[Cell Cycle 6:14, e1-e10, EPUB Ahead of Print: http://www.landesbioscience.com/journals/cc/abstract.php?

id=4434; 15 July 2007]; ©2007 Landes Bioscience

Report

Autophosphorylation Properties of Inactive and Active JNK2

Genaro Pimienta1 Abstract

Scott B. Ficarro3 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
Gustavo J. Gutierrez2

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

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Anindita Bhoumik2 or apoptosis. Active phospho JNK1 is the main in vivo kinase component of the JNK

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cascade, whereas JNK2 is presumed not to participate as a kinase during JNK signal‑
Eric C. Peters3 ling. However, there is evidence that JNK isoforms interact functionally in vivo. Also, a

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Ze’ev Ronai2 recent chemical genetics investigation has confirmed that JNK transient activation leads
to cellular proliferation, whereas a sustained one is pro‑apoptotic. Here we investigate
Jaime Pascual1,* the phosphorylation pattern of JNK2, with protein biochemistry tools and tandem mass

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1Inflammation and Infectious Diseases Center; 2Cancer Center; Burnham Institute spectrometry. We choose to focus on JNK2 because of its reported constitutive activity in
for Medical Research; La Jolla, California USA 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

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3Genomics Institute of the Novartis Research Foundation; San Diego, California
USA
pThr386 along its unique C‑terminal region. Upon UV stimulation, its phosphorylation

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stoichiometry is upregulated on the activation loop, generating a mixture of mono‑pTyr185
*Correspondence to: Jaime Pascual; Burnham Institute for Medical Research; and the expected dual‑pThr183/pTyr185 species, with the pThr386 specie present but
10901 North Torrey Pines Road; La Jolla, California 92037 USA; Tel.:
858.646.3100; Fax: 858.646.3195; Email: pascual@burnham.org
unaltered respect to the basal conditions.
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Original manuscript submitted: 04/23/07
Manuscript accepted: 05/10/07
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This manuscript has been published online, prior to printing for Cell Cycle, Volume Introduction
6, Issue 14. Definitive page numbers have not been assigned. The current citation is:
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Cell Cycle 2007; 6(14): The c‑Jun N‑terminal kinases (JNKs) are a subgroup of the mitogen activated protein
http://www.landesbioscience.com/journals/cc/abstract.php?id=4434 kinases (MAPKs), cloned initially from cDNA libraries of human liver1 and rat brain.2
Once the issue is complete and page numbers have been assigned, the citation
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will change accordingly. 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
Key words JNKs relay cellular signals conveyed by the MAPK cascade, when stimulated by physical
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stress, inflammatory cytokines, such as IL‑1 and TNF, or DNA‑damaging agents in the
JNK2/tandem mass spectrometry/autophos- form of UV or ionizing radiation.5 The JNK signalling cascade relies on protein scaffolds
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phorylation/time-dependent phosphorylation to achieve specificity and spatial localization of the transferred signal.6 It also functions
pattern within the context of other signalling systems, such as the NFB cascade7‑9 and those
orchestrated by the PKC.10,11 A consequence is that JNK signalling can initiate events of
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Acknowledgements cell cycle progression or apoptosis, depending on the cell‑type and the nature of a given
This work was partially funded by grant stimuli.12 Current models suggest that a transient stimulation of JNK generally leads to
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P01CA102583 to J.P. 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,
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Note whereas JNK3 is tissue‑specific and found mainly in the brain.14,15 Like other MAPKs,
Supplementary material can be found at: JNKs have a conserved protein kinase fold and a typical activation loop. This loop has the
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www,.landesbioscience.com/supplement/ sequence signature of Thr (T) ‑x‑ Tyr (Y) motif, where x is a Pro (P) in the case of JNKs.16
pimientaCC6-14-sup.pdf A hallmark in JNK activation is its induction by the dual phosphorylation (on T and Y) of
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the aforementioned motif.17,18 This event is orchestrated by two upstream dual‑specificity

