(Received for publication, January 23, 1997)
From the Several mitogen-activated protein kinase (MAPK)
cascades have been identified in eukaryotic cells. The activation of
MAPKs is carried out by distinct MAPK kinases (MEKs or MKKs), and
individual MAPKs have different substrate preferences. Here we have
examined how amino acid sequences encompassing the dual phosphorylation motif located in the loop 12 linker (L12) between kinase subdomains VII
and VIII and the length and amino acid sequence of L12 influence autophosphorylation, substrate specificity, and upstream kinase selectivity for the MAPK p38. Conversion of L12 of p38 to an
"ERK-like" structure was accomplished in several ways: (i) by
replacing glycine with glutamate in the dual phosphorylation site, (ii)
by placing a six-amino acid sequence present in L12 of ERK (but absent
in p38) into p38, and (iii) by mutations of amino acid residues in loop
12. Two predominant effects were noted: (i) the Xaa residue in the dual
phosphorylation motif Thr-Xaa-Tyr as well as the length of L12
influence p38 substrate specificity, and (ii) the length of L12 plays a
major role in controlling autophosphorylation. In contrast, these
modifications do not result in any change in the selection of p38 by
individual MAPK kinases.
Protein kinase cascades control a variety of functions in
eukaryotic cells (1). Key components of these protein kinase cascades
are members of the superfamily of mitogen-activated protein kinases
(MAPKs).1 These serine/threonine kinases
act by modulating the activity of cellular proteins such as
transcription factors, enzymes including other kinases, cell-surface
receptors, and structural proteins (2). In yeast, distinct signaling
pathways leading to MAPK activation have been defined using
biochemical and genetic approaches (3-5).
Several individual MAPK signal transduction pathways have been
characterized in mammalian cells. One pathway leads to activation of
the extracellular signal-regulated protein kinase (ERK) group of MAPKs,
and these enzymes have been shown to play an important role in
regulating cellular responses to growth factors (2, 6). A second MAPK
pathway is regulated by changes in the extracellular milieu induced by
physical-chemical changes or by proinflammatory cytokines leading to
phosphorylation of transcription factors such as c-Jun (7, 8). A third
MAPK pathway leads to activation of the p38 pathway by stimuli such as
UV light, increased extracellular osmolarity, proinflammatory
cytokines, or products of microbial pathogens (9-12). An additional
MAPK family member has been recently described termed ERK5 (13) or BMK1
(14). MAPK family members have marked sequence homologies, with an
overall sequence identity of >40%. Nonetheless, there are
specificities found at the level of the upstream activators, the MAPK
kinases (15-20) and the substrates phosphorylated by the active MAPKs
(7, 8, 10, 21, 22).
From the x-ray crystallographic structures of ERK2 (23) and p38 (24,
25) as well as from primary sequence comparisons of the members of the
MAPK family (9, 14), two structural domains of MAPK have been suggested
to be important in regulating function insofar as recognition by
upstream activators and substrate specificity are concerned.
Specifically, these are the Thr-Xaa-Tyr dual phosphorylation motif and
the loop 12 linker (L12) as defined by data from the three-dimensional
structure of ERK2 (23). Herein we have examined the influence of the
Xaa residue in the Thy-Xaa-Tyr dual phosphorylation motif and the role
of sequences that are part of L12 in several aspects of p38 function.
We provide evidence that the Xaa residue in the Thy-Xaa-Tyr motif and
the length of the loop are important for the substrate specificity of
p38. The length of L12 also plays an important role in controlling the extent of autophosphorylation. In contrast, modification of the L12
structure alone does not change the selectivity of the upstream MAPK
kinases.
A construct designed to express
murine p38 with His6 at its amino terminus was prepared by
ligating the p38 coding region, amplified from p38 cDNA in
BlueScript using the polymerase chain reaction (primers
gcagccatATGTCGCAGGAGGCC and ccggatccTCAGGACTCCATTTCTTC), with pET14b
vector (Novagen, Madison, WI), incorporating NdeI and
BamHI sites. A construct designed to express p38 with a flag tag at the amino terminus in a mammalian expression vector (pcDNA3) was prepared as described (10, 26). The p38 mutants shown in Fig. 1
were generated using a polymerase chain reaction-based approach as
described (27).2 The cDNA encoding each
of these mutants was ligated into pET14b vector using NdeI
and BamHI sites and pcDNA3 vector with
HindIII and XbaI sites as described above for
wild-type p38. Each construct was sequenced to ensure the fidelity of
the mutations.
