(Received for publication, January 6, 1996; and in revised form, February 14, 1996)
From the
CSBP p38 is a mitogen-activated protein kinase that is activated
in response to stress, endotoxin, interleukin 1, and tumor necrosis
factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as
the bait in a yeast two-hybrid screen, we have identified and cloned a
novel kinase which shares 70% amino acid identity to
mitogen-activated protein kinase-activated protein kinase (MAPKAP
kinase)-2, and thus was designated MAPKAP kinase-3. The binding of CSBP
to MAPKAP kinase-3 was confirmed in vitro by the precipitation
of epitope-tagged CSBP1, CSBP2, and CSBP2(D168A) and endogenous CSBP
from mammalian cells by a bacterially expressed GST-MAPKAP kinase-3
fusion protein and in vivo by co-precipitation of the
epitope-tagged proteins co-expressed in HeLa cells. MAPKAP kinase-3 was
phosphorylated by both CSBP1 and CSBP2 and was then able to
phosphorylate HSP27 in vitro. Treatment of HeLa cells with
sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and
activation of MAPKAP kinase-3 could be blocked by preincubation of
cells with SB203580, a specific inhibitor of CSBP kinase activity.
These data suggest that MAPKAP kinase-3 is activated by stress and
cytokines and is a novel substrate of CSBP both in vitro and in vivo.
One response to a variety of cellular stimuli involves a series
of protein kinase steps known as the mitogen-activated protein (MAP) ()kinase cascade(1, 2, 3) .
Several genetically distinct MAP kinase pathways have been defined in
yeast and at least three exist in mammalian
cells(1, 2) . The mammalian MAP kinases include the
extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal
kinases (JNKs), and the CSBP/p38/RK/Mpk2 kinases(2) . These
kinases are activated by distinct upstream dual specificity kinases
(MAP kinase kinases), which phosphorylate both threonine and tyrosine
in a regulatory TXY (Thr-Xaa-Tyr, where X is any
amino acid) loop present in all MAP kinases(4) . Once
activated, these MAP kinases phosphorylate their substrates on serine
and/or threonine residues with attendant effects on their activity. For
example, phosphorylation of c-Jun and ATF2 by JNK (5, 6) stimulates their transcriptional activity.
CSBP (also known as p38, RK, and mpk2) (7, 8, 9) is the mammalian homologue of the yeast Hog1 protein, which is required for growth of yeast in high osmolarity media(10) , and it can partially complement a hog1 deficiency in yeast(8, 11) . CSBP is activated in mammalian cells by environmental or chemical stress such as hyperosmolarity, UV light, heat shock, arsenite, and endotoxin or cytokines such as interleukin-1 and tumor necrosis factor (TNF)(7, 8, 9, 12, 13) . In response to stress, CSBP kinase activity is activated through phosphorylation by at least two MAP kinase kinases, MKK3 and MKK4 (also known as SEK)(14, 15) . Of the in vitro substrates of CSBP, which include MAPKAP kinase-2(9, 12, 16) , myelin basic protein(7, 11) , and ATF2(13) , only MAPKAP kinase-2 is known to be an in vivo substrate, since pretreatment of cells with SB203580, a specific inhibitor of CSBP, blocks the activation of MAPKAP kinase-2. In turn, MAPKAP kinase-2 phosphorylates the small heat shock proteins HSP25/27 in vitro and in vivo(9, 12, 16) . Inhibitors of CSBP also block the production of inflammatory cytokines from lipopolysaccharide-stimulated human monocytes (7) and interleukin-1-stimulated endothelial cells(17) , and more recently CSBP has been implicated in the apoptosis of neurons upon growth factor removal(18) .
Given the many signals that activate CSBP, and its potential involvement in several cellular responses, we were interested in discovering further activators and substrates. In this paper, we used human CSBP as the bait in a yeast two-hybrid screen (19) to identify a novel serine-threonine protein kinase, MAPKAP kinase-3, which binds to and is an in vivo and in vitro substrate of CSBP.
