(Received for publication, November 6, 1995; and in revised form, December 5, 1995)
From the
Mitogen-activated protein (MAP) kinases require dual phosphorylation on threonine and tyrosine residues in order to gain enzymatic activity. This activation is carried out by a family of enzymes known as MAP kinase kinases (MKKs or MEKs). It appears that there are at least four subgroups in this family; MEK1/MEK2 subgroup that activates ERK1/ERK2, MEK5 that activates ERK5/BMK1, MKK3 that activates p38, and MKK4 that activates p38 and Jun kinase. Here we describe the characteristics of a new MKK termed MKK6. The clones we isolated encode two splice isoforms of human MKK6 comprised of 278 and 334 amino acids, respectively, and one murine MKK6 with 237 amino acids. Sequence information derived from cDNA cloning indicated that MKK6 is most closely related to MKK3. The functional data revealed from co-transfection assays suggests that MKK6, like MKK3, selectively phosphorylates p38. Unlike the previously described MKKs (or MEKs), MKK6 exists in a variety of alternatively spliced isoforms with distinct patterns of tissue expression. This suggests novel mechanisms regulating activation and/or function of various forms of MKK6.
The signal transduction pathways that utilize mitogen-activated
protein (MAP) ()kinases have an important role in a variety
of cellular responses including growth factor-induced proliferation,
gene expression, and compensation for alterations in the extracellular
milleau induced by heat shock, UV light, increased extracellular
osmolarity etc.(1, 2, 3, 4) . Four
MAP kinase pathways have been defined in yeast(5) ; these
pathways are functionally independent and are regulated by distinct
protein kinase cascades. Each pathway results in activation of a
separate MAPK by a unique MAPK kinase (MKK or MEK). It is now clear
that higher eukaryotes also have distinct MAP kinase pathways; such
pathways are comprised of kinase cascades leading to the activation of
discrete MKKs and
MAPKs(5, 6, 7, 8, 9) .
Specifically four subgroups of MAPKs have been identified(5) :
the ERKs (extracellular signal-regulated
kinase)(10, 11) , Jun kinases (c-Jun amino-terminal
kinases (JNK) or stress-activated protein kinase
(SAPK))(12, 13) , p38 MAP kinase(14) , and
BMK1/Erk5(15, 16) . The signal transduction pathway
leading to p38 activation is related, in part, to a pathway in yeast
leading to activation of a MAP kinase known as Hog1p. To date the
activation of this yeast pathway has been shown to occur principally in
response to increased extracellular osmolarity(17) . Recently
Saito and colleagues (18, 19) have defined two
distinct pathways leading to Hog1p activation in Saccharomyces
cerevisiae.
In mammalian cells p38, the Hog1p homologue is activated by multiple stimuli acting through different receptors. For example Lee et al.(20) showed that p38 is involved in bacterial endotoxin (lipopolysaccharide)-induced cytokine production through the use of pharmacologic inhibitors that are specific for p38(20) . p38 is also activated by other bacterial components, proinflammatory cytokines, and physical-chemical changes in the extracellular milleau(21) .
There have been several distinct MKKs/MEKs identified in mammalian cells; one type, termed MEK1/MEK2, does not phosphorylate or activate p38 or JNK while in contrast is a strong activator of ERK1/ERK2(7, 9, 22) . In contrast, two other MKKs known as MKK3 and MKK4 activate p38 but not ERK(6, 7) ; MKK4 (also known as SEK1/JNKK1) also activates JNK/SAPK(6, 7, 8) . MKK3 and MKK4 are most closely related to PBS2p, the upstream activator of Hog1p(6, 7) .
p38 and JNK are often activated in parallel (21) but independent activation of p38 also has been observed(23) . Simultaneous activation of ERK and p38 also occurs when cells are exposed to a stimulus such as lipopolysaccharide or increased extracellular osmolarity(14, 24) . Unlike yeast MAP kinase pathways which appear to operate independently, cross-talk seems to occur between the mammalian MAP kinase pathways(6, 7, 25, 26) . Emerging data suggest that the MAPK signal transduction pathways in mammalian cells are much more complex than homologous systems in yeast. One level of such complexity might exist at the level of the MAPK kinases. Indeed multiple activators of p38 or JNK can be detected by fractionation of the fibroblasts activated by hyperosmolar media(27) . Thus there is a need to identify all of the p38 upstream activators in order to fully understand the regulation of p38. To isolate additional MKKs which function as activators of p38, we have employed a polymerase chain reaction (PCR)-based strategy with degenerate oligonucleotides based on conserved kinase domains present in the known members of the MKK family. This approach resulted in the isolation of two human cDNAs and one murine cDNA encoding closely related proteins of the MKK family. These clones are the different splice forms of one gene which we have designated MKK6, which is selectively expressed in different tissues. Amino acid sequence comparison revealed that the proteins encoded by these cDNAs are most closely related to MKK3, and co-transfection studies show that MKK6, like MKK3, activates p38 but fails to activate either ERK or JNK. In this article we also present data showing that unlike MKK4, MKK3 and MKK6 are not regulated by Rac or Cdc42.
