2 unlike JNK1 autophosphorylates in vitro. (A and B) Time

course of His‑JNK2 over‑expression in E. coli at 15˚C (A) and 37˚C (B).

The SDS‑PAGE gels were stained sequentially with Sypro Ruby (upper) and

ProQ‑Diamond (lower). In both cases the first aliquot was taken at time zero

after IPTG induction (lane 1). Subsequent time points were sampled every

30 (lanes 2‑5), or 60 minutes (lanes 6 and 7), and after an over‑night incu‑

bation (lane 8). C, Comparing JNK1 and JNK2 expression with SDS‑PAGE,

Sypro Ruby (upper) and ProQ‑Diamond (lower). Lanes 9–12 are respectively

His‑JNK1, His‑JNK2 WT, a JNK2 kinase dead mutant (K55R) as a negative

control, and a construct of JNK2 (1‑387) that lacks its distinctive C‑terminal

region. Each lane corresponds to a total protein aliquot taken after four hours

of IPTG induction at 37˚C.

packed with 8 cm 5 mm Monitor C18), and peptides were gradient

eluted (0–25% B in 30 minutes, 25–90% B in 5 minutes; A =

0.1 M acetic acid in water, B = 0.1 M acetic acid in acetonitrile) into

the mass spectrometer at a flow rate of approximately 100 nL/min.

Mapping JNK phosphorylation sites was achieved using two

approaches. First, digests were analyzed with a linear ion trap mass

spectrometer (LTQ, San Jose, California) operated in data-dependent mode, where the top 5 most abundant precursor ions were subjected to MS/MS (spray voltage = 2000 V, collision energy= 35%, isolation width = 3 Da). MS/MS spectra were matched to JNK sequences using the SEQUEST algorithm. Next, an equivalent amount of digest was analyzed using a 4000 QTRAP hybrid triple quadrupole/ linear ion trap mass spectrometer (Applied Biosystems, Foster City,


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Autophosphorylation Properties of Inactive and Active JNK2

Autophosphorylation Properties of Inactive and Active JNK2 Figure 3A. Ion chromatograms of the tryptic phosphopeptides

Figure 3A. Ion chromatograms of the tryptic phosphopeptides observed for the TPY motif. Fragmentation data corresponding to the three phospho‑peptides

identified for JNK2 activation loop. TACTNFMoxMoxpTPYVVTR. The ion chromatograms of the novel TP‑motifs, T243 and T386 we have identified, are

shown in the supplementary material. Asterisk marks refer to water loses.

CA). The instrument was programmed to perform a three second

precursor scan of m/z 79 in the negative ion mode from m/z 450

to 1800 (spray voltage = - 2200 V, curtain gas = 10, declustering

potential = 80, mode = peak hopping, step size = 1 Da, Q1 = low

resolution, Q3 = unit resolution) using a collision energy ramp of

65 to - 110 V. After the precursor scan, the top three ions above

5000 counts/sec were subjected to an enhanced resolution scan in the

positive ion mode (spray voltage = 2400 V, Q1 resolution= open,

scan rate = 250 amu/sec, dynamic fill time = on, TIC target = 5 x 1

e6 cps). These ions were then subjected to positive ion MS/MS using

a charge state dependent rolling collision energy (scan rate = 4000

amu/sec, Q1 resolution =low, fixed fill time of 40 ms, Q0 trapping =

on, Q3 entry barrier = -8 V). Peak lists were generated using Analyst

software and searched using Mascot (Matrix Science).

Once the sites of phosphorylation were identified, specific phos-

phopeptides were monitored in targeted experiments using one

of two approaches. When quantifying more than nine peptides,

multiple reaction monitoring (MRM) experiments were performed

on the QTRAP instrument (3–5 transitions/peptide) with a dwell time of 50 ms/transition, Q1 resolution set to low, Q3 resolution set to unit, and a collision energy derived from previous MS/MS experiments. When nine or fewer peptides needed to be monitored, targeted MS/MS experiments were performed on the LTQ linear ion trap mass spectrometer with an isolation width of 3 Da and a collision energy of 35%. Peptide and peptide fragment peak areas


were determined using XCalibur 2.0 or Analyst 1.4.1 software.

