(Received for publication, September 20, 1994; and in revised form, October 27, 1994)
From the
Mitogen-activated protein (MAP) kinase-activated protein kinase 2, a Ser/Thr kinase, is phosphorylated and activated by MAP kinase. Sequence analysis of a clone isolated from the human HL-60 cell line revealed a 370-amino acid protein with a proline-rich N terminus, a highly conserved catalytic domain, and a C-terminal region containing a MAP kinase phosphorylation site. To better understand how the kinase is regulated, mutation analysis was used to map the functional domain(s). The wild type recombinant kinase had a low basal activity as detected by phosphorylation of a substrate peptide derived from the N terminus of glycogen synthase. Deletion of the proline-rich N terminus showed little effect on the basal activity. Deletion of the C terminus resulted in a marked increase in catalytic activity either with or without the pretreatment of the kinase by MAP kinase. Further analysis indicated that amino acid residues 339-353 in the C-terminal region were acting as an autoinhibitory domain. A synthetic peptide (RVLKEDKERWEDVK-amide) derived from this autoinhibitory domain inhibited the kinase activity in a concentration-dependent manner. These results suggest a regulatory model for the kinase.
A variety of extracellular stimuli activates mitogen-activated
protein (MAP) ()kinases by an intracellular kinase
cascade(1, 2, 3, 4) , which includes
MAP kinase kinase
kinase(5, 6, 7, 8, 9, 10) and MAP kinase kinase(11, 12) . The
activated MAP kinases in turn regulate the phosphorylation and
activation of many substrates including cell surface molecules,
cytoskeletal proteins, transcription factors, and protein kinases
(reviewed in (3) and (4) ). Both ribosomal protein S6
kinase II (13, 14, 15) and MAP
kinase-activated protein (MAPKAP) kinase 2 are known as MAP kinase
substrates(16) . MAPKAP kinase 2 can be distinguished from the
ribosomal protein S6 kinase II by several means including its
resistance to the protein kinase inhibitor, H-7, its failure to
phosphorylate the peptides derived from the C terminus of ribosomal
protein S6, and its partial amino acid sequences(16) . MAPKAP
kinase 2 was originally identified by its ability to phosphorylate
rabbit skeletal muscle glycogen synthase at Ser-7 as well as the
equivalent serine in the synthesized peptide derived from the N
terminus of glycogen synthase(16) . MAPKAP kinase 2 also
phosphorylates the low molecular weight heat shock proteins (17) and tyrosine hydroxylase(18) .
Partial cDNA sequences of MAPKAP kinase 2 derived from mouse lung(19) , rabbit and human skeletal muscles(20) , and a full-length cDNA sequence derived from human HL-60 cells (21) have been reported. Sequence analysis of the MAPKAP kinase 2 derived from HL-60 cells revealed that it is a 370-amino acid protein containing a proline-rich N terminus, a well conserved catalytic domain in the central area, and a MAP kinase phosphorylation site in the C terminus(21) . In order to understand the possible mechanism of MAPKAP kinase 2 activation we have mapped its functional domains using mutation analysis. An autoinhibitory domain containing 15 amino acids within the C-terminal region was identified, and a regulatory model for the kinase was suggested.
The activities of cAMP-dependent protein kinase
(Sigma) and protein kinase C (Calbiochem) were evaluated using a
synthetic peptide substrate, RSRKRLSQDAYRRNSVRF-amide, that corresponds
to the amino acids 314-331 of
p47(26, 27) . In the protein kinase C
kinase reaction, additional components (1.6 mM CaCl
, 20 µg/ml phosphatidylserine, and 2 µg/ml
phorbol 12-myristate 13-acetate) were included. MAP kinase activity was
assessed using myelin basic protein as a
substrate(28, 29) .
Figure 1:
Deletion analysis
of C terminus. A, construction of the recombinant kinases.
MAPKAP kinase 2 has a proline-rich N terminus (amino acids 1-62)
containing two SH3 domain-binding motifs (), a well-conserved
catalytic domain (hatched box, amino acids 63-326), and
a C terminus (amino acids 327-370) containing a MAP kinase
phosphorylation site at threonine 334 (T334). The kinases were
prepared as glutathione S-transferase fusion proteins as
described under ``Experimental Procedures.'' Wild type (WT) kinase expresses the full-length cDNA of human MAPKAP
kinase 2 (amino acids 1-370) cloned from HL-60
cells(21) . Mutants CT327 and CT339 are two C-terminal
truncated kinases expressing amino acid residues 1-326 and
1-338 of MAPKAP kinase 2, respectively. B, enzymatic
analysis of the recombinant kinases. The relative enzymatic activity
(counts/min) of the kinases was examined by an in vitro protein kinase assay using a peptide substrate derived from the N
terminus of glycogen synthase(16) . Glutathione S-transferase vector protein was used as the kinase(-)
background control. In order to activate the kinases, samples were
pretreated with or without MAP kinase as indicated. This figure is
representative of three separate experiments. C, protein
phosphorylation of the recombinant kinases by MAP kinase. Purified
recombinant kinases were analyzed by 10% SDS-PAGE with Coomassie Blue
staining (lanes 1-6) and autoradiography (lanes
7-12). The kinases were phosphorylated with (lanes
4-6 and 10-12) or without (lanes
1-3 and 7-9) MAP kinase as described in the
text. Molecular weight standards are shown on the left. The
positions of the recombinant kinases and MAP kinase are indicated on
the right.
