(Received for publication, December 6, 1995; and in revised form, December 22, 1995)
From the
Mitogen-activated protein (MAP) kinases are involved in
controlling a cell's responses to a variety of stimuli and can be
activated by both protein tyrosine kinase and G protein-coupled
receptors. It was shown previously that Dictyostelium MAP
kinase ERK2 is required for normal activation of adenylyl cyclase and erk2 null cells are aggregation-deficient. In this manuscript,
we show that the Dictyostelium MAP kinase ERK2 is rapidly and
transiently activated in response to the chemoattractant cAMP. This
response requires cAMP receptors, but is independent of the coupled
G2 subunit and the only known G
subunit. These data indicate
that ligand-mediated receptor activation of adenylyl cyclase requires
two receptor-dependent pathways, one of which requires heterotrimeric G
proteins, including G
2 and the only known G
subunit, and the
second of which requires ERK2. Our results suggest that ERK2 may be
activated by a novel receptor-mediated pathway.
MAP ()kinases (MAPKs) or ERKs control a wide array of
cellular responses in eukaryotes and are stimulated by environmental
stress and through receptor tyrosine kinases and G
protein-linked/serpentine
receptors(1, 2, 3, 4, 5) .
In the yeast Saccharomyces cerevisiae alone, five distinct MAP
kinase activation pathways have been defined that control mating,
pseudohyphal growth, cell integrity, response to osmotic shock, and
sporulation(6) . Little is known about the diversity of
mechanisms that can lead to the activation of MAP kinase cascades. In S. cerevisiae and mammalian cells, some MAP kinase cascades
can be controlled, at least in part, through G
subunits(7, 8, 9) , while the pathway that
activates the MAP kinase Hog1 in response to osmotic shock is regulated
through a transmembrane histidine kinase(10) .
In Dictyostelium, the multicellular organism is formed via the
chemotactic aggregation of up to 10 cells. The
chemoattractant is extracellular cAMP that binds to the serpentine
receptors cAR1 or cAR3, which are coupled to the heterotrimeric G
protein containing the G
2 subunit, activating guanylyl and
adenylyl cyclases, triggering alterations in the cytoskeleton, and
inducing gene
expression(11, 12, 13, 14, 15) .
Recent results indicate that the G
subunits released by the
ligand activate adenylyl cyclase, whereas the coupled G
2 subunit
is thought to regulate a variety of other
effectors(11, 16, 17) . The aggregation-stage
adenylyl cyclase in Dictyostelium has a similar structure to
that of mammalian adenylyl cyclases(18) ; however, the
aggregation-stage adenylyl cyclase has a more complex activation
pathway compared with known activation pathways for mammalian adenylyl
cyclases(19) . In addition to the G
subunit, adenylyl
cyclase activation in Dictyostelium also requires a pleckstrin
homology domain containing protein designated
CRAC(20, 21) , and, surprisingly, a MAP kinase ERK2. erk2 null cells do not aggregate due to their deficiency in
the ability to activate adenylyl cyclase and relay the cAMP
signal(22) .
In this communication, we show that the MAP
kinase ERK2 is rapidly and transiently activated in response to cAMP.
The kinetics of this response are similar to those of the activation of
adenylyl cyclase. The response requires the cAMP receptor necessary for
mediating other cAMP-mediated responses, but is independent of the
coupled G2 and the only known G
subunit. Our data indicate
that receptor activation of adenylyl cyclase requires two
receptor-dependent pathways, one requiring G
and involving
ERK2.
Figure 1: cAMP stimulated ERK2 activation. In-gel assay of cAMP stimulation of ERK2 activity. The activation of ERK2 was measured in aggregation-competent cells (cells pulsed with nM cAMP for 4 h, see ``Materials and Methods''). The lanes on the right are ``normalization'' controls containing extracts of a time course that allows an internal standardization of the results between gels and between experiments. An aliquot is also taken to quantitate total protein for normalization of activity between different strains and experiments. The same amount of total extract protein is loaded in each lane. In general, the same amount of protein is loaded for each sample between the different experiments. The autoradiographs are quantitated by densitometry and relative activity is normalized using the internal controls and protein levels. The band corresponding to ERK2 is marked with a solid arrowhead. This band is missing in erk2 null cells. The 30-kDa kinase is labeled with an open arrowhead.