Normalization across time course samples was accomplished using

signals from nonphosphorylated peptides.


Recombinant JNK2 (p55) but not JNK1 (p46) autophosphory‑

lates during its overexpression in E. coli. Recent work has suggested

that JNK2 may be capable of autophosphorylation in vitro, 1,25,31 and

that this may in part explain why in certain types of cancer, JNK2

is constitutively active. 30,32 In accordance, we observe here that

E. coli-produced recombinant JNK2 is considerably active towards

its substrate c - Jun (Fig. 1). To monitor the kinase reactions, we have

used the set of chemiluminiscent custom dyes ProQ-Diamond and

Sypro Ruby. Pro-Q Diamond selectively stains phospho-proteins

on SDS-PAGE, which once destained and washed, can be restained

with Sypro Ruby to observe the total protein content. 34 This strategy

allowed us to find that E. coli-produced JNK2 is a phospho-protein

because it is recognized by ProQ-Diamond, already prior to its incubation in a kinase reaction assay (Fig. 1). As a proof of concept, ProQ-Diamond fails to recognize JNK2 on an SDS-PAGE if the protein is treated with (l-phosphatase, Fig. 1). The autophosphory- lation of recombinant MAPKs in E. coli has been previously shown not to be an artifact. 35 Therefore, we postulate that JNK2 auto- activates in vitro by means of an auto-phosphorylation event. We also

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Autophosphorylation Properties of Inactive and Active JNK2

Autophosphorylation Properties of Inactive and Active JNK2 Figure 3B. Ion chromatograms of the tryptic phosphopeptides

Figure 3B. Ion chromatograms of the tryptic phosphopeptides observed for the TPY motif. Fragmentation data corresponding to the three phospho‑peptides

identified for JNK2 activation loop. TACTNFMoxMoxTPpYVVTR. The ion chromatograms of the novel TP‑motifs, T243 and T386 we have identified, are

shown in the supplementary material. Asterisk marks refer to water loses.

reasoned that this happens during the overexpression of JNK2, inside

the E.coli milieu.

To explore this hypothesis, we traced the autophosphorylation of

recombinant JNK2 over-time, during its overexpression in E. coli.

JNK2 expresses well at 15˚C (Fig. 2A) and 37˚C (Fig. 2B), but its

autophosphorylation is observed with ProQ-Diamond only at 37˚C

(Fig. 2B). To validate our findings, we compared the expression

profile of JNK2 wild type versus poly-His-tag fusions of JNK1 wild

type, a well-documented JNK2 kinase dead mutant (K55R), and a

truncated JNK2 construct that lacks its unique C-terminal region

(a.a. 1-387) (Fig. 2C). We found that JNK1 is not phosphorylated.

K55R is a known kinase dead mutant that we use here as a negative

control. The failure of JNK1 to autoactivate in vitro is in agreement

with previous published work regarding the heterologous production

of active recombinant MAPKs. 36,37

Mass‑spectrometry analysis of phospho‑JNK2 overexpressed

in E. coli. We extended our investigation of JNK2 phosphoryla-

tion properties using LC/MS. For this purpose, recombinant JNK2

was over-expressed during 12 hours at 37˚C, to assure maximum

autophosphorylation. Pure JNK2 aliquots were digested with three

different enzymes, trypsin, Asp-N or chymotrypsin, to obtain a peptide mixture representing most of the protein sequence. Each protein digest set was then analyzed by LC/MS, using a data depen- dent tandem MS/MS protocol, and a precursor scanning method capable of selective phosphopeptide detection. The overall analysis resulted in the identification of 17 fragments (Supplementary Table S1), allowing the unequivocal assignment of 12 phospho-residues.