To examine MAP kinase-induced activation of MAPKAP kinase 2, the recombinant kinases were pretreated with MAP kinase prior to initiation of the kinase assay. Fig. 1B shows that MAP kinase treatment increased the enzymatic activity of the WT kinase 249%, but had little effect on either mutant CT339 (115%) or CT327 (82%). Phosphorylation of MAPKAP kinase 2 (with or without MAP kinase pretreatment) was analyzed by SDS-PAGE and autoradiography. As shown in Fig. 1C (lanes 7-12), MAP kinase phosphorylated both the WT kinase and the mutant CT339 to an approximately equivalent extent but not the mutant CT327 which lacked a MAP kinase phosphorylation site.
Human MAPKAP kinase 2 has two proline-rich regions in the N terminus (Fig. 1A). To examine the role of the N terminus in regulating the enzymatic activity, an N-terminal truncated mutant, NT47 (amino acids 48-370) which lacked the proline-rich regions was generated (Fig. 2A). Deletion of the N terminus resulted in a lower kinase activity (68% of the WT kinase). The result suggests that this region may have a role in maintaining the kinase activity but not in the autoinhibitory function (Fig. 2C).
Figure 2: Identification of an autoinhibitory domain. A, construction of mutant kinases. Mutants of the kinase were generated using the same strategy as described in Fig. 1. Mutant NT47 is an N-terminal truncated kinase encoding amino acid residues 48-370, which lacks the proline-rich region. Mutants NCT327, NCT339, and NCT354 are both N- and C-terminal truncated mutants of the kinase expressing amino acid residues 48-326, 48-338, and 48-353 of MAPKAP kinase 2, respectively. B, preparation of the recombinant kinases. The kinase were expressed in E. coli, purified on a glutathione-Sepharose column, and analyzed by 10% SDS-PAGE with Coomassie Blue staining. C, analysis of enzymatic activity. Relative kinase activities of mutants were examined by in vitro kinase assay as described in Fig. 1, and the percentages of enzymatic activity as compared to the wild type kinase (100%) are shown in the graph. This is representative of three separate experiments.
Figure 3:
Characterization of an autoinhibitory
domain-derived peptide, I-pp. A, design of I-pp. Based on the
sequence of the autoinhibitory domain (amino acids 339-353), a
14-amino acid peptide (underline) was synthesized and
designated as I-pp. B, effects of I-pp on protein kinases.
Various concentrations of I-pp were incubated with the kinases for 1
min prior to the initiation of kinase reaction as indicated. Kinase
assay was started by the addition of substrate peptides and ATP. For
cAMP-dependent protein kinase and protein kinase C, a synthetic
substrate peptide derived from the amino acid residues 314-331 of
p47 was used. For MAP kinase, myelin basic
protein was used as a substrate. C, inhibition of
p47
phosphorylation by I-pp. p47
(from left to right) was phosphorylated by
cAMP-dependent protein kinase in the presence of 0, 12.5, 25, 50, and
100 µM (final concentrations) I-pp, respectively.
Phosphorylated proteins were analyzed by 10% SDS-PAGE followed by
Coomassie Blue staining (upper panel) and autoradiography (lower panel).
Fig. 4shows the sequence alignment of I-pp with the
substrates and pseudosubstrates derived from glycogen
synthase(16) , cAMP-dependent protein kinase(33) , and
calcium/calmodulin-dependent protein kinase II (CaM kinase
II)(34) . The alignment indicates that the phosphorylated
serine residue of the peptide substrate for MAPKAP kinase 2 is
substituted with an aspartic acid in I-pp (pointed arrow). A
serine residue of the cAMP-dependent protein kinase substrate is
similarly replaced with an alanine in the pseudosubstrate, the
cAMP-dependentprotein kinase inhibitor. It is noteworthy that all of
the amino acid residues important for peptide recognition, which have
been identified for both the cAMP-dependent protein kinase inhibitor
and the pseudosubstrate of CaM kinase II
, are conserved in I-pp (bold type).
