(Received for publication, November 28, 1995; and in revised form, December 19, 1995)
From the
To discern MEK1 and MEK2 specificity for their substrate, extracellular signal-regulated kinase (ERK), site-directed mutagenesis was performed on the amino acid residues flanking the regulatory phosphorylation sites of ERK1. These ERK1 mutants were analyzed for the ability to act as a substrate for MEK1 and MEK2. Based on both phosphorylation and activation analyses, the mutants could be divided into four classes: 1) dramatically decreased phosphorylation and activation, 2) enhanced basal kinase activity, 3) preferentially enhanced phosphorylation of tyrosine and decreased phosphorylation of threonine, and 4) increased threonine phosphorylation with an increase in activation. In general, the residues proximal to the regulatory phosphorylation sites of ERK1 had greater influence on both phosphorylation and activation. This is consistent with the highly specific recognition of the ERK1 regulatory sites by MEK. Mutation of Arg-208 or Thr-207 to an alanine residue significantly altered the relative phosphorylation on Thr-202 and Tyr-204. The Arg-208 to alanine mutant increased the phosphorylation of Tyr-204 approximately 4-fold yet almost completely eliminated the phosphorylation on Thr-202. In contrast, mutation of Gly-199 to alanine resulted in an increased phosphorylation of Thr-202 relative to Tyr-204. This suggests that both Gly-199 and Arg-208 play important roles in determining the relative phosphorylation of Thr-202 and Tyr-204. Our results demonstrate that residues in the phosphorylation lip of ERK play an important role in the recognition and phosphorylation by MEK.
Mitogen-activated protein kinases (MAPKs) ()are
important components of various signaling pathways, phosphorylate
numerous substrates, and may be key components in linking growth factor
receptor activation to serine/threonine protein
phosphorylation(1, 2, 3) . MAPK activation
can be induced by numerous mitogenic stimuli including phorbol esters,
cytokines, T-cell antigens, and growth factors with tyrosine kinase
receptors such as insulin, epidermal growth factor, fibroblast growth
factor, and platelet-derived growth
factor(3, 4, 5, 6) . Stimulation of
MAPK in various non-lymphoid cell types leads to a multitude of
cellular responses including phosphorylation of microtubule-associated
proteins involved in microtubule rearrangement (7, 8) and phosphorylation resulting in subsequent
activation of transcription factors TCF and
STAT(9, 10, 11) . In lymphoid cells,
activation of MAPK results in stimulation of T-cells to produce
cytokines(12, 13, 14) . The involvement of
MAPK in such a variety of cells and cellular processes emphasizes a
potentially vital role of MAPK in mediating cellular signal
transduction. One of the most unique features of MAPK is that it must
be phosphorylated on both threonine and tyrosine to exhibit full
enzymatic activity(15) . Dephosphorylation of either
phosphothreonine or phosphotyrosine completely inactivates MAPK,
emphasizing the importance of both residues for
activation(16, 17, 18) .
MAPKs are also referred to as extracellular signal-regulated kinases (ERKs). The upstream activator of ERK is a dual specific kinase called MAPK or ERK kinase (MEK), which phosphorylates ERK2 on both threonine 183 and tyrosine 185(15) . Mammalian isoforms of MEK, two of which (MEK1 and MEK2) can phosphorylate and activate ERK, were identified by molecular cloning techniques(18, 19, 20, 21, 22, 23, 24) . ERK is the only known substrate for MEK, with all other proteins tested thus far being phosphorylated at least 1000-fold less efficiently by MEK(25) .
MEK is activated by a number of different serine/threonine kinases including the proteins encoded by the proto-oncogene c-raf(26, 27, 28, 29) . In fact, the regulatory phosphorylation sites in MEK were mapped using immunoprecipitated Raf from epidermal growth factor-stimulated Swiss 3T3 cells(30) . Activation was shown to occur through phosphorylation of 2 serine residues at positions 218 and 222 of MEK1(30) . Constitutively active MEK1 can be obtained by mutation of the regulatory phosphorylation sites to glutamic acid(31, 32) . Alternatively, if these sites are mutated to alanine (S218A/S222A) the resulting MEK mutant is incapable of being activated by Raf(30) . Phosphorylation and activation of MEK occur in response to the same mitogenic stimuli leading to ERK activation(2, 6, 33) , again providing indirect evidence of MEK's substrate specificity for ERK.
