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Biochimica et Biophysica Acta 1644 (2004) 61–69

Phosphorylation of MAP kinase-like proteins mediate the response of

the halotolerant alga Dunaliella viridis to hypertonic shock

Carlos Jimeneza, Tomas Berlb, Christopher J. Rivardb, Charles L. Edelsteinb, Juan M. Capassob,*

aDepartment of Ecology, Faculty of Science, University of Malaga, Malaga 29071, SpainbDivision of Nephrology, Department of Medicine, University of Colorado, School of Medicine, 4200 East 9th Avenue, Denver, CO 80262, USA

Received 24 February 2003; received in revised form 15 September 2003; accepted 31 October 2003

Abstract

The microalga Dunaliella viridis has the ability to adapt to a variety of environmental stresses including osmotic and thermal shocks, UV

irradiation and nitrogen starvation. Lacking a rigid cell wall, Dunaliella provides an excellent model to study stress signaling in eukaryotic

unicellular organisms. When exposed to hyperosmotic stress, UV irradiation or high temperature, a 57-kDa protein is recognized by

antibodies specific to mammalian p38, to its yeast homologue Hog1, and to the phospho-p38 MAP kinase motif. This 57-kDa protein appears

to be both up-regulated and phosphorylated. Three other proteins (50, 45, 43 kDa) were transiently phosphorylated under stress conditions as

detected with an antibody specific to the mammalian phospho c-Jun N-terminal kinase (JNK) motif. Treatment with specific inhibitors of p38

MAP kinase (SB203580) and JNK (SP600125) activities markedly impaired the adaptation of Dunaliella to osmotic stress. From an

evolutionary standpoint, these data strongly suggest that MAP kinase signaling pathways, other than ERK, were already operating in the

common ancestor of plant and animal kingdoms, probably as early as 1400 million years ago.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Microalgae; Hypertonicity; MAP kinase; Cell survival; Adaptation

1. Introduction

Mitogen-activated protein (MAP) kinases are highly

conserved serine/threonine kinases found in all eukaryotic

cells in combination with their upstream activators. These

kinases have been extensively studied in organisms from

yeast to humans as transducers of extracellular signals in a

variety of cytoplasmic and nuclear events [1]. In contrast, the

characterization of MAP kinases in plants is much more

limited but also of increasing interest [1–4]. Several studies

have established that plants express proteins homologous to

some of the components of the MAP kinase cascade de-

scribed in mammals [4]. Specifically, tobacco leaves stressed

by cutting activate a 46-kDa protein designated as PMSAP

[5]. Likewise, in this plant two MAP kinases, one induced by

salicylate/salcylic (SIPK) and a wound-induced protein ki-

nase (WIPK), have been described [6]. Also in tobacco, the

bacterial protein harpin induces activation of a 49-kDa

0167-4889/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbamcr.2003.10.009

* Corresponding author. Tel.: +1-303-315-6723; fax: +1-303-315-

4852.

E-mail address: juan.capasso@uchsc.edu (J.M. Capasso).

kinase [7] and the fungal elicitor cryptogein induces activa-

tion of both a 46- and a 50-kDa MAP kinase [8]. In tomato

leaves, wounds turn on a 48-kDa protein [9] and in alfalfa a

kinase designated as MKK4 is activated by cold stress and

drought [10]. Signal pathways have been explored in Arabi-

dopsis thaliana leaf cells exposed to cold, touch [11],

bacterial products [12,13] and oxidative stress [14].

Osmotic stress homologues to the yeast osmoreceptor

[15] have been sought in plants [12]. In A. thaliana a two-

component system was cloned [16] including the salt stress-

induced transcription of a gene and the expression of a

protein (ATMPK3) that is structurally related to MAP

kinases [11]. The aforementioned alfalfa MKK4 activated

by drought is not altered by salinity [10], but rather a 46-kDa

MAPK is salt induced (SIMK), which is localized in the

nucleus [17]. In tobacco plants placed under osmotic stress

conditions, three different kinases (44, 46 and 50 kDa) were

activated by tonicity [18], the latter two being identified as

the aforementioned tobacco SIPK and WIPK, respectively.

The 44-kDa protein does not appear to belong to the MAP

kinase family as it failed to be recognized by anti-human

extracellular signal-regulated kinase (ERK1/ERK2) antibody.