(S/T and Y) MAPK kinases, the MKK419,20 and MKK7,21,22 which in turn are activated
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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) with 20 mL of either Nickel‑NTA or Glutathione Sepharose High
suggest that active JNK1 is the main in vivo kinase, relaying the JNK Performance resin, for His or GST fusion proteins, respectively. After
cascade signal.26,27 This has lead to the model that JNK1 and JNK2 extensive washing (5 column volumes) with equilibration buffer
have distinct, rather opposite functional roles.28 This concept has (25 mM Tris‑HCl, pH 8.0, 500 mM NaCl, supplemented with
recently been challenged by a chemical genetics report, which shows 2.5 mM imidazole in the case of Nickel column), the bound proteins
that JNK1 and JNK2 display a functional in vivo cross talk and were eluted with a linear gradient of either 2.5–200 mM imidazole
rather have compensatory functional roles.29 A possible explanation for His fusion proteins or 0–50 mM reduced glutathione in the
is that the differences among JNKs are dictated at the post‑transla- case of GST fusion polypeptides. The elution peak was collected
tional level, in the form of a differential phosphorylation pattern. Ifand dialyzed over‑night against 2 L of 25 mM HEPES, pH 7.6,
true, this may underpin their in vivo functional cross‑talk. 50 mM NaCl and 10 mM DTT. Proteins were purified further by
Here we explore this hypothesis by investigating with biochemical anion exchange, with a manually packed column containing 20 mL
tools and tandem mass spectrometry (MS), the phosphorylation of Mono Q Sepharose resin, using a linear NaCl gradient 0‑1M,
pattern of JNK2 when overexpressed in E. coli and 293T cells. We with the same buffer conditions used to dialyze overnight. The pure

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choose to work with JNK2 (p55), because we find in the literature proteins were concentrated by centrifugation with an Amicon Ultra

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an emerging notion that JNK2 has autocatalytic properties.30‑32 concentrator (10,000 Da molecular weight cut off, MILLIPORE),

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Our results show that JNK2 but not JNK1 auto‑activates in vitro to about 1 mg/mL and used immediately or flash frozen with liquid
because it has dual‑specificity autophosphorylation properties. It nitrogen and stored at ~80˚C.

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phosphorylates itself on the T and Y residues of the activation loop. SDS‑PAGE and Western blotting protocols. SDS‑PAGE and
In addition we identify a previously unknown phosphorylation Western blot (WB) transfers were performed on preassembled Bio‑Rad
site, T386 that is an amino acid unique to JNK2 located along its systems, using standard protocols, with precast 4–20% Tris‑Glycine

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C‑terminal extension. minigels and polyvinylidene difluoride (PVDF) membranes, respec-
tively (Invitrogen). The samples were loaded with reducing buffer.

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Experimental Procedures SDS‑PAGE gels were stained and imaged with Pro‑Q Diamond and
Sypro Ruby as indicated by the manufacturer (Invitrogen). PVDF