The
pET14b vectors containing DNA encoding p38 or the p38 mutants were
transformed into the BL21(DE3) strain of Escherichia coli. E. coli cells were grown at 37 °C in Luria broth until
A600 = 1.0, at which time
isopropyl- HA (hemagglutinin)-tagged MKK3b or MKK6b
in pcDNA3 vector and HA-tagged MEK1 cDNA in pSR COS-7 (4 × 105) or CHO-K1 (6 × 105) cells (in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum) on 35-mm plates were
transfected with 1 µg (total) of one or more plasmid DNAs using
Lipofectamine (Life Technologies, Inc.). After 48 h, the cells
were treated with or without stimuli as described below. The tagged
proteins were immunoprecipitated with the appropriate monoclonal
antibody and quantitated by Western blot analysis as described
below.
HA-tagged MKK3b or
MKK6b in pcDNA3 vector and HA-tagged MEK1 cDNA in pSR GST fusion proteins of the
amino-terminal portion of activating transcription factor 2 (ATF2;
amino acids 1-109) (16), GST-ELK1 (30), and GST-c-Myc (31) were
prepared as described (32). PHAS-1 was obtained from Stratagene (San
Diego, CA). Recombinant His-ERK2 was prepared as described (23).
Western blotting was performed as
described (33). Anti-phosphotyrosine antibodies 4G10 (Upstate
Biotechnology, Inc., Lake Placid, NY) and FB2 (obtained from American
Type Culture Collection) was used to detect proteins with
phosphotyrosine; antibodies M2 and 12CA5 were used in Western blotting
for analysis of tagged proteins expressed in COS-7 and CHO-K1 cells.
Goat anti-mouse IgG conjugated to horseradish peroxidase (Cappel,
Durham, NC) was used as secondary antibody. Enhanced chemiluminescence
detection reagents (ECL, Amersham Corp.) were used according to the
manufacturer's instruction. Quantitation was done by densitometer
analysis.
Kinase assays using 0.2-0.4 µg of purified
p38, p38 mutants, or immunoprecipitates containing activated kinases
were performed at 37 °C for 20 min using 5 µg of substrate, 250 µM ATP, and 10 µCi of [ Sequence
comparisons of the known MAPKs revealed that each group has a distinct
phosphorylation lip (L12) structure (9, 14). To investigate how the L12
structure is involved in the function of p38, we have created a series
of p38 mutants with modifications in this region (shown schematically
in Fig. 1). Specifically, we introduced mutations to
make p38 more "ERK-like" in the L12 region. Thus, p38(E) contains
an ERK-like Thr-Glu-Tyr dual phosphorylation site; p38(6+) has an
additional six amino acids from ERK inserted after Glu178;
p38(6+E) has both the additional six-amino acid sequence of p38(6+) and
the Thr-Glu-Tyr dual phosphorylation site; p38(VAP) has
174HTDD177 substituted with the ERK2 sequence
VADP; p38(DL) has 178EM179 replaced with DL;
and p38(VAPD6+LE) has a loop 12 structure identical to that found in
ERK2.
Previous
studies have demonstrated that there are distinct MAPK kinases (MEKs or
MKKs) responsible for activating ERK, p38, or JNK (or stress-activated
protein kinase) (15). It has been suggested by others that the nature
of the second amino acid in the Thr-Xaa-Tyr dual phosphorylation motif
may play a role in determining the specificity of interactions between
MKK and its MAPK substrate (34, 35). To test this hypothesis, we
examined the activation of wild-type p38 and the panel of p38 mutants; MKK3b (36),3 MKK6b (16),3 or
MEK1 (37) was used for this experiment. Using an in vitro coupled kinase assay, we observed that the enzymatic activity of p38 or
p38 mutants was increased when MKK3b or MKK6b was included in the
kinase reaction, but not when we used MEK1 (Fig. 2). The extent of activation was nearly the same when each of the mutants was
compared with wild-type p38. As expected, MEK1 dramatically increased
the activity of ERK2 (Fig. 2, lower panel). In a second approach, we cotransfected MKKs and individual epitope-tagged p38 forms
in COS-7 cells to further examine the effects of modifying L12 on the
ability of MKKs to activate p38 (Fig. 3A).