C. Jimenez et al. / Biochimica et Biophysica Acta 1644 (2004) 61–6962

In tobacco plants a calcium-dependent serine/threonine ki-

nase is activated by hypotonicity [19].

Most plant MAP kinases have been described by their

ability to phosphorylate myelin basic protein [9] or by

genetic analysis [11]. Based on sequence analysis, the

presently known plant MAPKs are most similar to ERKs,

even though increasing evidence indicates that those kinases

are also involved in various forms of biotic and abiotic stress

responses. In other words, all plant MAPKs reported to date

possess the TEY motif [20,21].

Protein kinase activities have been demonstrated in the

green alga Dunaliella on exposure to osmotic stress [22,23].

However, these proteins appear also to belong to the ERK

subfamily, due to their ability to phosphorylate human ERK.

In addition, no recognition by specific MAP kinase anti-

bodies has been demonstrated previously. The present stud-

ies were performed in a microalgae of the genus Dunaliella

that lives in a hyperosmolar aquatic environment and which

needs to cope with rapid changes in salinity. The experiments

were designed to explore the possibility that these lower

organisms respond to changes in tonicity by activating MAP

kinase signaling pathways other than ERK, such as p38 and

JNK, and whether inhibiting these pathways compromises

the ability of these cells to adapt to environmental stresses.

2. Materials and methods

2.1. Algae culture

D. viridis Teodoresco was isolated from the athalassic lake

of Fuente de Piedra (Malaga, Spain). Dunaliella was grown

in batch culture as described by Jimenez and Niell [24], in

a basal medium containing 2 M NaCl and 5 mM NaNO3.

Cells were cultivated under continuous orbital shaking and

continuous illumination (150 Amol m� 2 s� 1) provided by

commercial cool-white fluorescent lamps, at a temperature of

25 jC.

2.2. Experimental conditions

Cells in their mid-exponential phase of growth were used

for experiments. They were subjected to three kinds of stress:

(i) osmotic stress, (ii) UV irradiation, and (iii) high temper-

ature. Osmotic stress consisted either of an increase or a

decrease of the osmotic pressure in the medium using either

NaCl or glycerol. Cells cultivated at 2 M NaCl were

concentrated by centrifugation (1500� g, 10 min) and the

pellets were resuspended in growth medium containing

either 4 M NaCl or 5.04 M glycerol for hyperosmotic shock,

1 M NaCl or 1.57 M glycerol for hypoosmotic shock or 2 M

NaCl or 2.81 M glycerol for control cultures. Osmotic

equivalence between NaCl and glycerol concentrations was

calculated according to the Handbook of Chemistry and

Physics [25]. The influence of UV irradiation was studied

by exposing cultures of D. viridis to 70 mJ cm� 2 of UV light

in the range 200–400 nm using a GS Gene Linker UV

chamber (Bio-Rad, Hercules, CA). Cultures were placed in

14-cm Petri dishes and, following UV exposure, were kept

under continuous orbital shaking with illumination [150

Amol m� 2 s� 1 of visible light (400–700 nm)]. For thermal

stress, cultures were incubated at 36 jC in 50-ml clear plastic

conical tubes in a temperature-controlled water bath with

continuous shaking and illumination. At fixed times, an

aliquot of culture was removed and centrifuged at

1500� g for 10 min. Cell pellets were resuspended in 1 ml

of MAP kinase lysis buffer [26] and kept at 4 jC for 1 h.

Samples were frozen in liquid N2 and kept at � 80 jC or

processed immediately. After thawing, samples were centri-

fuged at 100,000� g for 45 min in a Beckman L8-60M

ultracentrifuge (type Ti-70 rotor). Supernatants were frozen

at � 80 jC until further analysis.

2.3. Treatment with inhibitors

Aliquots of the same algae culture were transferred to 50-

ml conical tubes. Appropriate volumes of inhibitor solutions

in DMSO (1000� stock) were added to make the culture

media 20 AM in SB203580 or 1 AM in SP600125, both

inhibitors were provided by Calbiochem (La Jolla, CA). At

these concentrations these molecules are very selective inhib-

itors of the p38 MAPK [27] and JNK [28] pathways,

respectively. After 2 h of incubation with the inhibitor, cells

were challenged for increasing times with a hyperosmotic

shock (4 M NaCl) as described above.