The crystal structure of the unphosphorylated form of ERK2 reveals that Thr-183 and Tyr-185 are contained within a loop structure(34) . In this inactivated form, Tyr-185 is buried in a hydrophobic pocket and Thr-183 is exposed to the surface of the molecule. Since the active form of ERK2 is phosphorylated on both Tyr-185 and Thr-183, ERK2 must undergo a conformational change in this loop structure upon association with MEK. Although the exact mechanism of the MEK/ERK interaction is unknown it is highly likely that some feature of the looped structure is important for determining MEK specificity. Thus, it is quite probable that the amino acid residues contained within this phosphorylation lip contribute to both the conformational mobility of ERK and the specificity of ERK recognition by MEK.
In this study, we have performed numerous site-directed mutations in both regions flanking the regulatory phosphorylation sites of ERK1 to further define MEK's specificity for its substrate, ERK. The ERK mutants were tested for differences in phosphorylation and activation by GST-MEK1 or GST-MEK2. Results in this paper indicate that four classes of mutants have been obtained: the first exhibits dramatically decreased phosphorylation and activation, the second shows enhanced basal kinase activity of ERK, the third exhibits preferentially enhanced phosphorylation of tyrosine and substantially decreased phosphorylation of threonine, and the fourth revealed an increase in threonine phosphorylation and an increase in activation. Results in this paper further indicate that the specificity of MEK1 and MEK2 for their substrate ERK1 is rendered, in part, by the amino acid residues flanking the regulatory phosphorylation sites of ERK1. These flanking residues not only influence the overall effectiveness of ERK1 as a substrate of MEK1 and MEK2 but also affect the relative phosphorylation levels of Thr-202 versus Tyr-204.
Purification of MEKs was performed as stated above for the ERKs with the exception of the elimination of the thrombin cleavage procedure. Instead, GST-MEKs were eluted from the glutathione-agarose with 6 ml of Buffer C containing 5 mM glutathione. Samples were dialyzed with two exchanges of Buffer C. GST-MEKs were concentrated by a Centricon 30 according to the manufacturer's instructions. Purified proteins were frozen at -80 °C in 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 5 mM EDTA (pH 8.0).
ERK is activated by a dual specific kinase (MEK) by phosphorylation on specific threonine and tyrosine residues. The native form of ERK is the only substrate identified thus far for MEK(21) . This is based on the inability of MEK to phosphorylate various proteins or peptides known to act as substrates for either protein serine/threonine kinases or protein tyrosine kinases. Furthermore, two-dimensional phosphopeptide mapping in combination with mass spectrometry techniques has shown that, for ERK2, only the regulatory residues Thr-183 and Tyr-185 are phosphorylated upon activation by MEK (15) . The portion of the ERK2 sequence containing the phosphorylation sites is conserved in ERK1 and thus the same specificity is presumed to hold for residues Thr-202 and Tyr-204 of ERK1. The crystal structure of the unphosphorylated form of ERK2 reveals that Thr-183 and Tyr-185 are contained within a loop structure(34) . The Tyr-185 is buried in a hydrophobic pocket, and Thr-183 is exposed to the surface of the molecule. Since ERK2 is more highly phosphorylated on Tyr-185 it has been suggested that the protein must undergo both local and global conformational changes in order for phosphorylation of this residue to occur upon association with MEK(34) . Thus, it is conceivable that the amino acid residues flanking the regulatory phosphorylation sites in both ERK1 and ERK2 may contribute to interactions that are vital for their structural mobility. Furthermore, it is plausible to speculate that these flanking residues of the regulatory phosphorylation sites may contribute to the specificity of ERK activation by MEK. We have undertaken mutation assays of ERK1 to investigate this hypothesis.