Consistent with the in vitro experiments, the activity of
p38 or p38 mutants was enhanced similarly when cotransfected with MKK3b
or MKK6b, but not with MEK1. ERK2 activity was only enhanced when
cotransfected with MEK1. Equal amounts of wild-type or modified p38
protein were detected in the immunoprecipitates used for the kinase
assay (Fig. 3B).
We next asked whether this same pattern is observed in a setting where
activation is initiated by an extracellular stimuli. To investigate
this, CHO-K1 cells were transiently transfected with flag-tagged p38,
p38 mutants, or ERK2, and cells were treated with 0.4 M
sorbitol, UV light, epidermal growth factor + insulin, or 20% fetal
calf serum. The transiently expressed epitope-tagged MAPKs were
immunoprecipitated with the M2 antibody, and the resultant immunoprecipitates were analyzed for kinase activity. High osmolarity or UV light enhanced the kinase activity of p38 and all p38 mutants (Fig. 4A). Addition of an ERK-like loop
structure did not result in conversion of p38 to respond to growth
factor signals that activate ERK. Thus, the totality of these data
suggest that neither the Thr-Xaa-Tyr motif nor the L12 structure is a
critical structural feature directing the selectivity of the upstream
kinase.
Histidine-tagged
recombinant p38 expressed in E. coli appears to undergo an
autophosphorylation reaction since it is phosphorylated on tyrosine
(Fig. 5). Although at present this event cannot be linked to any physiologically relevant changes, the parameter of
autophosphorylation provides us with an additional way to evaluate structure-function relationships of p38. We used anti-phosphotyrosine antibody in Western blots to analyze the extent of tyrosine
phosphorylation of recombinant wild-type p38 or p38 mutants with the
modification in the L12 region. We observed that the proteins with
added amino acids in L12 have substantially more phosphotyrosine when
compared with equal amounts of wild-type p38 (Fig. 5, A and
B). The same results were obtained using two different
anti-phosphotyrosine monoclonal antibodies, 4G10 and FB2. The level of
phosphotyrosine in p38(6+), p38(6+E), or p38(VAPD6+LE) is similar to
that found in recombinant ERK2 expressed and isolated identically to
the means used to obtain p38 forms.
Autophosphorylation of p38 or its modified forms can also be detected
by incubation of recombinant protein with [ We next asked whether changes in L12 influence the
substrate specificity of p38. To investigate this, we performed
in vitro kinase assays using the following proteins as
substrates: ATF2 (38), ELK1 (30), c-Myc (31), PHAS-1 (39), and myelin
basic protein (MBP). Each of these proteins can be phosphorylated by p38 (Fig. 6), with the exception of GST used as a
control for the GST fusion proteins (data not shown). Equal quantities
of kinase (see Fig. 5A) and substrate protein (Fig.