2.4. Western blot analysis

Sample protein concentration was determined by the

bicinchoninic acid method (Pierce, Rockford, IL). Equal

amounts of protein were loaded per lane (50 Ag/lane) forSDS-PAGE. To ensure adequate data analysis several experi-

ments were run in duplicate with one of the gels stained with

Coomassie blue to confirm uniform protein loading. Electro-

phoresis conditions, electroblotting to PVDF membranes,

and immunodetection were performed as previously de-

scribed [27,29]. Antibodies were purchased from Cell Sig-

naling Technology (Beverly, MA) and Santa Cruz

Biotechnology (Santa Cruz, CA).

Antibody specificity: Both p38 and Hog1 antibodies detect

the presence of the corresponding kinases, regardless of their

phosphorylation state. These antibodies are specific for p38/

Hog1 and do not cross-react with JNK and ERK. The

reactivity represents a total concentration of all forms of the

protein. Hog1 from yeast has been previously demonstrated

to be functionally and structurally homologous to the mam-

malian p38 [30]. The phospho-p38 antibody detects only the

phosphorylated form of this kinase and is specific for the

antigen sequence T*GY* and does not cross-react with

similar sequences such as TPY (JNK) or TEY (ERK). This

phospho-p38 antibody does not react with either the non-

phosphorylated or single phosphorylated form of the protein.

C. Jimenez et al. / Biochimica et Biophysica Acta 1644 (2004) 61–69 63

At this time an antibody specific to the non-phosphorylated

form of p38 MAPK is not available although the non-

phosphorylated p38 pools may be inferred with respect to

the difference in signal kinetics from Western blots using the

two antibodies described above.

The phospho-JNK antibody used is specific for the T*PY*

sequence and only when double-phosphorylated. This anti-

body does not recognize either the non-phosphorylated or

single phosphorylated form of the protein. This antibody does

not cross-react with p38 or ERK. This antibody recognizes all

isoforms of the JNK protein. This information has been

provided by the manufacturers as well as corroborated by

the authors.

Band analysis was performed as described [26]. Analysis

of the same blot with different antibodies was done by

striping with Western Re-Probe (Geno Technology, St.

Louis, MO) as directed by the manufacturer.

2.5. Metabolic labeling of Dunaliella with radioactive

phosphate

To detect protein phosphorylation in Dunaliella in res-

ponse to stress, mid-log cultures growing at 2 M NaCl were

concentrated by centrifugation to 10% of the initial volume,

and incubated overnight in the presence of 2 mCi of 32P

(K2HPO4, SpAc 1 Ci/mM, NEX055, Perkin Elmer Life

Sciences, Boston, MA). Cells were osmotically challenged

by adding NaCl to a 4 M final concentration. Cells were

Fig. 1. Aliquots of mid log phase Dunaliella cultures were challenged as described

hypoosmotic conditions) for increasing time periods. Cell protein extracts were an

form of mammalian p38 MAP kinase. The graph depicts the MeanF S.E. of the 5

blot is also shown.

harvested by centrifugation after 4 h, and washed three

times in 4 M NaCl medium. Samples were prepared for

electrophoresis as above. Gels were stained with Coomassie

blue, digitized, dried and autoradiographed using Kodak

BioMax ML film with one intensifying screen for different

times.

2.6. Cell viability measurement

Algal cell viability was measured using CellTiter 96AQ

(Promega, Madison, WI), as described by Capasso et al.

[31].

2.7. Statistics

Results were analyzed by using the INSTAT software

package (GraphPad, San Diego CA). A value of P < 0.05

was considered significant.

3. Results

3.1. Effects of alterations in NaCl concentration on

proteins probed with a phosphorylated p38 MAP kinase

antibody

Cultures of D. viridis, growing under basal conditions (2

M NaCl), were exposed to either a hypertonic stress

in Section 2 with three different NaCl treatments (control, hyperosmotic and

alyzed by Western blot using an antibody raised against the phosphorylated

7-kDa band intensity from three independent experiments. A representative

Fig. 2. Cell cultures were subjected to thermal and UV stresses. Protein

extracts were analyzed by Western blot using antibodies specific for

phosphorylated p38 MAP kinase (as in Fig. 1). (A) Cells were exposed to

thermal shock by incubation at 36 jC, quantitative analysis of band intensity(n= 4) and one representative blot is shown. (B) Cells were exposed to

irradiation with UV light (70 mJ cm� 2/200–400 nm); the graph depicts the

MeanF S.E. of the 57-kDa band intensity (n= 3); a representative blot is

also shown. The difference between 0 and 15 min is highly significant

( P < 0.01).