Interestingly, we find that the phosphorylation of the TPY motif

(activation loop) is a mixture of one-site phosphorylated peptides

(pT183 or pY185), and the biologically relevant phosphorylation of

both sites (pT183/pY185) (Fig. 3).

It is well accepted that JNKs phosphorylate their substrates on

Ser/Thr residues N-terminal to a Pro (S/TP-motifs), and so referred

to as Pro-directed kinases. 17 JNK2 has four TP-motifs (T93, T183,

T243 and T386) along its protein sequence. Here we have obtained

peptide fragmentation data for the four of them and observe auto-

phosphorylation on T183 from the activation loop (as mentioned

above), and on T243 and T386. The other TP-motif in JNK2 (T93)

is not phosphorylated. We also find several phosphorylation-sites

(pT255, pS282, pS377 and pS388) located C-terminal, rather than

N-terminal, of a Pro residue (PS/T-motifs). S282 is found along the

sequence FPS(282)ES(284)ER and the proximity of another Ser

(S284), precluded the precise identification of the phosphorylated

residue. Three non-S/TP phosphosites were defined. These are

pS87 that is N-terminal to a Leu, S416 preceding a Thr, and T417

N-terminal to a Gly. Finally, two ambiguous phosphosites were

found within a Ser-rich sequence, DAAVp(SS)NApT(386)PSQp(S

SS)IN, from which pT386 is part. We estimate, based on the speci- ficity of the enzymes used, to have obtained a sequence coverage of 81% along the full protein (343 out of 424 residues), covering a total of 85% of the S/T residues. The activation loop controls the autophosphorylation of JNK2.

Our next step was to distinguish the specific autophosphoryla- tion events, from those that may have derived as artifacts from the


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Autophosphorylation Properties of Inactive and Active JNK2

Autophosphorylation Properties of Inactive and Active JNK2 Figure 3C. Ion chromatograms of the tryptic phosphopeptides

Figure 3C. Ion chromatograms of the tryptic phosphopeptides observed for the TPY motif. Fragmentation data corresponding to the three phospho‑peptides

identified for JNK2 activation loop. TACTNFMoxMoxpTPpYVVTR. The ion chromatograms of the novel TP‑motifs, T243 and T386 we have identified, are

shown in the supplementary material. Asterisk marks refer to water loses.

E.coli overexpression conditions. To do so, we sought to establish

the timing of appearance of the various autophosphorylation sites

during the over-expression of JNK2. The His-JNK2 expression levels

started to increase drastically at about 90 minutes post-induction

and its phosphorylation is observed with ProQ-Diamond signal at

about 150 minutes after IPTG induction (Fig. 2B). Based on this, we

sampled the JNK2 expression at discrete time points after induction

from minutes 90 to 240. The samples were immediately harvested

without membrane disruption prior to SDS-PAGE and LC/MS

analysis. The reason to analyze nondisrupted E. coli milieu, was based

on the assumption that physical and/or chemo-enzymatic factors that

follow cell membrane disruption, could promote undesirable JNK2

autophosphorylation events. By performing a relative quantification

analysis of the LC/MS data, we find that pY185 and the pTP motifs

(pT183, pT243 and pT386) appear early, upon IPTG induction,

and that with the exception of pY185, their stoichiometry increases

slowly and with a linear fashion until about minute 150. We observe

an exponential increase of T386 between minutes 150-180, and of

T183 and T243 between minutes 210-240 (Fig. 4). In the case of

the Tyr-containing phosphopeptides, pY185 remains rather constant,

with a moderate increase along the time course, whereas the double phosphosite pT183/pY185 is observed only at the last time point, minute 240 (Fig. 4). It is worth mentioning that the non-TP-motif phosphosites we had identified from a 12 hours expression experiment (Table S1) were not identified this time. We therefore suggest that the crowding conditions to which JNK2 is exposed at some point during its

