(Received for publication, July 6, 1995; and in revised form, September 7, 1995)
From the e 10, D-13122 Berlin,
Federal Republic of Germany
A recently described downstream target of mitogen-activated
protein kinases (MAPKs) is the MAPK-activated protein (MAPKAP) kinase 2
which has been shown to be responsible for small heat shock protein
phosphorylation. We have analyzed the mechanism of MAPKAP kinase 2
activation by MAPK phosphorylation using a recombinant MAPKAP kinase
2-fusion protein, p44 and p38/40
in
vitro and using an epitope-tagged MAPKAP kinase 2 in heat-shocked
NIH 3T3 cells. It is demonstrated that, in addition to the known
phosphorylation of the threonine residue carboxyl-terminal to the
catalytic domain, Thr-317, activation of MAPKAP kinase 2 in vitro and in vivo is dependent on phosphorylation of a second
threonine residue, Thr-205, which is located within the catalytic
domain and which is highly conserved in several protein kinases.
Constitutive activation of MAPKAP kinase 2 is obtained by replacement
of both of these threonine residues by glutamic acid. A constitutively
active form of MAPKAP kinase 2 is also obtained by deletion of a
carboxyl-terminal region containing Thr-317 and the A-helix motif or by
replacing the conserved residues of the A-helix. These data suggest a
dual mechanism of MAPKAP kinase 2 activation by phosphorylation of
Thr-205 inside the catalytic domain and by phosphorylation of Thr-317
outside the catalytic domain involving an autoinhibitory A-helix motif.
The network of mitogen-activated protein kinases is based on
subsequent activation of protein kinases by phosphorylation (for a
recent review, see (1) ). A major activator of the vertebrate
MAP ()kinases ERK1 and ERK2 has been identified as the
protein kinase MEK, a dual specific kinase which itself is activated by
protein kinases encoded by the proto-oncogenes raf1 or mos(2, 3) as well as by MEK kinase(4) . In
addition, stress-dependent signaling seems to proceed via parallel MAPK
cascades leading to activation of further subgroups of MEKs and
MAPKs(5, 6) . One of the MAPK subgroups is designated
stress-activated protein kinases (SAPKs) (7) and also termed
amino-terminal c-Jun kinases (JNKs)(8, 9) . Another
distinct subgroup covers the p38
and p40
,
including the reactivating kinase (RK) (10, 11, 12) , which are more similar to the
yeast MAPK homologue HOG1 (13) .
Signaling downstream of the
MAPKs proceeds by phosphorylation of several transcription factors and
of at least two different groups of MAPK-activated protein (MAPKAP)
kinases, the different isoforms of ribosomal S6 kinase II (RSK, MAPKAP
kinase 1) and the MAPKAP kinase 2. The latter enzyme has been shown to
be activated by the MAPK ERK1 and ERK2 (14) in vitro and by the p38/40 (RK) in
vivo(11, 12) . Interestingly, activation of this
kinase seems to be correlated to the phosphorylation of a threonine
residue in a MAP kinase recognition consensus sequence PXTP
located carboxyl-terminal to the catalytic domain of the
enzyme(14) . This would indicate a process of activation of
MAPKAP kinase 2 different from other protein kinases, which are
activated by phosphorylation within the catalytic domain in the
vicinity of the putative substrate binding site (reviewed in (15) and (44) ).
In this article we use a
recombinant glutathione S-transferase (GST)-MAPKAP kinase
2-fusion protein and various mutants to study the mechanism of
activation of MAPKAP kinase 2 by p44 (ERK1) and
p38/40
(RK) in vitro. Furthermore, we analyze
the stress-dependent activation of MAPKAP kinase 2 in vivo by
transfection experiments with an epitope-tagged enzyme and appropriate
mutants in NIH 3T3 cells. We provide evidence that, in addition to the
phosphorylation at Thr-317 outside the catalytic domain, activation of
MAPKAP kinase 2 by ERK1 proceeds through phosphorylation of a second
threonine residue Thr-205 inside the catalytic domain in vitro. Furthermore, the data presented indicate that there is no further
regulatory residue in MAPKAP kinase 2 phosphorylated in vitro by p38/40 or in vivo as a result of heat shock. Different
constitutively active mutants of MAPKAP kinase 2 are obtained by
replacement of the threonine residues at the phosphorylation sites by
glutamic acid, which mimics a negative phosphate charge. In addition,
constitutive activation of MAPKAP kinase 2 is reached by mutations of
the A-helix motif carboxyl-terminal to the catalytic domain. A model
for the mechanism of dual regulation of MAPKAP kinase 2 activity by
phosphorylation of Thr-205 inside the catalytic domain and by
phosphorylation of Thr-317 outside the catalytic domain involving the
A-helix motif is proposed.