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Protein expression constructs. The DNA expression constructs membranes were probed sequentially with primary rabbit anti‑JNK
encoding GST‑c‑Jun N‑terminal fragment (a.a. 1‑89), Flag‑JNK2, total, mouse anti‑pTP motif and mouse anti‑pY monoclonal anti-
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His‑JNK1 and His‑JNK2 are those described previously.10 Mutations bodies (Cell Signaling). For imaging, Alexa Fluor700‑conjugated
on His‑JNK2 were introduced by using the Quick Change anti‑mouse/rabbit secondary antibodies were used (Cell Signaling).
Site‑Directed Mutagenesis Kit (Stratagene) and confirmed by DNA An ODYSSEY infrared imaging system (LI‑COR) was utilized for
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sequencing. Four single point mutants (K55R, T183D, Y185E and blot documentation and analysis.
T243A) were used in this study, one double phosphomimic point In vitro kinase assays. Kinase assays were performed at 30˚C for
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mutant (T183D/Y185E) and a truncated form (a.a. 1‑387) of JNK2 an hour. Pure recombinant His‑JNK and its substrate GST‑c‑Jun
that overlaps with JNK1. N‑terminal were mixed at various ratios in reaction buffer (25 mM
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Protein expression in E. coli. The GST and 5xHis fusion HEPES, pH 7.4, 1 mM DTT, 25 mM MgCl2, 150 mM NaCl
proteins were produced growing bacteria in Luria‑Bertani Broth and 50 (M ATP). Kinase reactions were separated by SDS‑PAGE
(LB) supplemented with ampicillin at 37˚C. Typically, a fresh and documented with ProQ‑Diamond/Sypro Ruby staining or by
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colony transformed with a particular DNA construct, was picked exposure of the dried SDS‑PAGE on an X‑ray film (Fuji). When
up from a solid LB plate and used to inoculate 1–2 mL of liquid radioactivity was used, 5 (M of (32P)(‑ATP was added per reaction.
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LB and incubated overnight. Next, this preculture was diluted to Cell culture and transient transfection. Human embryonic
1 L of LB in a 2 L flask and cultured until the optical density of the kidney (HEK) 293T cells were maintained at 37˚C and 5% CO2, in
media measured at 600 nm reached a value of 0.6–0.9. Recombinant Dulbeccoís modified Eagle’s medium (DMEM), supplemented with
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protein over‑expression was induced with 0.25 mM of IPTG at this bovine serum (10%) and penicillin/streptomycin 1% (v/v). Cells were
point. Protein expression experiments lasted either 4 or 12 hours and transfected at 30–35% confluent with a Flag‑JNK2 DNA construct.
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were performed at 15˚C or 37˚C, depending on the experiment. See This was done by calcium phosphate or with the Lipofectamine
the results sections for details. Plus Reagent (Invitrogen), following the manufacturerís protocol. At
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Protein purification from E. coli. All recombinant proteins were 18 hours post‑transfection, the medium was aspirated and replaced
prepared in a similar way. Only the affinity column used to enrich with fresh one.
them was different, as specified below. For the liquid chromatog- UV‑stimulation of cells and time course analysis. At near 100%
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raphy (LC) protocols described here, we have used a ƒKTA‑prime confluent, cells were either exposed to UV (45J) and harvested or
fast performance liquid chromatography (FPLC) system, with chro- harvested nonstimulated. The medium from each plate was aspirated
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matographic columns (XK 16), affinity resins and other LC utilities, and the cells washed twice with ice‑cold phosphate buffered saline
from GE‑Amersham Biosciences. E. coli expression milieu was (PBS). The open plates where exposed to 45J of UV and immediately
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harvested at 4˚C by centrifugation (4500 rpm) during 30 minutes, incubated with fresh medium for a fixed amount of time (0.5/1/4/8
using a Sorvall SLA30 rotor. Cells were resuspended in ice‑cold lysis hours). Each UV‑stimulation time point was harvested and lysed,
buffer (25 mM Tris‑HCl, pH 8.0, 500 mM NaCl and 1 tablet/L of as indicated below. A control time point (time zero) of cells not
an EDTA‑free protease inhibitors cocktail from Roche) and lysed treated with UV was also prepared. For each time point, 5–10 plates
by sonication. Next, the lysate was centrifuged with a Sorvall SS34 (100 mL) were cultured and transfected to obtain sufficient Flag‑JNK2
rotator at 12,000 rpm/4˚C, to remove the insoluble cell debris. for proteomic analysis.
The resulting supernatant was loaded onto a 50 mL super‑loop Purification of flag‑tagged JNK2 from 293T cells. Confluent
and injected on a preequilibrated affinity column, manually packed ± UV treated cells were washed twice with ice‑cold PBS. The cells

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 Autophosphorylation Properties of Inactive and Active JNK2

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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

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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

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control. Lanes 6 and 7 are recombinant JNK2 before and after treatment
with l‑phosphatase.

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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
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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
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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,
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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
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hour, with smooth rocking at 4˚C. Next, the lysate was centrifuged Figure 2. JNK2 unlike JNK1 autophosphorylates in vitro. (A and B) Time
at 12,000 rpm/4˚C to remove the insoluble cell debris. The resulting course of His‑JNK2 over‑expression in E. coli at 15˚C (A) and 37˚C (B).
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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
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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‑

rpm/4˚C and washed 5 times with lysis buffer. Finally, Flag‑JNK2 bation (lane 8). C, Comparing JNK1 and JNK2 expression with SDS‑PAGE,
was eluted by pH disruption, with 100 mM Glycine pH 3.0. The Sypro Ruby (upper) and ProQ‑Diamond (lower). Lanes 9–12 are respectively
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eluted protein was immediately neutralized with 1M Tris‑HCl, His‑JNK1, His‑JNK2 WT, a JNK2 kinase dead mutant (K55R) as a negative
pH 8.0 and concentrated to a minimum volume by centrifugation control, and a construct of JNK2 (1‑387) that lacks its distinctive C‑terminal
with an Amicon Ultra concentrator (10,000 Da molecular weight region. Each lane corresponds to a total protein aliquot taken after four hours
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cut off, Millipore). The samples were either flash frozen with liquid of IPTG induction at 37˚C.
nitrogen and stored at ~80˚C or immediately analyzed by mass
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spectrometry (MS). packed with 8 cm 5 mm Monitor C18), and peptides were gradient
Mass spectrometry experiments. Recombinant JNK2 protein was eluted (0–25% B in 30 minutes, 25–90% B in 5 minutes; A =
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purified as described above from either E. coli or mammalian 293T 0.1 M acetic acid in water, B = 0.1 M acetic acid in acetonitrile) into
cells. Equal amounts of the pure protein were subject to SDS‑PAGE the mass spectrometer at a flow rate of approximately 100 nL/min.†
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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
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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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
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shown in the supplementary material. Asterisk marks refer to water loses.