overexpression, leads to its unspecific autophosphorylation of non-

TP-moieties. For this reason, we decided to focus further on the

4 phospho - sites appearing within the time frame 90 - 240 mins. To

dissect their importance in JNK2 autophorylation, we performed

a western blot (WB) experiment with anti-pTP motif, anti-pY and

anti-nonphosphorylated JNK monoclonal antibodies. We probed

these antibodies against JNK2 (WT), the alanine substitution of

the TP-motif T243A outside the activation loop, and several point

mutants that aimed at mimicking the activation loop phosphosites

(T183D, Y185E and T183D/Y185E). The mutant T386A was

not included in this experiment, because it did not express well in

E. coli (data not shown). As summarized in Table 1, we find that the

mutants T183D and T183D/Y185E fail to autophosphorylate on the

pTP motifs, whereas Y185E and T243A are active enough to do so.

Also only T183D/Y185E has a negative anti - pY signal (Fig. 5A). To

extend our investigation, we also tested these JNK2 constructs with

ProQ-Diamond. Here we found that from the two mutants with no

anti-pTP WB signal, T183D gives a positive ProQ-Diamond signal,

whereas T183D/Y185E does not (Fig. 5B). In the case of T183D,

the ProQ-Diamond signal is consistent with its anti-pY signal due to

the presence of pY185. As mentioned above, the heterologous production of MAPKs in their active form is only accomplished if an upstream kinase (i.e., MKK7 for JNK) is coexpressed with/or fused to a given MAPK. 36,37 Accordingly, the mutants T183D, Y185E and T183D/Y185E do not phosphorylate the bonafide JNK substrate c-Jun (Fig. 5C), meaning that they are not properly mimicking the conformation of the

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Autophosphorylation Properties of Inactive and Active JNK2

Autophosphorylation Properties of Inactive and Active JNK2 Figure 4. Time‑dependent phosphorylation of JNK2 in E.

Figure 4. Time‑dependent phosphorylation of JNK2 in E. coli. The time in minutes after IPTG‑induction (horizontal axis) is plotted against the relative amount,

in the form of normalized counts (vertical axis) of each pTP motif: pT183 (A), pT243 (B), pT386 (C), the mono‑pY185 (D) and dual‑pT183/pY185 species


activation loop. The Pro-Q Diamond signal observed for T183D and

Y185E suggests that the autophosphorylation and the phosphoryla-

tion of the substrate (c-Jun) are independent. Finally, the remaining

phosphorylated TP-motifs we observed may have a structural role

or represent protein-protein interaction sites. In agreement with

this, the mutant T183D prevents the appearance of the other two

pTP-motifs (pT243 and pT386).

JNK2 purified from nonstimulated 293T cells is a mixture of the monophosphosites pT183, pY185 and pT386. To corroborate the biological relevance of our findings in E. coli, we cultured 293T cells that were transiently transfected with Flag-tagged JNK2. For this, JNK2 was immunoprecipitated with agarose-bound anti-Flag monoclonal antibodies and subjected to biochemical inspec- tion. From its ProQ-Diamond signal, we find that Flag-JNK2 is

phosphorylated when isolated from nonstimulated 293T cells

(Figure S1, supplementary material). Analysis of the purified

samples by LC/MS shows that basal Flag-JNK2 is phosphorylated

on T183, Y185 and T386 (Fig. 6A–C). Previous studies show that

in nonstimulated KB and PC12 cells, MKKs are not active. 38 Thus,

the phospho-signal we observe for basal Flag-JNK2 derives most

probably from an autophosphorylation event. In addition, the fact

that one of the phospho-sites is Tyr185 implies that JNK2 has dual-specific (S/T and Y) auto-phosphorylation properties. This notion is compelling because, as stated above, JNKs had long been conceived as Pro-directed S/T kinases. Finally, T386 appears here as well, supporting its relevance (Fig. 6C). Mass‑spectrometry analysis of UV‑activated JNK2. From a large-scale purification protocol, we were able to obtain (mg amounts


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Autophosphorylation Properties of Inactive and Active JNK2