C. Jimenez et al. / Biochimica et Biophysica Acta 1644 (2004) 61–6964

(medium supplemented to 4 M NaCl) or to a hypotonic

stress (medium containing 1 M NaCl) for varying times

ranging from 0 to 4 h. Exposure to 4 M NaCl was

associated with a time-dependent increase in the level of a

protein band with an estimated molecular weight of 57 kDa

(Fig. 1). This band cross-reacts with a highly specific

antibody raised against the phosphorylated form of the

mammalian p38 MAP kinase (P-Thr/Gly/P-Tyr). This phos-

phorylated protein was not detected in control cells main-

tained at 2 M NaCl or cells exposed to a hypotonic stress

for as long as 4 h. The same result was obtained using

equivalent concentrations of glycerol instead of NaCl in

order to increase or diminish the medium tonicity (data not

shown).

3.2. Effect of UV-irradiation and thermal shock on the

phosphorylation of the 57-kDa protein

To assess whether alternative environmental stresses also

caused the phosphorylation of the 57-kDa protein, algal

cells were exposed to heat shock or to UV irradiation for

increasing times. Exposure of algal cells to elevated tem-

perature (36 jC) induced rapid appearance of the 57-kDa

phosphoprotein using the phosphorylated p38 MAP kinase

antibody (Fig. 2A). However, the time course of phosphor-

ylation was very different from that obtained during os-

motic shock. After only 30 min of incubation at 36 jCmore than 50% of the maximum level had been reached

and after 60 min the level had essentially reached saturation

(Fig. 1 vs. Fig. 2A). The effect of UV irradiation (70 mJ/

cm2) on algal cell cultures is shown in Fig. 2B. Similar to

the effect of thermal shock stress, a very rapid increase in

the 57-kDa phosphoprotein was detected upon irradiation

of the cells; the kinetics of the phosphorylation was slightly

different.

3.3. Effect of hypertonicity on the expression of a protein

that cross-reacts with non-phosphorylated p38 MAP kinase

and Hog1 antibodies

In view of the above results, we examined whether the

57-kDa protein could also be detected in algal cells using

antibodies specific for the mammalian p38 MAP kinase and/

or its yeast homologue, Hog1 kinase, and if so, whether its

expression is altered by exposure to hypertonicity. As

described in Section 2, these antibodies recognize the

presence of both phosphorylated and unphosphorylated

states of the kinase. Fig. 3 shows that a protein band of

57 kDa is detected using both the p38 MAP kinase antibody

(Fig. 3A) and the Hog1 antibody (Fig. 3B). These data

indicate that the protein band cross-reacts more strongly

with the yeast antibody than with the mammalian one. In

contrast to the observation in mammalian cells that p38

MAP kinase is present at a relatively constant level [26], the

57-kDa protein is present at a low level under basal

conditions and is up-regulated in a time-dependent manner

following osmotic stress (Fig. 3A). This indicates that in

Dunaliella, stress induces both phosphorylation and synthe-

sis of the p57 protein, suggesting that it is part of the stress-

induced response mechanism. Interestingly, changing the

culture medium by itself (involving centrifugation and

resuspension) caused a small time-dependent increase in

the protein level (Fig. 3A and B), but no phosphorylation of

this protein occurred (see control data in Fig. 1).

3.4. 32P metabolic labeling of Dunaliella cultures under

osmotic stress

Cell cultures of Dunaliella were metabolically labelled

with 32P, as described in Section 2, to confirm the phos-

phorylation of the p57 protein in response to osmotic stress.

Fig. 4 clearly depicts incorporation of radioactivity into

several protein bands in response to hyperosmotic stress.

The main ones had apparent molecular weight of 54, 51 and

48 kDa, and of special interest for this work is the band at

57 kDa.