CA). The instrument was programmed to perform a three second were determined using XCalibur 2.0 or Analyst 1.4.1 software.†
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precursor scan of m/z 79 in the negative ion mode from m/z 450 Normalization across time course samples was accomplished using
to 1800 (spray voltage = ‑2200 V, curtain gas = 10, declustering signals from nonphosphorylated peptides.†
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potential = 80, mode = peak hopping, step size = 1 Da, Q1 = low

resolution, Q3 = unit resolution) using a collision energy ramp of Results
‑65 to ‑110 V. After the precursor scan, the top three ions above
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5000 counts/sec were subjected to an enhanced resolution scan in the Recombinant JNK2 (p55) but not JNK1 (p46) autophosphory‑
positive ion mode (spray voltage = 2400 V, Q1 resolution= open, lates during its overexpression in E. coli. Recent work has suggested
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scan rate = 250 amu/sec, dynamic fill time = on, TIC target = 5 x 1 that JNK2 may be capable of autophosphorylation in vitro,1,25,31 and
e6 cps). These ions were then subjected to positive ion MS/MS using that this may in part explain why in certain types of cancer, JNK2
is constitutively active.30,32 In accordance, we observe here that
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a charge state dependent rolling collision energy (scan rate = 4000

amu/sec, Q1 resolution =low, fixed fill time of 40 ms, Q0 trapping = E. coli‑produced recombinant JNK2 is considerably active towards
on, Q3 entry barrier = ‑8 V). Peak lists were generated using Analyst its substrate c‑Jun (Fig. 1). To monitor the kinase reactions, we have
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software and searched using Mascot (Matrix Science). used the set of chemiluminiscent custom dyes ProQ‑Diamond and
Once the sites of phosphorylation were identified, specific phos- Sypro Ruby. Pro‑Q Diamond selectively stains phospho‑proteins
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phopeptides were monitored in targeted experiments using one on SDS‑PAGE, which once destained and washed, can be restained
of two approaches. When quantifying more than nine peptides, with Sypro Ruby to observe the total protein content.34 This strategy
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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
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
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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
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shown in the supplementary material. Asterisk marks refer to water loses.
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reasoned that this happens during the overexpression of JNK2, inside
the E.coli milieu.
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To explore this hypothesis, we traced the autophosphorylation of
recombinant JNK2 over‑time, during its overexpression in E. coli.
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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
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type, a well‑documented JNK2 kinase dead mutant (K55R), and a
truncated JNK2 construct that lacks its unique C‑terminal region
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(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
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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
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Mass‑spectrometry analysis of phospho‑JNK2 overexpressed
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in E. coli. We extended our investigation of JNK2 phosphoryla-
tion properties using LC/MS. For this purpose, recombinant JNK2
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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.
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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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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
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shown in the supplementary material. Asterisk marks refer to water loses.

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E.coli overexpression conditions. To do so, we sought to establish overexpression, leads to its unspecific autophosphorylation of non-
the timing of appearance of the various autophosphorylation sites TP‑moieties. For this reason, we decided to focus further on the
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during the over‑expression of JNK2. The His‑JNK2 expression levels 4 phospho‑sites appearing within the time frame 90‑240 mins. To
started to increase drastically at about 90 minutes post‑induction dissect their importance in JNK2 autophorylation, we performed
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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
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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