Immunoblot
detection of ERKs in the Mono Q fractions was performed using a mouse
monoclonal pan ERK antibody (Transduction Laboratories, Lexington) and
a secondary antibody conjugated to alkaline phosphatase (Promega).
Western blot detection of p38/40 (RK) was achieved with
a sheep antiserum against a carboxyl-terminal peptide from human RK
(kindly provided by P. Cohen, Dundee). Immunoprecipitation of
p38/40
(RK) was performed with a rabbit antiserum raised
against a carboxyl-terminal peptide from Xenopus Mpk2 as
described in (11) .
For
immunoblot detection of epitope-tagged MAPKAP kinase 2, 10 cells were lysed by boiling in SDS-electrophoresis loading
buffer, applied directly to SDS-PAGE, and blotted onto nitrocellulose.
Immunochemical detection was performed using monoclonal antibody 9E10
(European Collection of Animal Cell Culture, cell line 85102202) and an
anti-mouse immunoglobulin secondary antibody conjugated to alkaline
phosphatase (Promega).
Immunoprecipitation was carried out after 15
min of incubation of 10 cells in 80 µl of lysis buffer
L. Cell lysate was diluted with 500 µl of IP buffer (50 mM Tris/HCl, pH 7.4, 25 mM
-glycerophosphate, 25 mM NaF, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100)
and incubated with 25 µl of purified 9E10 antibody overnight at 4
°C. Precipitation was achieved by adding 25 µl of a 1:1 (v/v)
suspension of Protein A-Sepharose (Pharmacia) and a further incubation
for 1 h at 4 °C. Immunoprecipitate was washed four times with IP
buffer, and the pellet was redissolved in 25 µl of MAPKAP kinase 2
reaction mixture and analyzed as described above.
Figure 1:
In vitro reconstitution of MAPKAP kinase 2 activation and subsequent Hsp25
phosphorylation in dependence of MAPKAP kinase 2 phosphorylation site
mutations. A, schematic representation of the different
recombinant forms of MAPKAP kinase 2 used. The fusion proteins
GST-MAPKAP kinase 2 (GST-MK2) and the SH3-binding domain (3B) deletion mutant GST-MK2-3B show identical activation
in the assay and are referred to as wild type protein (WT).
Based on the wild type protein GST-MK2-
3B, the phosphorylation
site mutants T205A, T317A, and the double mutant T205A,T317A were
constructed.. (Catalytic, catalytic domain; NTS,
nuclear translocation signal). B, analysis of the wild type
form (WT) and phosphorylation mutants (T205A, T317A, T205A,T
317A) of MAPKAP kinase 2 for their ability to phosphorylate Hsp25 in
dependence on activation by pp44 ERK1 MAP kinase. C, sequence
alignment of the region of MAPKAP kinase 2 containing the newly
identified phosphorylation site Thr-205, to the region between
subdomains VII and VIII of the catalytic core of serine/threonine
protein kinases known to be phosphorylated and activated at similar
sites (MAPKAP kinase 1, ERK2 Cdk2, cAPK). Phosphorylation sites are
indicated by asterisks.
