(Received for publication, September 5, 1995)
From the
CSBP1 and CSBP2 are human homologues of the Saccharomyces
cerevisiae Hog1 mitogen-activated protein kinase which is required
for growth in high osmolarity media. Expression of CSBP1, but not
CSBP2, complemented a hog1 phenotype. A CSBP2 mutant
(A34V) that complements hog1
was isolated and found to
have
3-fold lower kinase activity than the wild-type CSBP2.
Further analysis revealed that both the kinase activity and tyrosine
phosphorylation of CSBP1 and CSBP2 (A34V) is regulated by salt. In
contrast, wild-type CSBP2 is constitutively active but dependent on the
upstream kinase, Pbs2. Mutagenesis studies showed that reduction or
elimination of CSBP2 kinase activity restores salt responsiveness as
measured by tyrosine phosphorylation suggesting that too high a level
of kinase activity can result in desensitization of the host cell and
inability to grow in high salt.
We recently reported the cloning of a pair of closely related
novel MAP ()kinase homologues, CSBP1 and CSBP2(1) .
These kinases were identified as a target of a series of pyridinyl
imidazoles, which inhibited cytokine production from human
monocytes(1) . The two proteins are splice variants and differ
only in an internal 25-amino acid sequence. The murine (p38) and Xenopus (Mpk2) homologues of CSBP2 also have been identified
and cloned(2, 3) .
At least three distinct MAP kinase pathways exist in mammalian cells, as exemplified by the extracellular signal regulated kinases (ERKs), the c-Jun amino terminus kinases (JNKs), and the CSBPs/p38/Mpk2(4) . Each of these kinases define a distinct signal transduction pathway and are characterized by the presence of a regulatory TXY (Thr-Xaa-Tyr, where X is any amino acid) motif. Phosphorylation of both the threonine and the tyrosine by a dual specificity kinase(s) is essential for activation of MAP kinase activity. They can be grouped according to X in the TXY motif: glutamic acid in ERKs, proline in JNKs, and glycine in CSBP/p38/Mpk2s(4) . The JNK and CSBP protein kinases are activated in response to inflammatory agents and environmental stress, whereas ERKs are stimulated primarily by growth factors and tumor promoters(4, 5) . Further functional separation of these kinases is illustrated by their distinct activators; SEK or MKKs for JNKs/CSBPs and MEKs for ERKs(6, 7, 8, 9) . While there is some overlap in the activating enzymes and in vitro substrates for JNKs and CSBPs, there are also some distinctions(8, 9) . Since there has been no comparative study, it is not known if there are any differences between CSBP1 and -2.
The CSBPs are human homologues of Saccharomyces
cerevisiae Hog1(10) , a MAP kinase required for growth
under high osmolarity conditions(10) . CSBP/p38 can also be
activated by high osmolarity and other environmental stress (11) suggesting that stress response pathway may be conserved
across species, and that CSBP might be an active kinase in yeast. In
support of this conservation, it has been reported that murine p38 can
partially complement a hog1 deletion in yeast(3) .
Neither CSBP1 nor CSBP2 has been tested in this system, nor have they
been individually compared. Furthermore, most studies of p38 in
mammalian cells have used polyclonal antipeptide antibodies which would
be expected to immunoprecipitate both CSBP1 and
CSBP2(11, 12) . As a means to provide rapid
structure-function information on CSBP1 and CSBP2, we used
site-directed mutagenesis to alter key residues and analyzed the
expressed mutant proteins for functional complementation in yeast, as
well as for tyrosine phosphorylation and kinase activity. We have found
that CSBP1 and CSBP2 differ in their ability to complement hog1 and are differentially activated by salt.