without membrane disruption prior to SDS‑PAGE and LC/MS mutants that aimed at mimicking the activation loop phosphosites
analysis. The reason to analyze nondisrupted E. coli milieu, was based (T183D, Y185E and T183D/Y185E). The mutant T386A was
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on the assumption that physical and/or chemo‑enzymatic factors that not included in this experiment, because it did not express well in
follow cell membrane disruption, could promote undesirable JNK2 E. coli (data not shown). As summarized in Table 1, we find that the
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autophosphorylation events. By performing a relative quantification mutants T183D and T183D/Y185E fail to autophosphorylate on the
analysis of the LC/MS data, we find that pY185 and the pTP motifs pTP motifs, whereas Y185E and T243A are active enough to do so.
(pT183, pT243 and pT386) appear early, upon IPTG induction, Also only T183D/Y185E has a negative anti‑pY signal (Fig. 5A). To
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and that with the exception of pY185, their stoichiometry increases extend our investigation, we also tested these JNK2 constructs with
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slowly and with a linear fashion until about minute 150. We observe ProQ‑Diamond. Here we found that from the two mutants with no
an exponential increase of T386 between minutes 150‑180, and of anti‑pTP WB signal, T183D gives a positive ProQ‑Diamond signal,
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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
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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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
(E).
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activation loop. The Pro‑Q Diamond signal observed for T183D and phosphorylated when isolated from nonstimulated 293T cells
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Y185E suggests that the autophosphorylation and the phosphoryla- (Figure S1, supplementary material). Analysis of the purified
tion of the substrate (c‑Jun) are independent. Finally, the remaining samples by LC/MS shows that basal Flag‑JNK2 is phosphorylated
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phosphorylated TP‑motifs we observed may have a structural role on T183, Y185 and T386 (Fig. 6A–C). Previous studies show that
or represent protein‑protein interaction sites. In agreement with in nonstimulated KB and PC12 cells, MKKs are not active.38 Thus,
©

this, the mutant T183D prevents the appearance of the other two the phospho‑signal we observe for basal Flag‑JNK2 derives most
pTP‑motifs (pT243 and pT386). probably from an autophosphorylation event. In addition, the fact
JNK2 purified from nonstimulated 293T cells is a mixture of that one of the phospho‑sites is Tyr185 implies that JNK2 has
the monophosphosites pT183, pY185 and pT386. To corroborate dual‑specific (S/T and Y) auto‑phosphorylation properties. This
the biological relevance of our findings in E. coli, we cultured 293T notion is compelling because, as stated above, JNKs had long been
cells that were transiently transfected with Flag‑tagged JNK2. For conceived as Pro‑directed S/T kinases. Finally, T386 appears here as
this, JNK2 was immunoprecipitated with agarose‑bound anti‑Flag well, supporting its relevance (Fig. 6C).
monoclonal antibodies and subjected to biochemical inspec- Mass‑spectrometry analysis of UV‑activated JNK2. From a
tion. From its ProQ‑Diamond signal, we find that Flag‑JNK2 is large‑scale purification protocol, we were able to obtain (mg amounts

e7 Cell Cycle 2007; Vol. 6 Issue 14

 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

WT T243A T183D Y185E T183D/Y185E

α- pTP-motif + + - + -
α- pY + + + + -
α-JNK + + + + +
ProQ-Diamond + + + + -

of Flag‑JNK2. This allowed us to investigate how the phosphorylation

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

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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,

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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

IST
from its unique C‑terminal region (Fig. 6A–D).
To establish how the different phospho‑species fluctuate as a func-

D
tion of time, we analyzed the phosphorylation state of active JNK2
at different time points (0.5, 1, 4 and 8 hours), after UV‑stimulation

OT
(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
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and stoichiometry. In contrast, pT183 decreased, whereas pT386
.D
population remained relatively constant along the time course. The
TP‑motif phospho‑site T243 that we found in active JNK2 obtained
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from bacteria, was not observed here. The appearance of active JNK2
as a mixture of pY185 and pT183/pY185 suggests the existence of a
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mixed protein population in active JNK2 in vivo. Also, the decay in Figure 5. The autophosphorylation of JNK2 in E. coli is controlled by its
the values of pT183 in parallel to the appearance of pT183/pY185 activation loop. The constructs tested in (A–C) are WT (lane 1), T243A (lane
indicates that the activation of JNK2 follows an ordered distributive 2), T183D (lane 3), Y185E (lane 4) and the double mutant T183D/Y185E
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(lane 5). (A) WB analysis with several commercially available monoclo‑

path, in which basal pT183 is converted to the fully active moiety nal antibodies, anti‑pTP motif (upper), anti‑pY (middle) and anti‑total JNK
pT183/pY185 upon UV‑light irradiation. As for pT386, a likely (lower). The same blot was stripped after each antibody reaction. As for
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explanation is to assume a structural role for this phosphosite, being the positive anti‑pY signal we see for Y185E, we speculate that this may
involved in either inter or intra protein‑protein interactions, and be either an unspecific recognition by the antibody or the presence of
inaccesible to phosphatases. Accordingly, recombinantly produced other pY residues besides Y185 that get phosphorylated unspecifically.
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T386A mutant is unstable when expressed in E. coli. (B) ProQ‑Diamond (upper) and Sypro Ruby (lower) staining analysis. (C) In
vitro kinase assay monitored with 32P‑l ATP (lanes 1 and 3–5 as above)
using c‑Jun N‑terminal as the substrate. Top gel is radioactive labeled, bottom
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Discussion gel Coomassie stained.