In order to understand the molecular
mechanism underlying the activation of mouse MAPKAP kinase 2 by single
MAPK phosphorylation carboxyl-terminal to the catalytic domain, we
replaced the amino acid residue Thr-317, which is homologous to the
only identified MAPK phosphorylation site of rabbit MAPKAP kinase
2(14) , by the amino acid alanine which cannot be
phosphorylated. The mutation of this phosphorylation site led to
decreased phosphorylation and activation of MAPKAP kinase 2 by ERK1
but, unexpectedly, the T317A mouse MAPKAP kinase 2 mutant (cf. Fig. 1A) could still be activated by ERK1 (Fig. 1B). This indicated that there is at least a
second regulatory phosphorylation site of MAPKAP kinase 2 for ERK1. To
identify further putative Ser/Thr phosphorylation sites of MAPKAP
kinase 2, we compared primary sequences of MAPKAP kinase 2, MAPKAP
kinase 1 (p90), ERK2, Cdk2, and cAPK. Fig. 1C demonstrates the conservation of threonine residues in the loop
between subdomains VII and VIII of their catalytic core. These
threonine residues are essential for the activation of these kinases:
MAPKAP kinase 1 is known to be activated by ERK2 phosphorylation at the
threonine residue which is nine residues amino-terminal to the
subdomain VIII (APE)(27) , MAPK ERK2 is phosphorylated by MEK1
both at the threonine and tyrosine in similar positions(28) ,
and comparable regulatory phosphorylation sites are also present in
Cdk2 or in cAMP-dependent protein kinase (29) (marked by asterisks in Fig. 1C). Interestingly, in both
MAPKAP kinases 1 and 2, the equivalent threonine is followed by a
proline, the minimum consensus sequence for phosphorylation by MAP
kinases(30) . Hence, a potential second phosphorylation site of
MAPKAP kinase 2 could be Thr-205, the phosphorylation site equivalent
to Thr-470, Thr-192, Thr-161, and Thr-197 of, respectively, MAPKAP
kinase 1, ERK2, Cdk2, and cAPK. To experimentally prove whether this
site is phosphorylated by MAPK, we substituted Thr-205 of mouse MAPKAP
kinase 2 with alanine (cf. Fig. 1A) and
analyzed the phosphorylation and activation of this mutant by ERK1. As
shown in Fig. 1B, the T205A mutant shows an activation
by ERK1 similar to the mutant T317A. There is still activation of the
mutant by ERK1, but not to the same degree as in the wild type enzyme.
Only the double mutant T205A,T317A, which shows a slightly increased
basal activity, could not be activated by ERK1 (cf. Fig. 1B), indicating that both phosphorylation sites
contribute to the in vitro activation of MAPKAP kinase 2 by
ERK1 and that these sites seem to be the major regulatory
phosphorylation sites of the enzyme.
Figure 2:
Analysis of constitutively active forms of
MAPKAP kinase 2. A, phosphorylation of Hsp25 by constitutively
activated single and double mutants (T205E, T317E, and T205E,T317E) of
MAPKAP kinase 2 in dependence on ERK1 phosphorylation. B,
quantitative evaluation of enzymatic activity of the constitutive
mutants compared to the wild type enzyme (WT) in dependence on ERK1
phosphorylation. P-Labeled Hsp25 was detected by the Bio
Imaging Analyzer BAS 2000 (Fuji), and labeling was quantified by
photostimulated luminescence (PSL). The data represent the mean value
of three independent experiments as shown in A.
Figure 3:
Partial purification of p38/40 (RK) from anisomycin treated EAT cells and in vitro reconstitution of MAPKAP kinase 2 activation by
p38/40
. A, stimulation of MAPKAP kinase 2
activity in EAT cells by treatment with 10 µg/ml anisomycin for 20
min (+A) compared to control EAT cells (-A). Activity shown was determined in unfractionated
EAT cell lysates. B, Mono Q fractionation of cell lysates from
anisomycin-stimulated EAT cells. Fractions were assayed for MAPKAP
kinase 2 activators using recombinant GST-MAPKAP kinase 2
3B and a
subsequent assay for MAPKAP kinase 2 activity with Hsp25 as substrate
(
). Since in this assay both MAPKAP kinase 2 activator and
endogenous MAPKAP kinase 2 were measured, fractions were also analyzed
in an assay omitting recombinant GST-MAPKAP kinase 2
3B which
detects MAPKAP kinase 2 activity only (
). In both assays, the
same amount of protein from the Mono Q fractions was analyzed. The
difference of both activity profiles clearly demonstrates an activator
of MAPKAP kinase 2 eluting in fraction 19 at about 350 mM NaCl, probably corresponding to p38/40
(RK). C, detection of p38/40
(RK) in fraction 19 by
immunoprecipitation of the enzyme with an anti-Mpk2 antiserum and
subsequent in vitro reconstitution of MAPKAP kinase 2
activation using wild type MAPKAP kinase 2: 19, peak fraction; 13, control fraction containing ERKs. The two left lanes are controls using the fractions without prior
immunoprecipitation. D, in vitro reconstitution of MAPKAP
kinase 2 activation by ERK1 and peak fraction 19 using wild type MAPKAP
kinase 2 (WT) and its phosphorylation mutant (T205E,T317E). C is a control assay where the in vitro reconstitution reaction with fraction 19 is carried out without
adding recombinant MAPKAP kinase 2. A further control which excludes
autophosphorylation and autoactivation of the recombinant MAPKAP kinase
2 during the in vitro reconstitution reaction is carried out
by incubation of the recombinant enzyme in the presence of MgATP (+MgATP) omitting MAPKs (-p38/40 RK, -ERK1). As in the other in vitro reconstitution
reactions, this autophosphorylation-permitting preincubation does not
influence the enzymatic activity of the recombinant
enzyme.