6B) were used in each kinase assay. The level of substrate
phosphorylation was observed to differ substantially when wild-type p38
and its modified forms were compared (Fig. 6A). MBP, Myc,
and ELK1 contain the consensus Pro-Xaa-Ser/Thr-Pro sequence, which has
been defined as the consensus recognition site for ERK1/2 (40-42). The
kinase activity of p38 toward these three proteins was increased by the insertion of six residues or by a Gly to Glu mutation. However, p38(6+E) and p38(VAPD6+LE) do not show this enhanced activity, but have
reduced substrate phosphorylation. ATF2 and PHAS-1, which have
different primary structure at the phosphorylation site when compared
with MBP, Myc, or ELK1, behaved differently insofar as p38-catalyzed
phosphorylation is concerned. p38(E) and p38(6+) each demonstrated
markedly reduced phosphorylation of ATF2 when compared with that
produced by wild-type p38. The p38(6+E) and p38(VAPD6+LE) mutants also
showed an even greater reduction in ATF2 phosphorylation, suggesting
that the two modifications may synergize in terms of their effect on
substrate recognition and/or catalysis; p38(6+) and p38(6+E) had
enhanced activity toward PHAS-1. The mutation of HTDD (p38(VAP)) or EM
(p38(DL)) does not lead to any change in the substrate specificity. The
differential phosphorylation of these substrates by p38 and some p38
mutants cannot be due to a general change in enzymatic activity because
differing effects on phosphorylation by p38 and its mutants were
substrate-dependent.
We next
investigated whether the increased autophosphorylation observed with
the p38 mutants p38(6+), p38(6+E), and p38(VAPD6+LE) is reflected in a
similar change in enzymatic activity toward specific protein
substrates. We found that a higher level autophosphorylation of
p38(6+), p38(6+E), and p38(VAPD6+LE) did not correlate with phosphorylation of MBP and PHAS-1 (Fig. 7). Similar
results were obtained with the other substrates shown in Fig. 6 (data
not shown). These data show that there are different mechanisms in
controlling the autophosphorylation and substrate
recognition/phosphorylation.
Although all MAPKs are regulated by dual phosphorylation on
adjacent Thr and Tyr residues, each has its distinct set of upstream activators. Whereas the members of this enzyme family have >40% overall identity, each displays very distinct substrate specificities. A structural basis for these upstream and downstream
specificities has not been currently determined. Here we have utilized
p38 and ERK2 to investigate structural features that may control
recognition by upstream activators or may determine substrate
specificity. Using recombinant DNA techniques, we have modified p38 in
the phosphorylation lip L12, the locus of major differences between p38
and ERK2, to an ERK-like structure. This permitted us to evaluate the
influence of such modifications on the activation and function of
p38 as well as on its ability to undergo autophosphorylation.
Since this structural domain contains the dual phosphorylation sites,
the target of the upstream MAPK kinase, this region of MAPK must
interact with the activating kinase. Differences in the L12 structure
of the known MAPKs suggested that the Xaa residue in the dual
phosphorylation motif may play a role in determining the specificity of
interactions with MKK (34, 35). Our data indicate that neither the Xaa
residue in the Thr-Xaa-Tyr motif in p38 nor the other residues present
in L12 are critical in controlling p38 activation regardless of whether
this is tested in fully in vitro experiments or in cells.
After submission of this paper, two other studies provided data
supporting this contention. Brunet and Pouyssegur (31) expressed a
p38-ERK chimeric protein that demonstrated that the amino terminus of
p38 plays a predominant role in determining interactions with the
upstream activator and that L12 has no role in this event. Robinson
et al. (43) showed that Xaa in the dual phosphorylation
motif of ERK does not have any role in the upstream kinase selectivity
and that the length of the lip of ERK2 has only a small effect on the
ability of MEK1 to phosphorylate ERK2. Therefore, although amino acid
residues in the phosphorylation lip interact with MKK, the MKK
specificity is determined by a p38-MKK interaction involving other
domain(s). These may include portions of the amino terminus of p38
(31).
