Summary:
MAPK (Mitogen Activated Protein Kinases) signaling pathways have been
extensively studied and are known to control multiple biological processes,
such as proliferation, apoptosis, differentiation and cellular homeostasis.
Based on the amino acid residue present in the conserved activation motif TxY,
MAPKs are divided in three families, the Extracellular Signal Regulated Kinases
(ERKs), the c-Jun N-terminal Kinases (JNKs) and the p38 kinases.
MAPKs are activated by a variety of stimuli to induce distinct cellular
responses. Environmental stressors have been shown to preferentially induce the
activation of JNK and p38 kinases, which together form the MAPK-subgroup of
Stress Activated Protein Kinases (SAPKs). The intensity and duration of MAPK
activation, amongst other factors, are known to modulate the final outcome of
the signaling. Dual Specificity Phosphatases (DUSPs) have emerged as key
regulators of MAPK-activity in development, cancer and environmental stress
conditions and are promising therapeutic targets for cancer treatment. DUSPs
belong to the super-family of Protein Tyrosine Phosphatases and are
cysteine-based enzymes able to remove phosphate groups from both
phospho-tyrosine and phospho-threonine residues. In mammals several members of
the DUSP-family have been shown to specifically target and inactivate MAPKs.
Analysis of the Drosophila genome, in combination with extensive
genetic studies, have provided proof for the main components of the MAPK
signaling pathways being highly conserved between mammals and Drosophila, thus
establishing the fly as a model organism for their study. DUSPs, as other
MAPK-regulators, are evolutionarily conserved in Drosophila melanogaster. In
particular, six DUSPs have been annotated in the Drosophila genome of which
Puckered is the most studied. Puckered was first identified as a JNK-specific
phosphatase involved in the repression of JNK signaling at the final steps of
dorsal closure, a late morphogenetic event of Drosophila embryogenesis. Later
studies also implicated Puckered in other developmental processes. In adult
flies Puckered has been shown to affect innate immunity, wound healing, stress
tolerance and longevity.
The aim of this study was the functional analysis of DUSPs in
Drosophila melanogaster. With respect to that, our efforts focused on two main
objectives. First, we were interested in elucidating the post-translational
regulatory mechanisms of Puckered activity, under oxidative stress conditions,
and second we aimed to address the in vivo role of Puckered in the development
and function of the Drosophila nervous system.
In silico analysis of the Puckered amino acid sequence revealed several
potential sites bearing the consensus phosphorylation motif for MAPKs, mapping
mainly to the C-terminus of the protein. To experimentally evaluate these
predictions, endogenous MAPK activation was induced by stimulating HEK293
cells, transiently expressing Puckered, with arsenite. Arsenite is a potent
activator of the JNK and p38 pathways in HEK293 cells, only inducing a moderate
activation of ERK1/2. Arsenite treatment of Puckered expressing HEK293 cells
resulted in a dose dependent up-shift of Puckered mobility in SDS-PAGE. The
observed shift of Puckered mobility was due to phosphorylation, since
lambda-phosphatase treatment of immunoprecipitated Puckered from extracts of
cells co-expressing Puckered with active JNK resulted in its abolition.
Co-expression of Puckered with the constitutively active forms of JNK and p38
in HEK293 cells induced Puckered phosphorylation, while ERK activation, upon
stimulation of the cells with EGF, had no impact on Puckered. In addition,
simultaneous inhibition of both JNK and p38, employing the chemical inhibitors
SP600125 and SB203580, resulted in an almost complete loss of the
arsenite-induced Puckered phosphorylation. In contrast, the MEK1 inhibitor
PD98059, while effectively preventing ERK activation, had no effect on Puckered
phosphorylation. From theses results we concluded that Puckered is
phosphorylated by JNK and p38 in response to arsenite induced oxidative stress.
Analysis of Puckered immuno-precipitates by mass spectrometry
identified the presence of phosphate on serine 413. While phosphorylation of
serine 413 was identified in Puckered from un-stimulated and arsenite
stimulated cells, it was not possible to quantify the relative abundance in
each case. The mobility shift of Puckered upon arsenite treatment could
therefore be due to phosphorylation at additional sites that escaped detection.
In order to determine the effects of phosphorylation on Puckered
function we examined both the interaction of Puckered with the SAPKs and the
ability of Puckered to attenuate arsenite-induced JNK activation. The
interaction of Puckered with the p38 kinase was detected both in the presence
and absence of oxidative stress conditions, although in oxidative stress
conditions the interaction was enhanced. In contrast, Puckered interaction with
JNK was detected only under oxidative stress conditions. Therefore, Puckered
interacts with both SAPKs and the interactions are enhanced by the induction of
oxidative stress.
Finally, the time-course stimulation of Puckered expressing HEK293 cells with
arsenite showed that Puckered phosphorylation occurs gradually, reaching its
highest level at 60 minutes of treatment. Quantification of active JNK levels,
in the presence or absence of Puckered, from extracts of cells subjected to the
same time course stimulation showed that the presence of Puckered induced a
significant reduction in JNK phosphorylation, evident up to 60 minutes of
treatment and inversely correlated with the level of Puckered phosphorylation.
This correlation suggests a role for phosphorylation in reducing Puckered
activity.
The second aim of this study was the evaluation of the in vivo role of
Puckered in the development of the nervous system of Drosophila melanogaster.
To facilitate this analysis we generated a series of transgenic fly-lines,
which would allow the spatiotemporal control of puckered gene silencing in
Drosophila. In addition, we developed an antibody against the C-terminal
portion of Puckered protein, in order to detect the endogenous molecule in
Drosophila tissues.
Silencing of puckered in all post-mitotic neurons of the fly, both
during embryonic development as well as in the stages of first and second
instar larva, resulted in 100% lethality of the organism. Characterization of
this embryonic lethal phenotype revealed abnormalities in the formation of the
axonal scaffold and the peripheral nerves. The restricted silencing of puckered
in the cells of the ventral midline gave similar results, while the genetic
dissection of this cell-group showed that puckered function is necessary in the
midline neurons but not in the midline glia cells.
These results were further supported by the analysis of puckered
expression pattern in the embryonic central nervous system, since puckered
expression was not detected in glial cells. Analysis of puckered reporter lines
revealed that puckered is present in distinct neuronal populations of both the
ventral nerve cord and the peripheral nervous system. In particular puckered
expression was detected in the aCC/RP2 and the U motor neurons, as well as the
pCC and some of the EL inter-neurons. Moreover, results from immunostaining
experiments with a Puckered antibody that we developed, revealed conspicuous
expression in the embryonic central nervous system and highlighted the
longitudinal and commissural axonal tracts, implying a potential function for
Puckered in axons. It should be noted that the puckered pattern of expression
in the ventral nerve cord was found to be dynamic, increasing its complexity in
late embryonic stages.
Finally, we characterized the expression pattern of puckered in the
adult central nervous system, where puckered was detected both in neuronal and
glial cells. Disruption of neuro-transmission within the puckered neuronal
circuit resulted in paralysis of the fly.