We then analyzed activation of recombinant wild type MAPKAP
kinase 2, the single mutants T205A and T317A, and the double mutant
T205E,T317E by the p38/40 peak fraction. As seen in Fig. 3D, wild type MAPKAP kinase 2 can be stimulated by
the p38/40
fraction in the same manner as by ERK1. This
is also the case for ERK1/p38/40
stimulation of the
single mutants T205A and T317A (not shown). In contrast, the
constitutively active mutant T205E,T317E shows no changes in activity
after treatment with the p38/40
fraction (Fig. 3D). These observations support the notion that
regulation of MAPKAP kinase 2 by p38/40
(RK) in
vitro proceeds also by dual phosphorylation and that there is no
further regulatory phosphorylation site for p38/40
in
MAPKAP kinase 2.
Figure 4: Expression of epitope-tagged MAPKAP kinase 2 and the phosphorylation site mutants T205A,T317A and T205E,T317E in NIH 3T3 cells and analysis of activity of epitope-tagged MAPKAP kinase 2 and its mutants before and after heat shock (HS). A, Western blot detection of expression of wild type epitope-tagged MAPKAP kinase 2 (WT) and mutants using the anti-Myc antibody 9E10. As a control (C), lysate of NIH 3T3 cells which were transfected with the expression vector pcDNA3 is applied to Western blot analysis. B, MAPKAP kinase 2 activity assay after immunoprecipitation from cells transfected with pcDNA3 (C), wild type MAPKAP kinase 2, mutants T205A,T317A and T205E,T317E before(-) and after heat shock (+) using the anti-Myc antibody.
Figure 5:
Characterization of the autoinhibitory
region of MAPKAP kinase 2. A, sequence alignment of the
autoinhibitory region of MAPKAP kinase 2, the MAPKAP kinase 2 consensus
substrate motif and the protein kinase A pseudosubstrate PKI as
supposed in (32) . In the consensus substrate motif, is
a large hydrophobic residue (F > L > V), and
represents a
hydrophobic or acidic residue(35) . Matching residues are in bold letters. The phosphorylated serine residue is underlined. The appropriate MAPKAP kinase 2 mutants, which
should have altered pseudosubstrate properties, R331K and K329L/D334S,
are indicated. B, sequence alignment of the putative A-helix
motif of MAPKAP kinase 2 with the A-helix of the catalytic subunit of
mammalian (cAPK) and Dictyostelium discoideum (dict)
cAMP-dependent protein kinase and of the protein tyrosine kinase lck (lck). The phosphorylation site Thr-317 of MAPKAP kinase 2 and
the conserved tryptophan and lysine residues of the A-helix are
indicated by asterisks. The appropriate MAPKAP kinase 2
mutants, which should have an altered A-helix motif, W332A and K326E,
are indicated. C, analysis of the wild type form (WT), deletion mutant (
PC;
amino acids
315-383), pseudosubstrate mutants R331K, K329L/D3345 and A-helix
mutants (W332A, K326E) of MAPKAP kinase 2 for their
ability to phosphorylate Hsp25 in dependence on activation by pp44 ERK1
MAP kinase. D, assay for autophosphorylation of MAPKAP kinase
2 and mutant K329L/D334S. As a control, MAPKAP kinase 2 and the mutant
were incubated in the presence of ERK1 leading to phosphorylation of
MAPKAP kinase 2 and to autophosphorylation of
ERK1.