Fig. 3. Aliquots of mid log phase Dunaliella cultures were challenged as

described in Section 2 under control and hyperosmotic conditions for

increasing time periods. Basal conditions refer to protein extracts from cells

that were not challenged but only exposed to a media change. (A) Western

blot analysis employed a specific mammalian p38 MAP kinase antibody;

the MeanF S.E. of the 57-kDa band intensity is shown (n= 2). Differences

between control and hyperosmotic shock were significant ( P < 0.02). A

representative blot is shown. (B) A similar experiment was performed

employing the yeast Hog1 antibody. The graph depicts the MeanF S.E. of

the 57-kDa band intensity (n= 4); a representative blot is also shown.

Differences between control and hyperosmotic stress were very significant

( P< 0.004).

Fig. 4. Autoradiography of protein extracts from metabolically labelled 32P

Dunaliella cultures. (A) Control cells, and (B) Cells subjected to

hyperosmotic shock (2! 4 M NaCl) for 4 h. One hundred micrograms

of protein was loaded in each lane. One of three independent experiments is

shown.

C. Jimenez et al. / Biochimica et Biophysica Acta 1644 (2004) 61–69 65

3.5. Effect of changes in tonicity and UV irradiation in

proteins probed with a phosphorylated JNK antibody

We examined whether exposure to non-isotonic condi-

tions altered the phosphorylation of proteins in Dunaliella

when probed with a specific antibody against another

member of the mammalian MAP kinase family, the double

phosphorylated form of JNK. As is depicted in Fig. 5, three

protein bands with apparent molecular weights of 50, 45,

and 43 kDa were evident. This band pattern is strikingly

similar to that obtained with mammalian renal cells [26].

Statistical data analysis (ANOVA) of the phosphorylation of

the 50-kDa band reveals no significant difference (P>0.21)

between the basal level and either the media change control

or hypoosmotically shocked cells. In contrast, exposure to

4M NaCl caused a rapid and transient increase in the

intensity of the 50-kDa protein band that peaked around

30 to 90 min and returned to basal levels after 4h following

osmotic shock. The 45- and 43-kDa bands follow the same

pattern although it was difficult to get reliable quantitative

data from such faint signals. A very similar result was

obtained upon UV irradiation. As shown in Fig. 6, a

transient increase in the level of the 50-kDa phosphoprotein

was detected. There were also some differences in the

kinetics of phosphorylation between osmotic and UV irra-

diation stresses (Fig. 5 vs. Fig. 6). Under UV stress, the 50-

kDa phosphoprotein peaked at 15 min and returned to basal

levels after 1 to 2 h. It is noteworthy that a basal level of

phosphorylated 50-kDa JNK-like protein was determined in

Dunaliella (Figs. 5 and 6). In contrast, phosphorylation of

the 57-kDa p38/Hog1-like protein was detected only under

stress.

3.6. Effect of inhibitors of p38 MAP kinase and JNK

activities on the adaptation of Dunaliella to osmotic shock

Specific inhibitors of p38 and JNK activities were

employed to demonstrate whether these MAP kinase-like

Fig. 5. Aliquots of mid log phase Dunaliella cultures were challenged as described in Section 2 with three different NaCl treatments (control, hyperosmotic and

hypoosmotic conditions) for increasing time periods. Cell protein extracts were analyzed by Western blot using an antibody raised against the phosphorylated

form of mammalian JNK. The graph depicts the MeanF S.E. of the 50-kDa band intensity from three independent experiments; a representative blot is also

shown. Basal refers to cells that were not manipulated. Differences in control and hyperosmotic stress conditions were significant between 30 and 90 min

( P< 0.02).

Fig. 6. Dunaliella cells were irradiated with UV light (70 mJ cm� 2 at 200–

400 nm), and protein extracts were obtained at increasing time periods after

shock and analyzed by Western blot using an antibody specific for

phosphorylated JNK as described in Section 2. The graph shows the band

intensity of the 50-kDa phosphoprotein (MeanF S.E., n= 3); a typical blot

is also shown. Differences in band intensity between 0 and 15 min were

very significant ( P< 0.01).