The amino terminus of CSBP2 was re-engineered to contain the IBI FLAG (Eastman Kodak) sequence to aid in immunoprecipitation. A BamHI-PstI linker (5`-GATCCTACCATGGATTATAAAGATGACGATGATAAATCTCAGGAAAGGCCCACGTTCTACCGGTCCCGGGCTGCA-3` and its complement, synthesized to contain sticky ends) was ligated into the unique BamHI and PstI sites of pBS (Stratagene). The resulting plasmid was digested with AgeI and PstI and ligated to the 1.7-kb BsrFI-PstI fragment of the CSBP1(1) cDNA. The NarI-KpnI region of this plasmid was replaced with the 1.1-kb NarI-KpnI (3`-polylinker site of pBS) of the CSBP2 cDNA, creating pBS/FLAG-CSBP2. The ORF-encoding FLAG-CSBP2 was then isolated as a 1.6-kb XhoI fragment and subcloned into the same site of p138NBU, creating p138NBU/FLAG-CSBP2. The FLAG-CSBP2 expression is driven by the copper-inducible CUP1 promoter. A similar construct was created for CSBP1 by replacing a 407-bp PvuII-BstXI fragment of p138NBU/FLAG-CSBP2 with a fragment with the same sites from the original CSBP1 clone; this resulted in switching the alternatively spliced region. For mutagenesis, the 1.6-kb BamHI-KpnI fragment of this plasmid was subcloned into the same sites of pAlter1, and site-directed mutagenesis was performed using the Altered Sites system (Promega, Madison, WI). The DNA sequence was altered to encode the amino acid changes indicated (for the mutants) and/or to add a 5` XhoI site in the polylinker.
Plasmids were introduced into S.
cerevisiae strains YPH499 (MAT a, ura3-52, lys2-801, ade2-101
, trp1-
63, his3
200, leu2
1),
YPH102 (MAT a, ura3-52, leu2
1, his3
200, lys2-801
, ade2-101
)(14) , JBY10 (YPH 499 + hog1::TRP1), or MAY1 (YPH102 +pbs2::LEU2) (10) using the lithium
acetate method(15) . Ura
prototrophs were
grown in synthetic complete minus uracil, SC-Ura, liquid media (16) at 30 °C and 225 rpm to A
,
induced for expression with 150 µM CuSO
, and
induced for the HOG pathway with 0.9 M KCl by adding an equal
volume of SC-Ura plus 1.8 M KCl and incubating for an
additional 10 min at 30 °C. Lysates were prepared by harvesting the
cells and vortexing at 4 °C in the presence of glass beads at
2
10
cells/ml in 150 mM NaCl, 20
mM Tris
HCl (pH 7.4), 1 mM MgCl
, 1
mM phenylmethylsulfonyl fluoride, 20 mM NaF, and 2
mM Na
VO
. Extracts were centrifuged at
1,500
g for 5 min at 4 °C, and supernatants were
recentrifuged at 12,000
g for 30 min at 4 °C.
Figure 1:
CSBP2 is toxic to yeast cells when
expressed constitutively. Cells of wild-type and pbs2 yeast strains were transformed with either yeast expression vector
alone or vector containing a full-length human CSBP2 cDNA, and cells
were plated on selective media. Growth after 5 days is
shown.
Figure 2:
Complementation of Saccharomyces
cerevisiae hog1 by CSBP1, CSBP2, CSBP2(A34V), and Hog1 under
normal (-KCl) or high salt (+KCl) conditions. All cDNAs were
expressed under the control of the copper-inducible CUP1 promoter.
Spots assay (increasing 1:10 dilution from left to right) were performed as described under ``Materials and
Methods.'' Growth after 5 days is
shown.
Figure 3:
Kinase activity and immunoblotting of
CSBPs and various CSBP2 mutants expressed in PBS2 and pbs2
yeast
cells. Kinase assays were performed using myelin basic protein (MBP) as the substrate. Western blotting was carried out using
a monoclonal anti-phosphotyrosine (Anti-PY) and a polyclonal
antibody generated against recombinant CSBP2 (Anti-CSBP). The first lane in each panel represents results from yeast
containing control vector.
Figure 4: A, kinase activity and immunoblotting of CSBPs and various mutants. The amount of total protein in each yeast lysate was adjusted to achieve comparable levels of expression for each protein. Kinase assay and Western blot were performed as in Fig. 3. The first two lanes represent results from yeast with control vector. B, the relative kinase activity of CSBPs and various mutants thereof (-salt, solid bars; +salt, empty bars) from A was quantitated in a PhosphorImager and is represented graphically. The kinase activity on the y axis is shown as arbitrary units, and the basal kinase activity of CSBP1 (-salt) is considered as 1.
Members of the MAP kinase family are characterized by a
conserved Thr-Xaa-Tyr motif, in which phosphorylation on Thr and Tyr in
response to extracellular stimuli leads to activation of their protein
kinase activity(4) . The two stress-activated MAP kinase
families represented by the JNKs and CSBPs have differing activation
motifs, TPY and TGY respectively, reflecting some differences in their
activation enzymes (4) . Thus, MKK3 is only able to activate
the CSBP family, whereas MKK4 can apparently activate both the CSBPs
and JNKs(8, 9) . It has been shown recently that both
JNK1 and p38, the murine homologue of CSBP, can complement to some
extent the hog1 deficiency in yeast, suggesting that the
stress response pathway is functionally conserved across
species(3, 18) . These results suggest that yeast
might be a host in which the activated form of CSBP could be expressed
in sufficient quantities for further studies.