We then analyzed enzymatic activity of the different pseudosubstrate and A-helix mutants (Fig. 5C) and the phosphorylation of the K329L/D334S mutant (Fig. 5D). As seen in Fig. 5C, only the two mutants affecting conserved residues of the A-helix motif lead to constitutive activation of the enzyme indicating that the A-helix motif contributes to suppress MAPKAP kinase 2 activity. However, the higher constitutive activity of the W332A mutant compared to the K326E mutant may indicate the central structural role of this tryptophan residue within the A-helix. In contrast, mutants constructed to change the pseudosubstrate properties of this region do not influence kinase activity. Not even the alteration of the pseudosubstrate motif to an ideal substrate for MAPKAP kinase 2 does increase kinase activity. Furthermore, there is no increased autophosphorylation of the enzyme carrying the phosphorylatable Ser-334 in the potential pseudosubstrate sequence (Fig. 5D), although the corresponding peptide KKLERWESVK-amide is efficiently phosphorylated by the mutant K329L/D334S (data not shown). Taken together, these data strongly indicate that the autoinhibitory region of MAPKAP kinase 2 does not function as a pseudosubstrate. Hence, it could be assumed that the A-helix motif does not directly bind to the peptide acceptor site within the catalytic cleft of MAPKAP kinase 2, but acts autoinhibitory by binding to some other region of the kinase. One potential binding region for the A-helix to the catalytic core could be the hydrophobic surface distal to the active site between the two lobes of the catalytic core as described for the A-helix of cAMP-dependent protein kinase(20) .
Figure 6: Molecular modeling of the interaction of the A-helix with the catalytic domain of MAPKAP kinase 2. The different colors help to identify the side chains of the hydrophobic region (pink) in the catalytic domain (gray) and the tryptophan and valine side chains (blue) of the A-helix (green). The plot was drawn with SETOR(45) . A, space-filling model of the catalytic domain to give an impression of the hydrophobic surface. B, a closer view with labeled side chains using the same coloring mode.
In this paper we identify a second regulatory phosphorylation
site of MAPKAP kinase 2 and provide experimental evidence that
stimulation of MAPKAP kinase 2 activity proceeds by MAPK
phosphorylation at two different regulatory sites. The evidence came
from the observation that single T205A and T317A MAPKAP kinase 2
mutants could still be activated by ERK1 and p38/40 (RK)
phosphorylation in vitro. Although there is a slight
difference between the basal activity of the double mutant T205A,T317A in vitro (detectable) and in vivo (not detectable),
which is probably due to the different expression systems, this mutant
cannot be stimulated either by ERK1 phosphorylation in vitro or by the heat shock-stimulated forms of MAPKs in vivo.
This finding indicates that both phosphorylation sites Thr-205 and
Thr-317 are necessary for MAPKAP kinase 2 activation.
In a second
approach we demonstrate that a constitutively active form of MAPKAP
kinase 2 could be obtained as a result of mimicking the negative
phosphate groups of phosphorylated Thr-205 and Thr-317 by replacement
with glutamic acid. The finding that the fully constitutively active
double mutant T205E,T317E cannot be further stimulated by ERK1 and
p38/40 (RK) phosphorylation in vitro and by
heat shock treatment in NIH 3T3 cells gives independent support to the
notion that these sites are the two major regulatory phosphorylation
sites of MAPKAP kinase 2.
Our results demonstrate that the mechanism
of activation of MAPKAP kinase 2 by ERK1 and p38/40 (RK)
is very similar and that MAPKAP kinase 2 activation by these enzymes
proceeds with comparable efficiency in vitro. However, in PC12
and A431 cells, ERKs fail to activate MAPKAP kinase 2, whereas
p38/40
(RK) is a major activator for this
enzyme(11, 12) . An explanation for this discrepancy
between in vitro and in vivo data could be a
different subcellular location of ERKs and MAPKAP kinase 2 in these
cells or a specific protein-protein interaction between MAPKAP kinase 2
and other unknown proteins, which prevent the contact to ERKs but
facilitate the binding to p38/40
(RK). The latter
explanation would be in agreement with the idea of the existence of
mammalian signal transduction particles tethered by ``scaffolding
proteins'' analogous to the yeast protein STE5(46) .
The replacement of regulatory phosphorylation sites by negatively charged residues from aspartate and glutamate to constitutively activate protein kinases has recently been used in the case of the MAPK kinase MEK1(37, 38, 39, 40) . Using this approach, it was possible to restore MEK activity independent of the upstream kinases and to analyze the role of activated MEK in growth, differentiation, and oncogenic transformation. The constitutively active form of MAPKAP kinase 2, which is in mitogenic signal transduction downstream of the bifurcation point of the MAPKs, will now open further ways to analyze the cellular role of MAPKAP kinase 2, as well as the role of the phosphorylation of its major substrate, the small mammalian heat shock protein.