Information from the three-dimensional structure of p38 suggests that a
surface groove of p38 is likely to be in contact with protein
substrates (24, 25). This groove is composed of several helices and the
carboxyl-terminal domain. Based on a cAMP-activated protein
kinase-peptide substrate model, the sequence amino-terminal to the
phosphorylation site of the substrate binds in the surface groove of
the kinase (44). Although L12 is not a part of this surface groove,
residues 170-178 in L12 were found to occupy the peptide-binding
groove in unphosphorylated p38 (24). Our data have demonstrated that
the changes in the L12 structure result in a change in substrate
specificity. Each MAPK is specific for proline in the P+1 site. The P+1
pocket (Val183-Arg189) is conserved in all
MAPKs and is adjacent to L12. It is possible that the phosphorylation
lip may be a part of the substrate-binding pocket in the phosphorylated
form of p38 or may contribute to the overall topology of the
substrate-binding groove. Analysis of the primary sequence around the
phosphorylation site of the substrates we used in the experiments
reveals that the changes in the substrate specificity of the p38
mutants are dependent on the primary amino acid sequence around the
phosphorylation site. The substrates that contain the
Pro-Xaa-Ser/Thr-Pro sequence (ELK1, c-Myc, and MBP) are phosphorylated
better by the p38(6+) and p38(E) mutants, while ATF2 and PHAS-1, with a
different sequence adjacent to the phosphorylation site, are recognized
differently by the p38 mutants. These data suggest that the L12
structure can influence the recognition of primary amino acid sequences by p38. Introducing six amino acids from the ERK sequence or
incorporating a Thr-Glu-Tyr dual phosphorylation site into p38 produced
an enhanced activity toward the consensus ERK phosphorylation site
Pro-Xaa-Ser/Thr-Pro. But completely converting L12 of p38 to that
contained in ERK2 does not change the substrate selection of p38. These
data suggest that interaction of the lip with other domains may occur
and that substrate recognition determinants may act in concert to
control substrate specificities. Nevertheless, our data provide
evidence for the first time supporting the contention that the
structure of the phosphorylation lip is a determinant of substrate
specificity.
We suggest that a physiological substrate of p38 should be a preferred
substrate for unmodified p38, but not for the L12 mutants. Therefore,
such p38 mutants may be useful tools to test the specificity of
suspected p38 substrates. For example, the stronger phosphorylation of
ATF2 by wild-type p38 than by p38 mutants suggests that there is a
specific interaction between this protein and p38.
In vitro autophosphorylation has been studied in the ERK
group of MAPKs, where it was shown to be a unimolecular reaction occurring primarily on tyrosine (45-52). From the results of
crystallographic studies, it is clear that the Thr residue in the
Thr-Glu-Tyr phosphorylation site of ERK is on the surface, while the
Tyr residue is buried in a large hydrophobic pocket. The Tyr residue is
located near a putative catalytic base, Asp147
(Asp150 in p38), and The insufficient activation of p38 by autophosphorylation suggests that
the phosphorylated amino acids may not reflect phosphorylations at all
critical regulatory sites; this is the case for ERK (46). Although this
study does not provide a physiological role for autophosphorylation, we
can speculate that the autophosphorylation may contribute to the
activation of MAPK in cells. Although it is well known that dual
specificity kinases are responsible for the activation of MAPKs (34,
53), it is still possible that a serine/threonine kinase can partially
activate MAPK when there is a prior autophosphorylation on tyrosine.
In summary, our data demonstrate that although the sequences in loop 12 account for major differences between p38 and ERK2, this
phosphorylation lip does not appear to direct MKK specificity. A change
in the length of L12 affects the structure of p38 in a way that alters
the level of autophosphorylation. In addition, modifications of the lip
sequence lead to specific changes in substrate selectivity. These data
suggest that the phosphorylation lip may be involved in p38-substrate
interaction. This finding would not have been predicted from what is
known about the cyclic AMP-dependent protein
kinase-substrate interaction (44). How the phosphorylation lip is
involved in substrate recognition awaits further investigation.
We thank Drs. E. Goldsmith and Z. Wang
(University of Texas Southwestern Medical Center) for recombinant rat
ERK2, Dr. M. Karin (University of California, San Diego) and Dr. R. Davis (Howard Hughes Medical Institute) for providing MKK3 and MKK4
(JNKK1) cDNAs, and Dr. J. Wilson (Scripps Research Institute) for
the 12CA5 antibody. We also thank Betty Chastain for excellent
secretarial assistance.
Department of Immunology,
Department of Biological Chemistry,
University of Michigan Medical School,
Ann Arbor, Michigan 48109
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Mutagenesis of Murine p38
Fig. 1.
Loop 12 sequence of wild-type p38 (9), p38
mutants, and ERK2 (54). The amino acids are presented in single
letter code. The Roman numerals indicate the positions of
protein kinase domains, and the boldface italic letters
designate modified/added amino acid residues.