Figure 1: Primary structure of human MKK6, MKK6b, and MuMKK6c obtained from cDNA cloning. A, the cDNA sequence and deduced amino acid sequence of MKK6 and MKK6b. The protein sequence are presented in single letter code. The first upstream in-frame stop-codon is underlined. B, sequence comparison of human MKK6 and MKK6b with the human MKKs. The identical and conserved amino acid sequences are boxed. The PILE-UP program (Wisconsin Genetics Computer Group) was used for the alignment; gaps were introduced into sequences to optimize alignments. C, the relation between members of the human MKK family is presented as a dendrogram created by the unweighted pair group method with the use of arithmetic averages (PILE-UP program). D, the cDNA sequence and deduced amino acid sequence of murine MKK6c. The nucleotides are numbered based on the alignment with human MKK6/6b.
We believe that the cDNAs for MKK6 and MKK6b contain full-coding regions because both clones contain in-frame stop codons 5` upstream of the first ATG sequence. The first in-frame ATG of MKK6 has been designated as position 1. Since it is the first ATG codon and the typical Kozak sequence, it should be the authentic start site for MKK6. The first ATG found in MKK6b is at position -168. Although not a typical Kozak sequence, translation of MKK6b mRNA most likely begins at this position. The murine clone (GenBank U93066, Fig. 1D) which is closely related to MKK6b has the latter ATG sequence but not an equivalent ATG at position 1. Thus we predict that MKK6 contains 278 amino acids while MKK6b contains 334 amino acid residues. Of interest are differences between MKK6 and MKK6b in DNA sequence before position -154; this may be due to differential splicing. A dendrogram created by progressive pairwise alignment comparison of the MKK family is shown in Fig. 1C.
When we compared the sequence of the murine cDNA clone we isolated with the sequence of human MKK6 or MKK6b we noted that the murine clone has a 62-base pair deletion covering the region from residues 16 to 77. Due to this deletion, the ATG at position 122 is most likely to be the starting codon. We predict that this murine clone contains 237 amino acids. Although at the DNA level, this murine clone is most closely related to human MKK6b, it has a completely different 5` sequence beginning with position -219 and a unique 3` sequence starting from position 1056 when compared with MKK6b. This may result from differential splicing, although additional studies are required to firmly establish this. On the basis of these differences we term this murine cDNA, murine MKK6c. The sequence of murine MKK6c is shown in Fig. 1D. Further investigation is required to determine whether a comparable form exists in human. Amino acid sequence comparison reveals that the murine MKK6c is 97.6% identical to human MKK6/6b in the overlapping region. Conservation of the amino acid sequence between species has been found in other MKKs and may indicate the importance of this family proteins(9, 22) .
Figure 2:
Tissue distribution of MKK6 mRNA detected
with different probes. A blot containing poly(A) RNA
isolated from various human tissues was hybridized with a probe
specific for the 5`-end of MKK6 (A), or the 5`-end of MKK6b/6c (B), or the region of the cDNA of all the splice forms of MKK
that displayed the highest degree of homology (C).
Figure 3:
MKK6/6b are p38 activators. COS-7 cells
were transfected with epitope-tagged p38, Erk1, or JNK1 together with
an expression vector encoding MKK3, MKK4, MKK6, MKK6b, or control DNA
represented by empty vector; or transfected with a signal expression
plasmid of MKK6 or MKK6 mutated at ATP binding site (M mutants),
phosphorylation sites (A mutant and E mutant), or empty vector. Some of
the cultured cells transfected with control empty plasmid were exposed
to UV radiation (50 J/m); p38, Erk1, or JNK1 were isolated
by immunoprecipitation with anti-epitope antibody. The protein kinase
activity was measured in the immunocomplex with
[
-
P]ATP and MBP (A) or GST-ATF2 (B) as substrate. The coupled kinase assay was used to test
the kinase activity of MKK6 mutants by including recombinant p38 and
MBP in the kinase reaction (C). The product of the
phosphorylation reactions was visualized after SDS-PAGE by
autoradiography.