Recombinant MEK proteins were overexpressed as GST fusion proteins (GST-MEK1 or GST-MEK2) and purified by glutathione-agarose affinity chromatography for use in ERK activation studies. Since both MEKs have molecular weights similar to that of ERK1, MEKs were not cleaved with thrombin but were purified as GST fusion proteins. This aided in separation on SDS-PAGE, as GST-MEK1 or GST-MEK2 migrated at molecular masses of 69 and 70 kDa, respectively(37) .
Figure 1: Diagram of the mutations performed in human ERK1. Site-directed mutagenesis was performed on the conserved TEY sequence and both flanking sequences as illustrated. Kinase-impaired mutants contain the conserved lysine (Lys-71) substituted by an arginine (ERK1*). The phosphorylation target threonine and tyrosine residues are denoted by asterisks.
ERK1* mutants were assayed for in vitro phosphorylation by GST-MEK1 or GST-MEK2. In vitro kinase assays were performed on catalytically active ERK1 mutants to determine whether this activation by GST-MEK1 or GST-MEK2 correlated with ERK1* phosphorylation. Mutants that revealed interesting phosphorylation and activation results were further analyzed by phosphoamino analysis.
Figure 2:
Phosphoamino acid analysis of ERK1*
mutants. Phosphorylated ERK1* mutants were resolved on SDS-PAGE and
transferred to an Immobilon-P membrane. P incorporated
bands were excised and hydrolyzed in 6 N HCl for 2 h at 110
°C. Phosphoamino acid analysis was performed by separation using
one-dimensional electrophoresis on cellulose plates (Kodak) and
visualized by autoradiography. Phosphothreonine, phosphotyrosine,
phosphoserine, origin, and unincorporated phosphate are indicated by pT, pY, pS, O, and Pi,
respectively. Lanes correspond to the following proteins: lane 1, ERK1*; lane 2, ERK1*Y204S; lane 3,
ERK1*T202S; and lane 4, ERK1*Y204F. The weak spot between
P
and phosphotyrosine in ERK1*T202S did not co-migrate with
the phosphoserine standard.
To determine whether the high substrate specificity of MEK2 is influenced by residues flanking the phosphorylation sites of ERK1, each amino acid in both flanking regions was initially substituted with an alanine residue. In general, residues in closer proximity to the phosphorylation sites have a more prominent effect than distal residues (Fig. 3). The only exception to this was mutant W209A, which had less than 30% phosphorylation. Mutants L201A and V205A showed less than 50% phosphorylation; residues F200A, E203Q, and Y204F had less than 25% phosphorylation. Interestingly, mutation of residue Arg-208 to alanine dramatically increased the phosphorylation of ERK1* by MEK2 approximately 4-fold. These results indicated that residues flanking Thr-202 and Tyr-204 have a significant influence on the ability of ERK1 to be phosphorylated by MEK2. However, to determine the specific features of this flanking region important for substrate recognition, further mutation analyses were performed on selected residues.
Figure 3:
In vitro phosphorylation of ERK1*
mutants in the presence of GST-MEK2. Catalytically impaired ERK1*
mutants (0.3 µg) and GST-MEK2 (0.3 µg) in buffer containing 18
mM Hepes (pH 7.5), 20 µM ATP, and 10 mM magnesium acetate were incubated for 30 min at 30 °C in the
presence of [-
P]ATP. Samples were analyzed
by SDS-PAGE, transferred to Immobilon-P, and visualized by
autoradiography. Bands were quantitated by a Molecular Dynamics, Inc.
PhosphorImager. The percent of phosphorylation for ERK1*R208A was 406
± 131. Error bars denote standard deviations of two to
four experiments.