In this article we have combined protein biochemistry and

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tandem MS to investigate the auto‑phosphorylation pattern of pY185. We think that the pT183 form is converted to pT183/
recombinant JNK2. With a combination of ProQ‑Diamond and pY185, because its stoichiometry decreases during the time frame in
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Sypro Ruby staining of reducing electrophoretic gels, the detected which active JNK2 is upregulated and then slowly revertes to its basal
JNK2 autophosphorylation in the E. coli milieu happens at 37˚C but levels as active JNK2 decays. Also, the presence of mono‑pY185 in
20

not at 15˚C, suggesting that our observations derive from a catalysed the total amount of post‑activation JNK2, leads to a net low molarity
chemical process. In agreement, Flag‑JNK2 immuno‑precipitated or “dilution” of fully active JNK2 (pT183/pY185). We envisage that
©

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/
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

www.landesbioscience.com Cell Cycle e8

 Autophosphorylation Properties of Inactive and Active JNK2

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.

E .
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D IST
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ON
.D
CE
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SC
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ES
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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‑
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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.
07

the pT386 site by interacting with other JNKs or with putative corroborate the early prediction that JNKs exhibit an ultra‑sensitive
protein scaffolds, becomes solvent inaccessible and thus incapable response pattern when activated.42 In addition, while it is well
20

of downregulation by phosphatases. This argument would explain 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
©

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
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

e9 Cell Cycle 2007; Vol. 6 Issue 14

 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.
References
1. Kyriakis JM, Avruch J. pp54 microtubule‑associated protein 2 kinase: A novel serine/threo-
nine protein kinase regulated by phosphorylation and stimulated by poly‑L‑lysine. J Biol
Chem 1990; 265:17355‑63.
2. Derijard B, Hibi M, Wu IH, Barrett T, Su B, Deng T, Karin M, Davis RJ. JNK1: A protein
kinase stimulated by UV light and Ha‑Ras that binds and phosphorylates the c‑Jun activa-
tion domain. Cell 1994; 76:1025‑37.
3. Hibi M, Lin A, Smeal T, Minden A, Karin M. Identification of an oncoprotein‑ and
UV‑responsive protein kinase that binds and potentiates the c‑Jun activation domain. Genes
Dev 1993; 7:2135‑48.
4. Kallunki T, Su B, Tsigelny I, Sluss HK, Derijard B, Moore G, Davis R, Karin M. JNK2
28. Sabapathy K, Wagner EF. JNK2: A negative regulator of cellular proliferation. Cell Cycle
2004; 3:1520‑3.
29. Jaeschke A, Karasarides M, Ventura JJ, Ehrhardt A, Zhang C, Flavell RA, Shokat KM,
Davis RJ. JNK2 is a positive regulator of the cJun transcription factor. Mol Cell 2006;
23:899‑911.
30. Tsuiki H, Tnani M, Okamoto I, Kenyon LC, Emlet DR, Holgado‑Madruga M, Lanham
IS, Joynes CJ, Vo KT, Wong AJ. Constitutively active forms of c‑Jun NH2‑terminal kinase
are expressed in primary glial tumors. Cancer Res 2003; 63:250‑5.
31. Cui J, Holgado‑Madruga M, Su W, Tsuiki H, Wedegaertner P, Wong AJ. Identification of
a specific domain responsible for JNK2alpha2 autophosphorylation. J Biol Chem 2005;
280:9913‑20.
32. Cui J, Han SY, Wang C, Su W, Harshyne L, Holgado‑Madruga M, Wong AJ. c‑Jun
NH(2)‑terminal kinase 2alpha2 promotes the tumorigenicity of human glioblastoma cells.
Cancer Res 2006; 66:10024‑31.
33. Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins
silver‑stained polyacrylamide gels. Anal Chem 1996; 68:850‑8.
34. Steinberg TH, Agnew BJ, Gee KR, Leung WY, Goodman T, Schulenberg B, Hendrickson J,

.
contains a specificity‑determining region responsible for efficient c‑Jun binding and phos- Beechem JM, Haugland RP, Patton WF. Global quantitative phosphoprotein analysis using

E
phorylation. Genes Dev 1994; 8:2996‑3007. Multiplexed Proteomics technology. Proteomics 2003; 3:1128‑44.