To confirm that cAMP-stimulated MBP
phosphorylating activity was ERK2, we examined the activation profile
in erk2 null cells and cells overexpressing ERK2. No
stimulation of this activity was observed in erk2 null cells,
while expression of ERK2 in the erk2 null background
(complemented erk2 null cells) resulted in the restoration of
the activity (Fig. 1). In addition, these erk2 null
complemented cells showed normal aggregation and morphological
differentiation that was indistinguishable from that of wild-type cells ((30) ; data not shown). The maximum level of kinase activity
in the erk2 null complemented cells was higher than that in
wild-type cells, probably due to an overexpression of the kinase (see
below). Overexpression of ERK2 in wild-type cells resulted in an
4-fold higher level of stimulated kinase activity (data not
shown). When the samples were analyzed by Western blot with a rabbit
polyclonal anti-ERK2 antibody made against a GST-ERK2 fusion (see
``Materials and Methods''), a band of 42 kDa was observed
that was absent in erk2 null cells (Fig. 2A).
A higher level of ERK2 was observed in the ERK2 overexpressor strain (Fig. 2A). This antibody did not interact with the
other known Dictyostelium MAP kinase, ERK1 (Fig. 2B), or with other proteins with the same
mobility as ERK2, although it did interact with some other proteins (Fig. 2, A and B). In the complemented null
cells overexpressing ERK2, the increased protein was greater than the
increased maximal level of activity seen in this assay (Fig. 1).
We assume that either not all of the kinase could be activated or the
assay was not linear at high kinase levels.
Figure 2: Western blot analysis with immunopurified anti-ERK2 antibodies. A, Western blot of erk2 null, wild-type, and ERK2 overexpressing cells. B, Western blot of glutathione-Sepharose-purified GST, GST-ERK2 fusion protein, and GST-ERK1 fusion proteins expressed in E. coli. See ``Materials and Methods.''
A background activity at
the same molecular weight as ERK2 was seen in erk2 null cells
and in unstimulated wild-type cells (Fig. 1). Notably, this
unstimulated activity was not detectably increased by overexpression of
ERK2, suggesting that the kinase activity at this mobility may not be
ERK2 and that ERK2 basal activity was very low. There is at least one
other MAP kinase, ERK1, in Dictyostelium and may be
responsible for the background activity in these assays. ERK1 has a
similar electrophoretic mobility to that of ERK2 on these gels, uses
MBP as a substrate, and is active at the same time in
development(26) . The absence of stimulation of the band at
this mobility in erk2 null cells suggests ERK1 is not
stimulated in response to cAMP, consistent with previous results (26) . When the ERK2 stimulation in response to cAMP in
wild-type cells was quantitated, we observed an 40-fold increase
in the level of kinase activity at this mobility. If the background
band observed in erk2 null cells were subtracted, the relative
level of stimulation would be higher.
Figure 3:
cAMP stimulation of ERK2 activity in
mutant strains. Assays were done as described above. The g null and g
2 null cells carry a vector that constitutively
expresses cAR1 from an actin (Act15) promoter, since these
strains express a lower number of receptors than wild-type cells after
cAMP stimulation.
To examine the possible role of heterotrimeric G proteins in ERK2
activation, g2 and g
null
strains(14, 39) were first transformed with a plasmid
that constitutively expresses cAR1 during growth and development. This
measure ensured that these cells had appropriate levels of receptors.
Surprisingly, in g
2 and g
null cells
constitutively expressing cAR1, the ERK2 pathway was also stimulated.
However, the onset of activation was delayed, and its peak was reduced
compared to that in wild-type cells. When quantified, the maximal level
of activation in the g
2 and g
null strains
varied between
40 and 80% of that observed in wild-type cells (six
separate experiments) and between
50 and 60% of that in wild-type
cells constitutively expressing cAR1 (data not shown).