C. Jimenez et al. / Biochimica et Biophysica Acta 1644 (2004) 61–6966

signaling pathways are present and physiologically re-

levant for adaptation of Dunaliella cells. For this purpose

hyperosmotic shock experiments were performed using

the specific p38 MAP kinase inhibitor SB203580 and

JNK pathway inhibitor SP600125. Cell cultures were

incubated for 2 h with 20 AM SB203580 (standard

concentration for mammalian cell experiments [27]) prior

to a 4 M NaCl shock. The change in osmolality pro-

duces a well-known and profound decrease in the

physiological function of the cells with recovery occur-

ring over the ensuing 48 h [32]. However, this recovery

was markedly impaired in cells exposed to the p38 MAP

kinase inhibitor (Fig. 7A). This effect was neither a

consequence of the solvent (dimethyl sulfoxide) (Fig.

7A) nor a nonspecific toxic effect of SB203580 per se,

since it had no effect on cells maintained at 2 M NaCl

(Fig. 7B). As shown in Fig. 7A, a more profound effect

was noted when Dunaliella cells were challenged with

hyperosmotic stress after preincubation for 2 h with 1

AM of the JNK pathway inhibitor SP600125. The

recovery of algal cells after hyperosmotic shock was

completely abolished by the JNK inhibitor, although

Fig. 7. Cell cultures were preincubated for 2 h with MAP kinase

inhibitors or with the same volume of solvent (DMSO) before being

subjected to (A) hyperosmotic stress (4 M NaCl) or (B) allowed to

remain at 2M NaCl. Culture samples were removed at increasing times to

assess cell viability by the CellTiter 96 assay as described in Section 2.

(A) Depicts the effect of the p38 MAP kinase inhibitor SB203580 (20

AM) on cell viability after osmotic shock. Data points are the

MeanF S.E. of five independent experiments. Differences between

control and inhibitor treatment were significant at 30 h ( P < 0.014) and

very significant at 48 h ( P< 0.001). Preincubation with the JNK inhibitor,

SP600125, demonstrated more profound effects. The carrier DMSO alone

had no effect on Dunaliella response to hypertonic conditions. (B) shows

the effect of the specific MAP kinase inhibitors on non-stressed cell

cultures.

C. Jimenez et al. / Biochimica et Biophysica Acta 1644 (2004) 61–69 67

the presence of inhibitor did not adversely affect control

cells (Fig. 7B).

4. Discussion

Recent studies indicate that MAP kinase signaling cas-

cades which have been extensively described in organisms

from yeast to mammals [1] are also present in vascular

plants such as Arabidopsis, tobacco, and alfalfa [20].

However, while three MAPK subfamilies (ERK, JNK, and

p38 MAP kinase) are present in animal cells, plant kinase

genes appear to belong solely to the ERK subfamily [20].

The presence of MAP kinase pathways in aquatic cellular

plants such as green algae (from which higher plants

evolved in the transition to terrestrial life) has not been

previously described. The use of Dunaliella as a model to

study signaling systems in unicellular algae is based on the

lack of a cell wall and its ability to adapt to environmental

stresses. The lack of a cell wall allows for the use of

common protocols used for mammalian cells to study

genetic and protein systems. This study reveals that the

microalgae Dunaliella possess a 57-kDa protein that is

induced in response to increasing tonicity, but not by

hypotonic stress. This 57-kDa protein was detected by

cross-reactivity with mammalian p38 MAP kinase antibody

and even more robustly with the antibody against the yeast

homologue, Hog1, an osmo-sensitive kinase. Furthermore,

exposure of algal cells to hypertonicity is rapidly followed

by phosphorylation of the protein, which is detected by a

highly specific antibody raised against the phospho p38

MAP kinase motif (T*GY*). This up-regulation and phos-

phorylation of the 57-kDa protein is not a specific NaCl

effect but rather an osmotic effect, since equivalent

increases in osmolality using glycerol had an almost iden-

tical effect. In addition, this protein is also up-regulated and

phosphorylated by non-osmotic stresses such as UV irradi-

ation and thermal shock. It is also of interest that the

phosphoprotein was not detected under basal conditions

(i.e., 2 M NaCl). Confirmation of the presence of the p57

kDa phosphoprotein was validated through metabolic label-

ing experiments, in which clear phosphorylation of a 57-

kDa protein band appeared in response to hyperosmotic

stress.