UT
5. Weston CR, Davis RJ. The JNK signal transduction pathway. Curr Opin Genet Dev 2002; 35. Seger R, Ahn NG, Boulton TG, Yancopoulos GD, Panayotatos N, Radziejewska E, Ericsson
12:14‑21. L, Bratlien RL, Cobb MH, Krebs EG. Microtubule‑associated protein 2 kinases, ERK1 and
6. Morrison DK, Davis RJ. Regulation of MAP kinase signaling modules by scaffold proteins ERK2, undergo autophosphorylation on both tyrosine and threonine residues: Implications

RIB
in mammals. Annu Rev Cell Dev Biol 2003; 19:91‑118. for their mechanism of activation. Proc Natl Acad Sci USA 1991; 88:6142‑6.
7. Tang G, Minemoto Y, Dibling B, Purcell NH, Li Z, Karin M, Lin A. Inhibition of JNK 36. Khokhlatchev A, Xu S, English J, Wu P, Schaefer E, Cobb MH. Reconstitution of mito-
activation through NF‑kappaB target genes. Nature 2001; 414:313‑7. gen‑activated protein kinase phosphorylation cascades in bacteria. Efficient synthesis of

8. Tang F, Tang G, Xiang J, Dai Q, Rosner MR, Lin A. The absence of NF‑kappaB‑mediated
inhibition of c‑Jun N‑terminal kinase activation contributes to tumor necrosis factor
alpha‑induced apoptosis. Mol Cell Biol 2002; 22:8571‑9.
9. Nakano H, Nakajima A, Sakon‑Komazawa S, Piao JH, Xue X, Okumura K. Reactive
IST
active protein kinases. J Biol Chem 1997; 272:11057‑62.
37. Zheng C, Xiang J, Hunter T, Lin A. The JNKK2‑JNK1 fusion protein acts as a constitu-
tively active c‑Jun kinase that stimulates c‑Jun transcription activity. J Biol Chem 1999;

D
274:28966‑71.
oxygen species mediate crosstalk between NF‑kappaB and JNK. Cell Death Differ 2006; 38. Meier R, Rouse J, Cuenda A, Nebreda AR, Cohen P. Cellular stresses and cytokines activate
13:730‑7. multiple mitogen‑activated‑protein kinase kinase homologues in PC12 and KB cells. Eur J

OT
10. Lopez‑Bergami P, Habelhah H, Bhoumik A, Zhang W, Wang LH, Ronai Z. RACK1 medi- Biochem 1996; 236:796‑805.
ates activation of JNK by protein kinase C [corrected]. Mol Cell 2005; 19:309‑20. 39. Kishimoto H, Nakagawa K, Watanabe T, Kitagawa D, Momose H, Seo J, Nishitai G,
11. Liu J, Yang D, Minemoto Y, Leitges M, Rosner MR, Lin A. NF‑kappaB is required for
UV‑induced JNK activation via induction of PKCdelta. Mol Cell 2006; 21:467‑80. ON
12. Lin A. Activation of the JNK signaling pathway: Breaking the brake on apoptosis. Bioessays
2003; 25:17‑24.
Shimizu N, Ohata S, Tanemura S, Asaka S, Goto T, Fukushi H, Yoshida H, Suzuki A, Sasaki
T, Wada T, Penninger JM, Nishina H, Katada T. Different properties of SEK1 and MKK7
in dual phosphorylation of stress‑induced activated protein kinase SAPK/JNK in embryonic
stem cells. J Biol Chem 2003; 278:16595‑601.
.D
13. Ventura JJ, Hubner A, Zhang C, Flavell RA, Shokat KM, Davis RJ. Chemical genetic 40. Yaffe MB, Elia AE. Phosphoserine/threonine‑binding domains. Curr Opin Cell Biol 2001;
analysis of the time course of signal transduction by JNK. Mol Cell 2006; 21:701‑10. 13:131‑8.
14. Gupta S, Barrett T, Whitmarsh AJ, Cavanagh J, Sluss HK, Derijard B, Davis RJ. Selective 41. Remenyi A, Good MC, Lim WA. Docking interactions in protein kinase and phosphatase
CE