Mutation of the glutamic acid at position 203 to a glutamine (E203Q) was performed to determine what effect the negative charge at this position has on the ability of MEK to phosphorylate human ERK1. In addition, residue Glu-203 was mutated to an aspartic acid (E203D) to determine the effects of side chain length. ERK1*E203Q exhibited a 4-fold decrease in the level of phosphorylation, and even the more conservative mutation, E203D, showed a similar 4-fold decrease in phosphorylation. The dramatic effect of this relatively minor change would seem to indicate a pivotal role for Glu-203 in ERK1 phosphorylation beyond simply providing a negatively charged center. This central amino acid of the dual phosphorylation site motif is not fully conserved among the MAPK family members. In the ERKs and yeast Saccharomyces cerevisiae proteins, encoded by the genes KSS1, FUS3, and MPK1, the motif is TEY (40, 41) . However, this site differs in the stress-activated MAPKs, HOG1 and p38 (TGY) and JNK (TPY) (42, 43, 44) . With the regulatory phosphorylation sites being invariant among the MAPK family members, it is of interest that the residue in between these two sites is not also highly conserved. It may be that this residue is at least partly responsible for conferring specificity for the various MEK isoforms.
Due to the dramatic 4-fold decrease in the phosphorylation level of ERK1*F200A it was decided to make an additional conservative mutation of Phe-200 to a tyrosine in order to assess any role of the aromatic side chain in this position. The ERK1*F200Y mutant also exhibited a 3-fold decrease in the level of phosphorylation. The role of Phe-200 is also unclear, but it may be involved in the conformational integrity of ERK1 enabling Thr-202 and Tyr-204 to be accessible for phosphorylation by MEK.
As expected, mutation of either Thr-202 or Tyr-204 eliminated the activation of ERK1 by MEK2. The phosphorylation level of T202S was nearly equal to that of ERK1*; however, it was not an effective substrate for activation by MEK2. These results were consistent with the 6-fold higher level of Tyr-204 versus Thr-202 phosphorylation in wild type ERK1. That is, threonine phosphorylation represents only about 14% of the total ERK1 phosphorylation and will therefore have little effect on the overall phosphorylation level of the protein. However, as phosphorylation of both Thr-202 and Tyr-204 is necessary for activation, this lack of threonine phosphorylation results in a marked decrease in activation for the T202S mutant protein. The comparable substitutions of Ser-218 and Ser-222 of MEK1 by threonine did not eliminate phosphorylation of MEK1 by Raf, indicating that MEK has a higher substrate specificity than Raf(30) .
In general, mutants revealed significantly decreased activation by MEK2 in correlation with decreased phosphorylation in vitro (Fig. 4). This attenuated activation was also generally more severe than the phosphorylation decrease. This may be explained by the fact that ERK must adopt an altered conformation for MEK to bind, leading to subsequent phosphorylation of both tyrosine and threonine. Thus, it may be possible that even though phosphorylation was detected, indicating interaction with MEK, the ERK1 mutants may not be able to adopt some optimal conformation necessary for its full activity. However, some notable exceptions were observed as described below.
Figure 4:
Activation of ERK1 mutants by GST-MEK2. To
activate ERK1, recombinant GST-MEK2 was incubated for 20 min at 30
°C in a 30-µl reaction volume containing 18 mM Hepes
(pH 7.5), 20 µM ATP, and 10 mM magnesium acetate.
To initiate the kinase reaction, myelin basic protein and
[-
P]ATP (5000 cpm/pmol) were added in the
same reaction buffer and incubated for an additional 30 min at 30
°C. Half of the reaction volume (20 µl) was applied to P81
phosphocellulose filters. The filters were washed with 180 mM phosphoric acid (5 times) and rinsed with 95% ethanol. Filters
were quantitated by liquid scintillation counting. Each sample was
performed in duplicate; error bars denote the standard
deviation of two to four experiments.