Table 1

Specific monoclonal-antibody reactivity and Pro-Q Diamond staining output for His-JNK2 WT and various site-specific mutants






α‑ pTP‑motif




α‑ pY
















Flag-JNK2. This allowed us to investigate how the phosphorylation

pattern of active JNK2 fluctuates over time in 293T cells. We chose

to irradiate the cells with UV, because this is by far, the physical agent

that activates JNKs the most. We found that upon UV-treatment,

active JNK2 is a mixture of four phosphorylated species. These were:


mono-pT183, mono-pY185, the expected double phospho-specie

pT183/pY185 on the activation loop, and the pT386 signal coming

from its unique C-terminal region (Fig. 6A–D).

To establish how the different phospho - species fluctuate as a func -

tion of time, we analyzed the phosphorylation state of active JNK2


(Fig. 6A–D). As expected from the available literature, 17,18 the

double-phospho moiety pT183/pY185 reached a maximum

30–60 minutes after UV-stimulation and then slowly decayed. This

was also the case for pY185, upregulated at about the same time

and stoichiometry. In contrast, pT183 decreased, whereas pT386

population remained relatively constant along the time course. The

TP-motif phospho-site T243 that we found in active JNK2 obtained

from bacteria, was not observed here. The appearance of active JNK2

as a mixture of pY185 and pT183/pY185 suggests the existence of a

mixed protein population in active JNK2 in vivo. Also, the decay in

the values of pT183 in parallel to the appearance of pT183/pY185

different time points (0.5, 1, 4 and 8 hours), after UV-stimulation

indicates that the activation of JNK2 follows an ordered distributive

path, in which basal pT183 is converted to the fully active moiety

pT183/pY185 upon UV-light irradiation. As for pT386, a likely

explanation is to assume a structural role for this phosphosite, being

involved in either inter or intra protein-protein interactions, and

inaccesible to phosphatases. Accordingly, recombinantly produced

T386A mutant is unstable when expressed in E. coli.


In this article we have combined protein biochemistry and

tandem MS to investigate the auto-phosphorylation pattern of

recombinant JNK2. With a combination of ProQ-Diamond and

Sypro Ruby staining of reducing electrophoretic gels, the detected

JNK2 autophosphorylation in the E. coli milieu happens at 37˚C but

not at 15˚C, suggesting that our observations derive from a catalysed

chemical process. In agreement, Flag-JNK2 immuno-precipitated

from nonstimulated 293T cells, is stoichiometrically auto-phosphor-

ylated on T183, Y185 and T386. It is well established that JNKs become fully active only when phosphorylated on both T183 and Y185 of their TPY motif. 17,18 In the case of JNK1, it has been suggested that its full activation requires first the phosphorylation at position Y185 followed by that of the Thr. 39 From our LC/MS results on JNK2, the main event

that follows UV radiation is the net increase of the pY185 signal as

a mixture of mono-pY185 and the fully active form dual-pT183/

mixture of mono-pY185 and the fully active form dual-pT183/ Figure 5. The autophosphorylation of JNK2 in

Figure 5. The autophosphorylation of JNK2 in E. coli is controlled by its

activation loop. The constructs tested in (A–C) are WT (lane 1), T243A (lane

2), T183D (lane 3), Y185E (lane 4) and the double mutant T183D/Y185E

(lane 5). (A) WB analysis with several commercially available monoclo‑

nal antibodies, anti‑pTP motif (upper), anti‑pY (middle) and anti‑total JNK

(lower). The same blot was stripped after each antibody reaction. As for

the positive anti‑pY signal we see for Y185E, we speculate that this may

be either an unspecific recognition by the antibody or the presence of

other pY residues besides Y185 that get phosphorylated unspecifically.