interaction of JNK protein kinase isoforms with transcription factors. Embo J 1996; networks. Curr Opin Struct Biol 2006; 16:676‑85.
15:2760‑70. 42. Ferrell Jr JE. Tripping the switch fantastic: How a protein kinase cascade can convert graded
15. Finch A, Davis W, Carter WG, Saklatvala J. Analysis of mitogen‑activated protein kinase inputs into switch‑like outputs. Trends Biochem Sci 1996; 21:460‑6.
IEN

pathways used by interleukin 1 in tissues in vivo: Activation of hepatic c‑Jun N‑terminal 43. Markevich NI, Hoek JB, Kholodenko BN. Signaling switches and bistability arising from
kinases 1 and 2, and mitogen‑activated protein kinase kinases 4 and 7. Biochem J 2001; multisite phosphorylation in protein kinase cascades. J Cell Biol 2004; 164:353‑9.
353:275‑81. 44. Hornberg JJ, Binder B, Bruggeman FJ, Schoeberl B, Heinrich R, Westerhoff HV. Control of
SC

16. Chen Z, Gibson TB, Robinson F, Silvestro L, Pearson G, Xu B, Wright A, Vanderbilt C, MAPK signalling: From complexity to what really matters. Oncogene 2005; 24:5533‑42.
Cobb MH. MAP kinases. Chem Rev 2001; 101:2449‑76. 45. Bagowski CP, Ferrell Jr JE. Bistability in the JNK cascade. Curr Biol 2001; 11:1176‑82.
17. Kyriakis JM, Avruch J. Mammalian mitogen‑activated protein kinase signal transduction
BIO

46. Bagowski CP, Besser J, Frey CR, Ferrell Jr JE. The JNK cascade as a biochemical switch in
pathways activated by stress and inflammation. Physiol Rev 2001; 81:807‑69. mammalian cells: Ultrasensitive and all‑or‑none responses. Curr Biol 2003; 13:315‑20.
18. Karin M, Gallagher E. From JNK to pay dirt: Jun kinases, their biochemistry, physiology
and clinical importance. IUBMB Life 2005; 57:283‑95.
19. Derijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ, Davis RJ. Independent
ES

human MAP‑kinase signal transduction pathways defined by MEK and MKK isoforms.
Science 1995; 267:682‑5.
20. Lin A, Minden A, Martinetto H, Claret FX, Lange‑Carter C, Mercurio F, Johnson GL,
ND

Karin M. Identification of a dual specificity kinase that activates the Jun kinases and
p38‑Mpk2. Science 1995; 268:286‑90.
21. Moriguchi T, Toyoshima F, Masuyama N, Hanafusa H, Gotoh Y, Nishida E. A novel
LA

SAPK/JNK kinase, MKK7, stimulated by TNFalpha and cellular stresses. Embo J 1997;
16:7045‑53.
22. Tournier C, Dong C, Turner TK, Jones SN, Flavell RA, Davis RJ. MKK7 is an essential
07

component of the JNK signal transduction pathway activated by proinflammatory cyto-

kines. Genes Dev 2001; 15:1419‑26.
23. Lawler S, Fleming Y, Goedert M, Cohen P. Synergistic activation of SAPK1/JNK1 by two
20

MAP kinase kinases in vitro. Curr Biol 1998; 8:1387‑90.

24. Lisnock J, Griffin P, Calaycay J, Frantz B, Parsons J, O’Keefe SJ, LoGrasso P. Activation of
©

JNK3 alpha 1 requires both MKK4 and MKK7: Kinetic characterization of in vitro phos-
phorylated JNK3 alpha 1. Biochemistry 2000; 39:3141‑8.
25. Fleming Y, Armstrong CG, Morrice N, Paterson A, Goedert M, Cohen P. Synergistic
activation of stress‑activated protein kinase 1/c‑Jun N‑terminal kinase (SAPK1/JNK) iso-
forms by mitogen‑activated protein kinase kinase 4 (MKK4) and MKK7. Biochem J 2000;
352:145‑54.
26. Liu J, Minemoto Y, Lin A. c‑Jun N‑terminal protein kinase 1 (JNK1), but not JNK2, is
essential for tumor necrosis factor alpha‑induced c‑Jun kinase activation and apoptosis. Mol
Cell Biol 2004; 24:10844‑56.
27. Sabapathy K, Hochedlinger K, Nam SY, Bauer A, Karin M, Wagner EF. Distinct roles for
JNK1 and JNK2 in regulating JNK activity and c‑Jun‑dependent cell proliferation. Mol
Cell 2004; 15:713‑25.

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