We also explored the possibility that another member of

the MAPK family, the c-Jun N-terminal kinases (JNKs), was

activated in Dunaliella by the same stresses that activate

them in mammalian cells. In fact, we were able to detect a

pattern of three protein bands similar to that found in

IMCD3 (inner medullary collecting duct) cells using a

highly specific antibody raised against the phospho JNK

motif (T*PY*). These bands, as occurs in mammalian cells,

were present at basal conditions in the algae, and their

intensity was also greatly and transiently increased under

hyperosmotic shock and UV irradiation.

The most compelling evidence for the presence and the

physiological role of p38-like and JNK-like pathways in

Dunaliella is provided by using highly specific inhibitors of

the p38 and JNK signaling pathways at standard concen-

trations. The relevance of the activation of these pathways in

the adaptation and survival of mammalian cells in response

to stress has been well established. For instance, in MDCK

osmotically stressed cells, inhibition of the p38 MAP kinase

with SB203580 was associated with loss of induction of

aldose reductase, an enzyme that generates sorbitol, which

in turn is an osmolyte involved in hypertonic adaptation

[33]. Also, inhibition of the JNK pathway with SP600125

C. Jimenez et al. / Biochimica et Biophysica Acta 1644 (2004) 61–6968

results in a significant decrease in cell survival after hyper-

osmotic shock in IMCD3 cells [34]. To date, despite the

increasing evidence of protein phosphorylation in response

to a great variety of stresses in plants, no indisputable direct

evidence for its physiological role has been provided. The

present work gives strong evidence for the relationship

between activation of the signaling pathways and cell

adaptation and survival upon hyperosmotic stress. We

propose that the p38-like kinase signaling cascade is in-

volved in the late response to environmental stress in

Dunaliella as protein synthesis is involved. It is well known

that the early response (glycerol synthesis) is independent of

protein synthesis. This de novo protein synthesis of the

signaling kinase is dissimilar to that in mammalian cells

[26]. However, Dunaliella’s decrease in adaptability and,

eventually, in cell survival in the presence of the specific

inhibitors of the p38 and JNK signaling pathways strongly

suggests the existence of signaling mechanisms in these

unicellular microalgae similar to those found in mammalian

cells.

Cloning of some of the components of these putative

signaling pathways will have very interesting evolutionary

implications. The fact that we have found p38-like and c-

Jun-like MAP kinases in an unicellular green microalga

suggests that these pathways were already present in the

common ancestor of the three main eukaryotic lines (i.e.

green plants, including both the green algae and the higher

plants, eukaryotic fungi of the phylum Ascomycota which

includes yeasts and molds, and the metazoan animals).

According to these results, we hypothesize that higher plants

are expected to have p38-like and c-Jun-like MAP kinases.

At present, none of them have yet been described. On the

other hand, the fact that no c-Jun-like MAP kinase has been

found in yeast, even though at least 12 complete yeast

genomes are known, would indicate an evolutionary loss of

this signaling pathway. Analysis of the 18S and 28S rRNA

nucleotide sequences shows that the ancestors of all the

main eukaryotic evolutionary lines were probably unicellu-

lar flagellates, represented in the fossil record by acritarchs

during Neoproterozoic times, some 1400 million years ago

[35,36]. This close phylogenetic relationship between the

green algae and the higher animals has also been confirmed

by comparisons of several gene and amino acid sequences

[37,38].

In summary, these data identify, for the first time, the

presence of p38- and c-Jun-like MAP kinase proteins in

algae and their phosphorylation in response to increments in

osmolality and other stress conditions. Also, these data

reveal that specific chemical agents that inhibit the activity

of these enzymes have markedly deleterious effects on the

ability of the algae to overcome hyperosmotic stress, there-

by revealing their critical role in adaptation and survival of

Dunaliella under such environmental conditions. Finally,

the data suggest that the origin of these MAP kinase

signaling pathways predates the divergence of the plant

and the fungi/animal lineages.

Acknowledgements

We thank Zafie Craft for excellent secretarial assistance.

This work was supported by a grant from the National

Institute of Health, DK-19928 to TB. C.J. was supported by

Research Project REN2002-00340/MAR of the Spanish

Ministry of Science and Technology, and by a Research

Grant from the University of Malaga. We thank Professors

Lynn Heasley and Uri Pick for critical reading of the

manuscript.

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