[View Larger Version of this Image (29K GIF file)]
-D-thiogalactopyranoside (1 mM final concentration) was added for an additional 2 h. The cells were then collected by centrifugation at 8000 × g for
10 min. For each 100 ml of original bacterial culture, the bacterial
pellet was resuspended in 5 ml of binding buffer (5 mM
imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH
7.9). The bacteria were lysed by sonication using a cell disrupter
(W-375, Ultrasonics, Inc.). Insoluble materials were removed by
centrifugation at 10,000 × g for 30 min. The
supernatant was applied to a Ni2+-nitrilotriacetic
acid-agarose column (0.5 ml; Novagen); the column was washed with 10 ml
of washing buffer (60 mM imidazole, 500 mM
NaCl, and 20 mM Tris-HCl, pH 7.9); and the bound protein
was eluted with elution buffer (1 M imidazole, 500 mM NaCl, and 20 mM Tis-HCl, pH 7.9). The
expressed p38 and altered proteins were soluble and were recovered with
a yield of ~5 µg/1 ml of bacteria culture. When analyzed by
SDS-PAGE, the proteins appeared to be >90% pure after purification on
an affinity column selective for His-tagged recombinant proteins (data
not shown).
vector were
constructed as described (16, 28, 29).
vector
(1 µg) were transfected into COS-7 cells as described above. 48 h after transfection, the cells were lysed by incubation at 4 °C for
15 min in lysis buffer (20 mM Tris-HCl, 120 mM
NaCl, 10% glycerol, 1 mM Na3VO4, 2 mM EDTA, 1% Triton X-100, and 1 mM
phenylmethylsulfonyl fluoride, pH 7.5), and the HA-tagged protein was
immunoprecipitated with anti-HA monoclonal antibody 12CA5 as described
(15). Quantification of MKKs in the immunoprecipitates was accomplished
by Western blotting using monoclonal antibody 12CA5; this quantitation
provided us with a basis for normalization in subsequent in
vitro kinase assays.
-32P]ATP in 20 µl of kinase buffer (20 mM Hepes, pH 7.6, 20 mM MgCl2, 25 mM
-glycerophosphate, 0.1 mM
Na3VO4, and 2 mM dithiothreitol). The reactions were terminated with Laemmli sample buffer, and the
products were resolved by SDS-PAGE and analyzed by autoradiography. The
extent of protein phosphorylation was quantified by phosphoimaging.
p38 Mutants with Modifications in Loop 12
Fig. 2.
In vitro activation of p38 and p38
mutants by various MKKs. A coupled kinase assay was performed by
incubating MBP and p38, p38 mutants, or ERK2 with no additional kinases
or with MKK3b, MKK6b, or MEK1. Phosphorylation of MBP was determined by autoradiography after SDS-PAGE. Mutations in L12 of p38 do not lead to
any change in the selection of p38 by specific MKKs.
[View Larger Version of this Image (32K GIF file)]
Fig. 3.
In vivo activation of p38 and p38
mutants by various MKKs. COS-7 cells were cotransfected with
flag-tagged p38, p38 mutants, or ERK2 with MKK3b, MKK6b, MEK1, or
control empty vector. The kinase activity of p38, p38 mutants, or ERK2
isolated by immunoprecipitation was measured using MBP as substrate.
Quantitation of MBP phosphorylation was done by PhosphorImager. The
quantity of flag-tagged protein in the immunoprecipitate was analyzed
by Western blotting using the M2 antibody, as shown in
B.
[View Larger Version of this Image (43K GIF file)]
Fig. 4.
Activation of p38 and p38 mutants by various
extracellular stimuli. CHO-K1 cells were transiently transfected
with flag-tagged p38 or its mutants or ERK2. 36 h after
transfection, cells were serum-starved for 8 h and then treated
with sorbitol (0.4 M), epidermal growth factor
(EGF; 10 nM) plus insulin (0.25 units/ml), or
20% fetal calf serum (FCS) for 30 min or with UV light (50 J/m2), followed by a 30-min incubation at 37 °C. The
kinase activity of p38, p38 mutants, or ERK2 isolated by
immunoprecipitation was measured using MBP as substrate. The
flag-tagged protein in the immunoprecipitate was quantitated by Western
blotting using the M2 antibody, as shown in B. Modification
of L12 in p38 to an ERK-like sequence does not result in p38 activation
by signals that activate ERK.