Figure 1: Sequence and alignment of human MAPKAP kinases-2 and -3. The predicted amino acid sequence of MAPKAP kinase-3 and alignment with MAPKAP kinase-2A (27) and MAPKAP kinase-2B (35) . The alignment was performed using MEGALIGN (DNASTAR, Inc.). Roman numerals indicate various kinase subdomains(4) . The proline-rich motif in the N terminus and the putative nuclear localization signal at the C terminus are boxed. Residues phosphorylated by CSBP/p38 in MAPKAP kinase-2, as well as the autophosphorylation sites (33) are shown by an asterisk and a dot, respectively, below the sequence.
Northern blot analysis showed a predominant mRNA of 3.5 kb and minor mRNA of 2 kb in most tissues except brain, with highest abundance in heart and skeletal muscle (data not shown).
Figure 2: GST-MAPKAP kinase-3 binds CSBP from COS and HeLa cells. A, GST or GST-MAPKAP kinase-3 (5 µg) loaded Sepharose beads (20 µl) were mixed with COS cell lysates (100 µg) expressing FLAG-tagged CSBP1, CSBP2, or CSBP2(D168A), respectively. After incubation for 2 h at 4 °C, beads were pelleted and washed extensively with lysis buffer and analyzed by immunoblotting with anti-FLAG antibody. Lane 7 (labeled C) represents a control COS cell lysate expressing CSBP immunoprecipitated with anti-CSBP. The same experiment was also repeated with HeLa lysate (100 µg) with or without salt activation as a source of endogenous CSBP, and blotted with anti-CSBP antibody (B) or with anti-phosphotyrosine antibody (C). The position of molecular mass markers (kDa) is indicated on the left.
The presence of small amounts of a slower migrating form of CSBP in precipitates (e.g.Fig. 2A), which corresponds to the activated, tyrosine-phosphorylated form of the enzyme, suggests that both activated and unactivated forms of CSBP can bind. This was further demonstrated by the experiments illustrated in Fig. 2(B and C), where GST-MAPKAP kinase-3 bound and precipitated both tyrosine-phosphorylated and non-tyrosine-phosphorylated CSBP from HeLa cells treated with or without sorbitol. In additional experiments, we are able to show that purified recombinant CSBP binds to purified GST-MAPKAP kinase-3, indicating that the association is direct and does not depend on any other proteins present in the yeast or mammalian cell lysates (data not shown).
Figure 3:
MAPKAP kinase-3 is a substrate of CSBP,
and HSP27 is a substrate of MAPKAP kinase-3 in vitro. A, E. coli-expressed GST or GST-MAPKAP kinase-3 (100
µg/ml) was used a substrate in an immune complex kinase assay with
either CSBP1, CSBP2, or CSBP2(D168A) from transfected and activated COS
cells or endogenous CSBP from HeLa cells as indicated. HSP27 (120
µg/ml) was included in lanes 8 and 9, and
GST-MAPKAP kinase-3 was omitted from lane 9. The position of
molecular mass markers (kDa) is indicated on the left. B, 50 ng of purified GST-Erk1 expressed in E. coli (lane 1) or 50 ng of purified p44 from sea star (lane 2) was used to phosphorylate
GST-MAPKAP kinase-3.
Since Hsp27 is a known substrate of MAPKAP kinase-2 in vitro and in vivo, we wanted to determine if it was also a substrate of MAPKAP kinase-3. Inclusion of HSP27 in the CSBP/GST-MAPKAP kinase-3 immune complex kinase reaction resulted in its phosphorylation (Fig. 3, lane 8), whereas CSBP alone or unactivated MAPKAP kinase-3 did not phosphorylate Hsp27 (Fig. 3, lane 9; data not shown), indicating that Hsp27 is a substrate of MAPKAP kinase-3.