Sequence
comparison of MKK6 with other MKKs suggested that Ser and
Thr
may be phosphorylation sites required for enzymatic
activity. To investigate this we modified the MKK6 cDNA by replacing
Ser
and Thr
with Ala (A mutant) or Glu (E
mutant). An additional mutant was made by changing Lys
to
Met to delete the ATP binding site (M mutant). Epitope-tagged versions
of these mutants were made by adding an HA-tag to the amino-terminal of
MKK6; the proteins encoded by these cDNAs were expressed in COS-7
cells. HA-tagged proteins were immunoprecipitated using anti-HA
monoclonal antibody 12CA5. The immunoprecipitates were used in a
coupled MKK assay to determine the activity of MKK6 or its mutants by
including recombinant His-p38 and MBP in the kinase reaction mixture (Fig. 3C). All three mutants fail to activate p38;
previous studies with MEK where A and M mutants were created also
resulted in a loss of activity(33) . Here the E mutant also
failed to activate p38 while in contrast, the analogous structural
change in MEK produced an active enzyme(33) .
Figure 4:
Regulation of the activation of MKK3,
MKK4, and MKK6. COS-7 cells were transfected with epitope-tagged MKK6,
MKK4, or MKK3 together with empty expression vector or an expression
vector encoding active-form MEKK1, Rac1, Cdc42, Ras, RhoA, and Raf.
Some of the cell cultures were exposed to UV radiation (50
J/m). The MKK6, MKK4, or MKK3 was isolated by
immunoprecipitation with the use of anti-epitope antibody. The kinase
activity was measured in the immunocomplex with
[
-
P]ATP and recombinant p38 as substrate.
The product of the phosphorylation reactions was visualized after
SDS-PAGE by autoradiography.
Herein we describe the properties of a newly identified member of a family of enzymes known as MAP kinase kinases (MKK). Nucleotide sequence data indicates that the human cDNA clones we isolated encode two proteins generated from one gene; the distinct mRNA species are produced through alternative splicing from a single gene encoding a MAP kinase kinase, here termed MKK6. There is tissue-specific expression of the isoforms of MKK6. Functional studies indicate that MKK6 can activate p38 but not ERK or JNK.
In contrast to the previously described members of this family, which are expressed in almost all tissues with a single transcript at the mRNA level(7, 8, 9, 22) , numerous splice variants of this gene exist. One splice form, MKK6, appears restricted to expression in skeletal muscle. In contrast, MKK6b is present in various forms in multiple tissues. Thus this represents the first report suggesting there are tissue-specific isoforms of the MKK family. Unique functions for tissue-specific splice forms of MKK6 is not provided by our work. However, demonstration of the presence of such forms suggests the possibility of regulation that is tissue and/or cell specific.
Here we also determined that Ser and
Thr
are likely to be important phosphorylation sites
since mutation of these residues to alanine prevented activation of
MKK6. This region is analogous to one established to be important by
others for MEK(33) . We also attempted to replace Ser
and Thr
with Glu to simulate the negative charge
resulting from phosphorylation. This approach did result in a
gain-of-function mutation for MEK(33) . Here we failed to
observe a similar effect. This may be due to the fact that the
phosphorylation sites of MEK are both Ser while in MKK6 the site is
determined by a Ser and Thr. The Glu may not mimic phosphothreonine in
the same way that it does phosphoserine.
Insofar as the MAP kinase family is concerned it was not surprising that MKK6 and MKK3 are quite similar in substrate specificity. MKK6 and MKK3 can be classified into a subgroup distinct from others members of the MKK family. This is justified based on sequence homologies and ability to activate p38 without activating ERK or JNK. Moreover here we showed that regulators of MKK4, namely Rac1 and Cdc42, apparently do not control activation of MKK6. Thus there may be two distinct pathways leading to p38 activation; one via MKK6/3 and another involving MKK4. Interestingly the studies of Saito and colleagues (18, 19) with S. cerevisiae revealed two distinct pathways leading to Hog1p activation. One involves a two-component histidine kinase system leading to activation of the MKK encoded by the PBS2 gene (18) . In contrast, an alternative pathway leading to PBS2p activation was discovered involving a membrane protein termed Sho1 that directly activates PBS2 through SH3 interactions(19) .
Given the distinct expression patterns of MKK6, it is likely that this MKK acts as a regulator of p38 activation depending on expression in a given tissue and/or cell type. A future challenge will be to define the specific function of individual p38 activators and how these proteins interact with other components of the signal machinery to transduce extracellular information into cellular responses. Gene targeting of individual MKKs in cultured cells and mice should shed light on this issue.
We suggest that multiple signaling pathways can control activation of p38. A model which takes into account the data presented here as well as the findings of others (7, 8, 26, 34, 35) is provided in Fig. 5. Investigations that may lead to the elucidation of alternative pathways for p38 activation via MKK3 and/or MKK6 are currently underway.
Figure 5: Proposed signaling pathways for activation of p38.