Figure 5:
Increased threonine phosphorylation in
ERK1*G199A and decreased threonine phosphorylation in ERK1*T207A and
ERK1*R208A mutants. Phosphorylated ERK1* mutants were resolved on
SDS-PAGE and transferred to an Immobilon-P membrane. P
incorporated bands were excised and hydrolyzed in 6 N HCl for
2 h at 110 °C. Phosphoamino acid analysis was performed by
separation using one-dimensional electrophoresis on cellulose plates
(Kodak) and visualized by autoradiography. Phosphothreonine,
phosphotyrosine, phosphoserine, origin and unincorporated phosphate are
indicated by pT, pY, pS, O, and Pi, respectively. Lanes correspond to the following
proteins: A) lane 1, ERK1*G199A, lane 2,
ERK1*; and B) lane 1, ERK1*, lane 2,
ERK1*T207A, and lane 3, ERK1*R208A. Similar results were
obtained in duplicate experiments.
To determine whether mutations at the phosphorylation sites and flanking residues of ERK1 have the same effect on both MEK1 and MEK2, ERK1 mutants were assayed for phosphorylation and activation by MEK1 as described previously for MEK2. The phosphorylation results obtained were similar to those for MEK2 (Fig. 6); mutation of the flanking residues generally decreased the phosphorylation of ERK1* by MEK1. These data suggest that a common sequence motif in ERK is recognized by both MEK1 and MEK2; however, noticeable differences were observed. For example, the ERK1*R208A mutant showed an increase in phosphorylation by MEK2 of 4-fold as compared with 2-fold for MEK1.
Figure 6:
In vitro phosphorylation of ERK1*
mutants in the presence of GST-MEK1. Catalytically impaired ERK1*
mutants (0.3 µg) and GST-MEK1 (0.3 µg) in buffer containing 18
mM Hepes (pH 7.5), 20 µM ATP, and 10 mM magnesium acetate were incubated for 30 min at 30 °C in the
presence of [-
P]ATP. Samples were analyzed
by SDS-PAGE, transferred to Immobilon-P, and visualized by
autoradiography. Bands were quantitated by a Molecular Dynamics, Inc.
PhosphorImager. The percent of phosphorylation for ERK1*R208A was 248
± 79. Error bars denote standard deviations of two to
eight experiments.
Activation of ERK1 mutants by MEK1 was also comparable with that by MEK2 (Fig. 7). Mutation of flanking residues dramatically reduced the activation potential of ERK1 by both MEK1 and MEK2. Among all the mutants, G199A was the only one to exhibit less than a 70% decrease in activation by MEK1.
Figure 7:
Activation of ERK1 mutants by GST-MEK1. To
activate ERK1, recombinant GST-MEK1 was incubated for 20 min at 30
°C in a 30-µl reaction volume containing 18 mM Hepes
(pH 7.5), 20 µM ATP, and 10 mM magnesium acetate.
To initiate the kinase reaction myelin basic protein and
[-
P]ATP (5000 cpm/pmol) were added in the
same reaction buffer and incubated for an additional 30 min at 30
°C. Half of the reaction volume (20 µl) was applied to P81
phosphocellulose filters. The filters were washed with 180 mM phosphoric acid (5 times) and rinsed with 95% ethanol. Filters
were quantitated by liquid scintillation counting. Each sample was
performed in duplicate; error bars denote the standard
deviation of two to three experiments.
Activation of MAPKs is a common event in many signal transduction pathways. At least six identified pathways in the budding yeast S. cerevisiae utilize different MAPK family members for their signal transduction(50) . These pathways have very unique functions, which include the mating pheromone response, osmolarity regulation, cell wall construction, pseudohyphal development and spore formation in diploid strains, and invasiveness in haploid strains(50) . The number of MAPKs and their upstream activators involved in such a variety of vital functions of the organism raises important questions on how the different isoforms of MAPK are regulated. The results of the mutation experiments presented in this paper provide additional information toward understanding the mechanisms of MAPK activation and the selectivity of MEK. Furthermore, these data will provide valuable insight toward the complete interpretation of the crystal structures of the tyrosine-phosphorylated and dual phosphorylated MAPK in terms of the mechanisms of MAPK dual phosphorylation. Understanding the mechanisms of activation for the different isoforms of MAPK and the specificity of the various activators will indeed lead to an improved general understanding of the mechanisms of regulation involved in the cell signaling pathways in mammalian systems.