(B) ProQ‑Diamond (upper) and Sypro Ruby (lower) staining analysis. (C) In

vitro kinase assay monitored with 32 P‑l ATP (lanes 1 and 3–5 as above)

using c‑Jun N‑terminal as the substrate. Top gel is radioactive labeled, bottom

gel Coomassie stained.

pY185. We think that the pT183 form is converted to pT183/

pY185, because its stoichiometry decreases during the time frame in

which active JNK2 is upregulated and then slowly revertes to its basal

levels as active JNK2 decays. Also, the presence of mono-pY185 in

the total amount of post-activation JNK2, leads to a net low molarity

or “dilution” of fully active JNK2 (pT183/pY185). We envisage that

in the case of JNK1, the absence in basal conditions of the mono

phosphorylated species may in turn lead to a higher net molarity of

pT183/pY185 JNK1 relative to active JNK2 (Fig. 7). Regarding the auto-phosphorylation of JNK2 on T386, this phospho-site may also have a biological meaning, yet to be elucidated. The reason for this is that TP-motifs are bona-fide protein- protein interaction sites. 40,41 In agreement, the C-terminal region

of JNK2, where T386 is located, has been proposed to function as

a substrate-binding scaffold. 4 A plausible scenario would be that

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Autophosphorylation Properties of Inactive and Active JNK2

Autophosphorylation Properties of Inactive and Active JNK2 Figure 6. Phosphorylation pattern of Flag‑JNK2 inactive

Figure 6. Phosphorylation pattern of Flag‑JNK2 inactive and active isolated from 293T cells. The LC/MS data for each phosphorylation site identified was analyzed by relative quantifica‑ tion. The resulting values plotted as counts normalized per phosphorylation site (vertical axis) versus time after UV‑light irradiation (hori‑ zontal axis) are shown. (A) pT183; (B) pY185; (C) pT386 and (D) pT183/pY185.

shown. (A) pT183; (B) pY185; (C) pT386 and (D) pT183/pY185. Figure 7. JNK2 (p55), as opposed

Figure 7. JNK2 (p55), as opposed to JNK1 (p46), is a phospho‑protein in basal conditions. Based on the observed basal autophosphorylation of

JNK2, we propose that during a total JNK response curve, JNK2 is activated first but with an attenuated net activity, whereas JNK1 with no basal

autophosphorylation will be activated later during the response curve but with a higher net activity. For simplicity, we assume in our model that the phospho‑

site pT386 has a structural role and is therefore present in all the protein components of the JNK2 pool. See the Discussion section for details.

the pT386 site by interacting with other JNKs or with putative

protein scaffolds, becomes solvent inaccessible and thus incapable

of downregulation by phosphatases. This argument would explain

why the stoichiometry of the pT386 specie does not fluctuate

upon UV-activation and why the mutant T386A is unstable when

over-expressed in E. coli. An emerging concept is that MAPKs convert graded stimuli into switch like responses, because they operate at near saturation stoichiometries of either the activating upstream kinases and/or the inactivating phosphatases. 42-44 It has been shown experimentally that the JNK signaling cascade exerts a positive feedback loop, resulting from a still unidentified autocatalytic behaviour. 45,46 These findings

corroborate the early prediction that JNKs exhibit an ultra-sensitive

response pattern when activated. 42 In addition, while it is well

established that active JNK1 is the main in vivo kinase component

of the JNK cascade, 17,18 JNK2 has been described as having a futile

activity perhaps masked by an intracellular inhibitor. 26 From our

experimental findings, we propose that active JNK2, through its stoichiometrical pY185 component, may be responsible for the saturation conditions in which both the activating kinases and the phosphatases are predicted to operate. Also, since we expect the 10 JNK isoforms to have different kinetic rate constants, a poten- tial compensatory equilibrium between them is likely to occur (Fig. 7). This prediction may underpin the recently proposed


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Autophosphorylation Properties of Inactive and Active JNK2

functional cross-talk amongst JNKs, 29 where a rapid but attenuated activation of JNK2 may pose an ultra-sensitive factor to the response curve, while the lagging activation of JNK1 may provide the func- tional output threshold.


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