Our analysis
has shown that the MAP kinase ERK2 is transiently activated in response
to chemotactic signaling by cAMP and that this activation is dependent
on cARs but is independent of G2, the known G
subunit that
couples to cAR1 and cAR3. Further, the activation of ERK2 occurred
independently of the only known Dictyostelium G
subunit,
that like G
2, is required for the in vivo activation of
adenylyl cyclase. The slower kinetics and the reduced level of
activation of ERK2 in g
and g
2 null cells
compared with wild-type cells nevertheless suggest that a G
protein-mediated pathway is important in obtaining a completely
wild-type response.
erk2 null cells express adenylyl cyclase aggregation-stage adenylyl cyclase and CRAC, another required component for this pathway (22) , indicating that ERK2 is not required for the expression of these genes. The kinetics of activation and deactivation parallel those of adenylyl cyclase, suggesting that activated ERK2 may be required continuously for the activation of adenylyl cyclase. Other results have shown that receptor-mediated activation of at least some aggregation-stage genes in response to cAMP pulses is normal in erk2 null cells(30) . However, we cannot exclude that ERK2 is required for the expression of some unknown gene and that the kinetics of activation are fortuitously similar to those of adenylyl cyclase.
Two different receptor-mediated pathways
are involved in cAMP activation of adenylyl cyclase; one requires the
G2 and G
subunits and the other requires ERK2 (11, 12, 22) (see model in Fig. 4). We
now find that ERK2 is activated by cAMP and that this requires cAMP
receptors, but is independent of either G
2 or the known G
that are also required for adenylyl cyclase activation. While the
possibility of another G
subunit can never be completely excluded,
there is significant biochemical evidence that Dictyostelium has only one G
subunit as described above (17, 39) . On the weight of this evidence, we
entertain the intriguing possibility that ERK2 is activated by a
pathway that may be independent of heterotrimeric G proteins. One model
to accommodate our observations is that the receptor functions as a
docking site for proteins that stimulate downstream pathways, one of
which leads to the stimulation of the ERK2 activation cascade (Fig. 4). This model has precedent in the known docking of
different effectors to activated receptor tyrosine kinases. The
underlying mechanism for docking to serpentine receptors such as cAR1
could be similar to the binding mechanisms that allow heterotrimeric G
proteins to interact with ligand-bound (``activated'')
receptors or receptor kinases to interact with and phosphorylate the
ligand-bound but not free receptors(40, 41) .
Figure 4:
Model for cAMP stimulation of adenylyl
cyclase and ERK2. Upper panel shows proposed location of
relevant components in an unstimulated cell. The bottom panel shows the G protein-dependent (G) pathway, CRAC, and
possible input of ERK2. It is expected that ERK2 is activated by a
conserved upstream activating cascade containing a MEK and MEKK (shown
as speckled ovals). The mechanism of activation of this
putative MAP kinase cascade is not known but it is independent of the
G
subunit required for interactions with CRAC and
aggregation-stage adenylyl cyclase. One model proposes that the
receptor acts as a docking site for the unknown required components, as
tyrosine kinase receptors act as a docking site. In addition, the
activation of calcium influx is also a receptor-dependent, but is
independent of the G
subunit. Whether this functions with another
receptor-mediated process or directly to promote the activation of the
MAP kinase cascade is not known. A relevant example is the
ligand-dependent but G protein-independent binding of both
ARK and
rhodopsin kinase to their respective receptors (see
text).
There
is evidence for G protein-independent pathways mediated through
serpentine receptors in mammalian cells. As described above, the
binding and phosphorylation of rhodopsin and -adrenergic receptors
by their respective kinases in mammals requires ligand binding, but is
independent of G proteins(40, 41) . In Dictyostelium, cAMP stimulation of Ca
influx
and cAMP-mediated activation of post-aggregative gene expression in the
multicellular stages (which is activated through the transcription
factor GBF and a high, continuous level of cAMP) are known to be
independent of the identified G
subunit and have been proposed to
be G protein-independent(42, 43) . Both G proteins and
receptor kinases interact with receptors in response to ligands, and
recent evidence indicates that STAT transcription factors are activated
through the angiotensin II receptor (44) , although it is not
known whether a JAK kinase directly couples to this receptor. With the
biochemical evidence that receptor kinase binding and phosphorylation
is ligand-dependent but G protein-independent, it is possible that
other heterotrimeric G protein-independent pathways will be identified
as future genetic analysis permits further dissection of pathways
controlled via serpentine receptors.