Figure 4: Sorbitol- and TNF-mediated cell stimulation results in CSBP and MAPKAP kinase-3 activation. A, HeLa cells were transfected with HA-tagged MAPKAP kinase-3 and pretreated with 10 µM SB203580 and/or treated with 50 ng/ml PMA, 0.4 M sorbitol or 20 ng/ml TNF for 10 min as indicated. After activation, the cells were lysed and HA-MAPKAP kinase-3 was immunoprecipitated with anti-HA antibody and an immune complex kinase assay was performed with HSP27 as a substrate (indicated by an arrow). B, HeLa cells were either co-transfected with FLAG-CSBP2 and HA-MAPKAP kinase-3 (lanes 1 and 2) or with vector alone (lanes 3 and 4), activated with 0.4 M sorbitol for 10 min, and immunoprecipitated with anti-HA antibody. The immunoprecipitate was analyzed by immunoblot using anti-FLAG antibody to detect co-precipitated FLAG-CSBP (indicated by an arrow). Arrowheads indicate the heavy and light chain of anti-HA antibody used for immunoprecipitation.
To confirm the association of CSBP and MAPKAP kinase-3 in vivo, we co-expressed FLAG-tagged CSBP2 and HA-tagged MAPKAP kinase-3 in HeLa cells. Immunoprecipitation of MAPKAP kinase-3 with anti-HA antibody resulted in co-precipitation of CSBP in cells transfected with both cDNAs but not from cells transfected with vector alone, as determined by immunoblotting with anti-FLAG antibodies (Fig. 4B, lanes 1-4). Like the data in Fig. 2B, the amount of CSBP co-precipitated from sorbitol-activated cells was lower than that from unactivated cells, suggesting weaker association of the CSBP-MAPKAP kinase-3 complex. These data indicate that MAPKAP kinase-3 is functionally similar to MAPKAP kinase-2, in that they are both in vivo substrates of CSBP/p38, and can phosphorylate Hsp27 in vitro. It is not clear whether both kinases contribute to phosphorylation of hsp27 in vivo.
The finding of association between CSBP and its
substrate MAPKAP kinase-3 is reminiscent of that between ERK2 and
p90 (also known as MAPKAP kinase-1)(28) , in
which the two kinases were found to be in a stable 110-kDa complex
within unactivated Xenopus oocytes. Upon activation, ERK
dissociates from the complex and is predominantly monomeric. Similarly,
in the present case a stable complex between CSBP and MAPKAP kinase-3
exists in vivo, and the preliminary data in Fig. 2B and Fig. 4B suggest some dissociation of the
activated enzymes, although further experiments will be needed to
establish this.
There are several reports of MAP kinases binding to other proteins. ERK has been reported to interact with the transcription factor Elk1 (29, 30) and the high and low affinity nerve growth factor receptors (31) by co-immunoprecipitation from mammalian cells, and the protein kinase MEK1 in a yeast two-hybrid screen(32) . JNK binds to the transcription factors c-Jun and ATF2 in vitro(5) . Formation of these complexes may serve to restrict cross-talk between different MAP kinase pathways. For example, both ERK and CSBP can activate MAPKAP kinase-2 and -3 in vitro, but only CSBP appears to activate these kinases in vivo(9, 27, 33) . JNK, another stress- and cytokine-activated MAP kinase, did not phosphorylate MAPKAP kinase-3 in vitro (data not shown). Thus, MAPKAP kinase-3 appears to be a specific substrate of CSBP.
Recently, the sites of
MAPKAP kinase-2 phosphorylation by CSBP/RK in vitro and in
vivo were determined (33) and are illustrated in Fig. 1. The three key phosphorylation sites, Thr-222, Ser-272
and Thr-334, any two of which must be phosphorylated for maximal
activation of MAPKAP kinase-2 activity, are conserved in MAPKAP
kinase-3. An additional in vivo autophosphorylation site is
also conserved (Thr-338), while a second site is changed from Ser to
Thr (Ser-9), and a third is not conserved (Thr-25). Another component
of MAPKAP kinase-2 activation conserved in MAPKAP kinase-3 is an
autoinhibitory -helix near the C terminus, which mimics the
substrate and when deleted produces a constitutively active MAPKAP
kinase-2(34) . It is likely that these conserved features play
a role in the activation of MAPKAP kinase-3.
Given the apparent similarity in activation and substrate activity of MAPKAP kinase-2 and MAPKAP kinase-3, it will be of interest to determine if these two closely related kinases play different roles in stress and cytokine stimulated cells. The availability of purified CSBP and MAPKAP kinase-3 and their cDNAs will allow further characterization of their binding interface.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U43784[GenBank].