[View Larger Version of this Image (45K GIF file)]
Fig. 5.
Autophosphorylation of p38 and p38
mutants. Purified recombinant proteins (0.4 µg/lane) were
resolved by SDS-PAGE and stained with Coomassie Blue (A) or
immunoblotted with anti-phosphotyrosine monoclonal antibody FB2
(B) or 4G10 (data not shown). In vitro autophosphorylation of p38 and p38 mutants was analyzed using the
kinase assay with [-32P]ATP and detected by
autoradiography after SDS-PAGE (C). The length of L12
influences p38 autophosphorylation.
[View Larger Version of this Image (28K GIF file)]
-32P]ATP
under conditions of an in vitro kinase assay; p38(6+),
p38(6+E), and p38(VAPD6+LE) display enhanced autophosphorylation when
compared with wild-type p38 or the other p38 mutants (Fig.
5C). Similar results were obtained when recombinant proteins
expressed in mammalian cells were used (data not shown). These data
suggest that the length of L12 controls the structure of p38 in ways
that regulate the autophosphorylation reaction, while the nature of the
Xaa residue in the Thr-Xaa-Tyr dual phosphorylation site has little or
no influence on this activity of p38.
Fig. 6.
Substrate specificity of p38 and p38 mutants.
In vitro kinase assays were performed using recombinant
wild-type p38 or p38 mutants as kinase with the following proteins:
MBP, GST-c-Myc, GST-ELK1, GST-ATF2, and PHAS-1. The kinase reaction was
stopped by SDS sample buffer, and the resultant mixture was analyzed by SDS-PAGE. Phosphorylation of the substrates was detected by
autoradiography. Quantitation of the substrate used in the experiments
is shown in B. Modification of loop 12 of p38 has an effect
on the substrate specificity.
[View Larger Version of this Image (55K GIF file)]
Fig. 7.
Autophosphorylation and enzymatic activity of
p38. In vitro kinase reactions were performed by incubating
p38 or p38 mutants with MBP (A) or PHAS-1 (B).
The phosphorylation of the enzyme itself and the substrate was analyzed
by SDS-PAGE and visualized by autoradiography. The levels of
autophosphorylation do not correlate with the levels of substrate
phosphorylation.
[View Larger Version of this Image (52K GIF file)]
-phosphate of bound ATP. This may
account for the ability of ERK to catalyze its efficient
autophosphorylation. The shortened length of L12 in p38 results in the
Thr-Gly-Tyr dual phosphorylation site being present on the surface of
the protein. This may cause a decrease in the flexibility of the loop that prevents the conformation change required to allow the Tyr residue
to be accessed.
*
This work was supported by National Institutes of Health
Grants GM51417 (to J. H.) and GM37696 and AI15136 (to R. J. U.) and American Heart Association Grant-in-aid 95007690 (to J. H.). This is
Publication 9797-IMM from the Department of Immunology, Scripps Research Institute.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
Established Investigator of the American Heart Association. To whom
correspondence should be addressed: Dept. of Immunology, Scripps
Research Inst., 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.:
619-784-8704; Fax: 619-784-8239.
1
The abbreviations used are: MAPKs,
mitogen-activated protein kinases; ERK, extracellular signal-regulated
protein kinase; L12, linker loop 12 between kinase subdomains VII and
VIII; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; MKK
or MEK, MAPK/ERK kinase; GST, glutathione S-transferase;
ATF2, activating transcription factor 2; JNK, c-Jun N-terminal kinase;
MBP, myelin basic protein.
2
The sequences of the primers used for generating
the mutations will be provided upon request.
3
MKK3b is a 347-amino acid spliced form of MKK3;
MKK6b is a 334-amino acid spliced form of MKK6.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.