Figure 4:
Sequence alignment of substrates and
inhibitory domains of protein kinases. The sequence of the
autoinhibitory domain of MAPKAP kinase 2 (amino acid residues
339-353) is listed based on the present study. The sequence of
the substrate peptide of MAPKAP kinase 2 is the amino acid residues
1-13 of glycogen synthase(16) . The sequence of the
pseudosubstrate of cAMP-dependent protein kinase is a segment of the
cAMP-dependent protein kinase inhibitor PKI(43) . The sequence
of the substrate of cAMP-dependent protein kinase is the in vivo phosphorylation site of pyruvate kinase(44) . The sequence
of the autoinhibitory peptide of CaM kinase II is the amino acid
residues 288-306 of CaM kinase II
(34) . The
alignment was made based on the substrate phosphorylation sites (pointed arrow) and the amino acid residues important for
peptide recognition (bold
type)(33) .
Using mutational analysis we have studied the roles of the N- and C-terminal regions of MAPKAP kinase 2 in regulating its enzymatic activity. Stepwise deletion of the C terminus revealed an autoinhibitory domain. Deletion of this domain resulted in a marked increase in kinase activity (431% of the wild type kinase). In contrast, deletion of the N terminus resulted in a slight decrease in kinase activity. In addition, the detailed mutational analysis demonstrated that the autoinhibitory domain was located within amino acids 339-353. An autoinhibitory domain-derived peptide (I-pp) inhibited the activity of MAPKAP kinase 2.
Based on the results of this study, we suggest the following regulatory model for MAPKAP kinase 2 (Fig. 5): first, extracellular stimuli induce the activation of the MAP kinase cascade; in turn, activated MAP kinases phosphorylate MAPKAP kinase 2 at Thr-334; the phosphorylation of Thr-334 results in a conformational change and subsequent release of MAPKAP kinase 2 from the repression of its autoinhibitory domain; and finally, MAPKAP kinase 2 becomes activated. Competing with the phosphorylation are protein phosphatases, which dephosphorylate MAPKAP kinase 2 at Thr-334 and return the kinase to its inactive state.
Figure 5: Regulatory model for MAPKAP kinase 2. An extracellular signal triggers a kinase cascade leading to the phosphorylation of MAPKAP kinase 2 at Thr-334. This phosphorylation event may result in a conformational change of MAPKAP kinase 2 and may release it from the repression of its autoinhibitory domain located in the C-terminal region. The activated MAPKAP kinase 2 regulates the activities of its substrates by phosphorylation. To restore the balance, protein phosphatases may inactivate MAPKAP kinase 2 by dephosphorylation of Thr-334.
Kinetic studies of the
substrate specificity of MAPKAP kinase 2 indicate that the minimum
sequence required for efficient phosphorylation is X-X-Hyd-X-Arg-X-X-Ser/Thr-X-X(20) . A hydrophobic residue (Hyd) at position n - 5 is required for efficient binding. In contrast, the
residue at n - 5 of I-pp derived from the autoinhibitory
domain of MAPKAP kinase 2 is lysine (K), a basic residue. This residue
is conserved in the pseudosubstrates of cAMP-dependent protein kinase,
CaM kinase II (Fig. 4), and MAPKAP kinase 1(20) .
The existence of this basic residue at n - 5 of I-pp may
explain why I-pp also inhibited cAMP-dependent protein kinase and
protein kinase C (Fig. 3B). At present, we are not sure
that I-pp is a true pseudosubstrate of MAPKAP kinase 2.
To date, only a few proteins, such as glycogen synthase(16) , low molecular weight heat shock proteins (17, 19, 35, and 36), and tyrosine hydroxylase (18) have been reported to be substrates for MAPKAP kinase 2. The active mutants of MAPKAP kinase 2 reported in this work should facilitate the search for new substrates. Using the active mutants, we have demonstrated that the major substrate for MAPKAP kinase 2 in an HL-60 cell lysate is a 27-kDa protein with mobility in SDS-PAGE similar to that of the heat shock protein, Hsp27 (data not shown).
The proline-rich N terminus of MAPKAP kinase 2 may interact with proteins that contain SH3 domain(s)(31, 32) . The protein-protein interaction between the SH3 domain and the proline-rich domain may play an important role in signal transduction(37, 38) . Our results indicate that the N terminus of MAPKAP kinase 2 is not involved in the autoinhibitory event. Identification of the proteins whose SH3 domain can bind specifically to the proline-rich domain of MAPKAP kinase 2 remains to be done.
While this paper was under review, Rouse et al. (39) and Freshney et al. (40) reported a novel protein kinase cascade triggered by interleukin-1, stress, and heat shock that stimulates MAPKAP kinase 2 and phosphorylation of the small heat shock protein. In intact cells, the kinase cascade that leads to the activation of MAPKAP kinase 2 is distinct from the classical pathway leading to the activation of MAP kinase. The upstream kinase which regulates MAPKAP kinase 2 was identified as a novel kinase termed RK (39) or p40(40) . RK is related to the yeast kinase HOG1 and the mammalian kinases JnK1, p38, and SAPKs(41, 42) . Further studies on the signal transduction pathway leading to the activation of MAPKAP kinase 2 should provide important insights into the intracellular action of interleukin-1, endotoxin, stress, and heat shock as well as the mechanism of cell volume control.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U12779[GenBank].