The identification of the phosphorylation sites of MAPKAP kinase 2 yields new insight into the mechanism of the regulation of protein kinase activity. The phosphorylation site identified in this report, Thr-205, in the loop between subdomains VII and VIII of the catalytic domain is homologous to regulatory phosphorylation sites of several other protein kinases involved in mitogenic signal transduction (cf. Fig. 2) and places the regulation of MAPKAP kinase 2 in one line with the emerging common mechanism of activation of many protein kinases. These phosphorylation sites are in the activation loop of the kinase and could regulate the accessibility of the substrate binding sites and/or the relative location of the amino- and carboxyl-terminal lobes of the catalytic core, leading to correct alignment of the different catalytic residues of these kinases (44) .
The second regulatory phosphorylation site of MAPKAP kinase 2, Thr-317, has been identified outside the catalytic domain, indicating an indirect influence of this phosphorylation on the catalytic properties of the kinase. Besides the direct activation of protein kinases through phosphorylation within the catalytic domain, several cases of regulation of protein kinase activity by intrasteric inhibition of catalytic activity due to autoinhibitory ``pseudosubstrate'' regions have been described. These autoinhibitory domains could be regulated by allosteric factors such as calcium/calmodulin (CaM) in the case of the CaM-dependent kinases or phospholipid diacylglycerol in the case of protein kinase C and, probably, also by phosphorylation (for reviews see (42) and (43) ). A second, recently described common sequence motif of protein kinases which has a regulative potential is the amphiphilic A-helix(33, 34) . The A-helix has been described originally as a stabilizing element of protein kinase structure which binds to a hydrophobic pocket present in most protein kinases between the two lobes of the catalytic core on the surface opposite to the catalytic cleft opening(44) .
In this paper we first provide
evidence that an A-helix can act as an autoinhibitory element in MAPKAP
kinase 2. Deleting the carboxyl-terminal region containing the A-helix
motif and even changing the conserved tryptophan and lysine residues of
the A-helix led to activation of the MAPKAP kinase 2, indicating that
the presence of a functional A-helix can suppress the activity of the
enzyme. This is in agreement with the recent finding that an
amphiphilic A-helix-like motif can also suppress the catalytic activity
of the protein kinase MEK(39) . The mechanism by which the
A-helix inhibits the kinase activity and by which phosphorylation may
regulate this inhibition is still unclear. An action of the A-helix as
a pseudosubstrate for the kinase seems unlikely, since alteration of
the conserved arginine residue of the pseudosubstrate motif and
modification of this motif to an ideal substrate does not influence
kinase activity or its autophosphorylation. However, it seems likely
that this mechanism is based on complex intramolecular interactions,
since an A-helix motif-derived peptide CVLKEDKERWEDVK and a GST-fusion
protein containing the carboxyl-terminal part of MAPKAP kinase 2 were
not able to specifically inhibit MAPKAP kinase 2 activity of the wild
type protein purified from rabbit muscle (generous gift of P. Cohen,
Dundee) or of the constitutively active A-helix deletion mutant
PC. (
)
By molecular modeling, we have shown a possible interaction of the A-helix motif of MAPKAP kinase 2 with the catalytic core. Interestingly, even in a protein kinase without an A-helix motif, as in the MAPK ERK2, the hydrophobic pocket between the lobes opposite to the catalytic cleft is filled by hydrophobic residues of the non-core sequences which are located approximately 30 residues downstream to the subdomain XI(41, 44) . This distance is similar to the distance of the tryptophan residue of the A-helix from the subdomain XI in MAPKAP kinase 2 and supports the notion that the A-helix of MAPKAP kinase 2 could also bind to this hydrophobic pocket between the lobes. Although binding of the A-helix of MAPKAP kinase 2 to other regions of the enzyme could not be excluded, molecular modeling supports binding of the A-helix to the hydrophobic pocket between the two lobes of MAPKAP kinase 2 predominantly based on interaction of the central tryptophan residue of the A-helix. Hence, a mechanism proposed to contribute to the regulation of MAPKAP kinase 2 is the binding of the A-helix to the hydrophobic pocket between the two lobes which could affect catalysis. Phosphorylation of Thr-317 at the proposed beginning of the A-helix may destabilize the A-helix itself and/or its binding to the hydrophobic cleft and by that activates MAPKAP kinase 2. Further studies to resolve the phosphorylation-dependent three-dimensional structure of MAPKAP kinase 2 will prove whether the proposed model describes the molecular mechanism underlying MAPKAP kinase 2 activation.