We, therefore,
expressed both CSBP1 and CSBP2 in a hog1 strain of yeast
and were surprised to find that CSBP1, but not CSBP2, could complement
the hog1
phenotype. As expected, increased salt led to
increased tyrosine phosphorylation and kinase activity of CSBP1, both
of which were dependent on the presence of the activating kinase Pbs2.
In contrast, CSBP2 expressed similarly was constitutively active and
tyrosine-phosphorylated both in the presence and absence of
hyperosmolarity despite its failure to complement. Both activity and
tyrosine phosphorylation, however, were dependent on Pbs2, suggesting
that constitutive expression of active CSBP2 may be desensitizing the
host toward a further response to high salt. In support of this,
mutants of CSBP2 with reduced (A34V) or absent (K53R,D168A) kinase
activity had restored salt responsiveness with respect to tyrosine
phosphorylation. Furthermore, reduction of the intrinsic kinase
activity of CSBP2(A34V) led to a mutant which was now able to
complement the hog1
phenotype. However, catalytically
inactive CSBP2 mutants (T180E, T180A, T180E/Y182E, Y182F, K53R, and
D168A) failed to complement the hog1
phenotype. The
ability to complement in yeast, therefore, is in part a function of the
absolute kinase activity of the expressed CSBP prior to salt treatment.
Differences in basal kinase activity may also explain the different
complementation potential seen with CSBP1 and CSBP2. We have shown that
this is not due to differences in expression levels (Fig. 4);
rather, it is more likely to be due to differences in the extent of
activation by Pbs2, sensitivity to phosphatases, or differences in
substrate specificity. These differences may also explain the ability
of the murine CSBP2 homologue, p38, which differs in only two amino
acids from the human protein, to complement hog1,
although we cannot rule out differences in expression levels in this
case(3) . However, we did show that expression of human CSBP2
using the same constitutive promoter was toxic to yeast. Differences in
kinase activity may also be the basis for the ability of JNK1, but not
JNK2, to complement hog1
. JNK1 is known to have a 10-fold
lower activity toward one of its known substrates, c-Jun(18) .
These results emphasize that while some elements of the stress-activated kinase cascade have been conserved between yeast and mammals, others may not. The results suggest that for the stress-activated pathway to function with heterologous kinases in yeast, the basal kinase activity must be tightly regulated to keep it below a certain threshold. Above that threshold, it leads to a desensitization of the host toward extracellular stimulation, presumably due to interference with some upstream component(s) of the signaling pathway leading to CSBP activation. Since CSBP2 and CSBP2(A34V) are not likely to differ in recognizing the substrate(s), and yet only CSBP2(A34V) complements, it is likely that the high basal kinase activity of CSBP2 affects more than the pathway leading directly to CSBP2, and perhaps includes other pathway(s) that are required for complementation. Whether this desensitization is a general feature of stress-activated kinases or an artifact of heterologous expression is not clear. Hog1 expressed in the same vector system was able to complement as well as the endogenous Hog1. Furthermore, differences in substrate specificity may also be important as evidenced by the failure of Hog1 to phosphorylate the CSBP substrate MBP.
The expression of
active CSBP2 in yeast also allowed us to dissect the role of the
TXY regulatory loop in CSBP2. As expected from other MAP
kinases and previous reports with
p38(4, 11, 12) , mutations in either
Thr or Tyr
resulted in loss of kinase
activity. Unlike ERK2, however, mutation of Thr
to Glu,
which can sometimes mimic a phosphorylated threonine(17) , did
not lead to a partially active kinase, even when it was phosphorylated
on Tyr
in response to salt. This suggests that
phosphorylation on both Thr
and Tyr
is
absolutely required for activity. Furthermore, it suggests that
tyrosine phosphorylation can occur in the absence of threonine
phosphorylation.
The differing ability of CSBP1 and CSBP2 to
complement the hog1 phenotype suggests that these two
kinases may have different properties and roles in mammalian cells as
well. Use of yeast-expressed CSBP may enable us to further understand
differences between the two in parallel with further work to dissect
the role of these kinases in mammalian cells.