(Received for publication, September 1, 1995; and in revised form, November 27, 1995)
From the
Mitogen-activated protein/ERK kinase kinases (MEKKs)
phosphorylate and activate protein kinases which in turn phosphorylate
and activate the p42/44 mitogen-activated protein kinase (MAPK),
c-Jun/stress-activated protein kinases (JNKs), and p38/Hog1 kinase. We
have isolated the cDNAs for two novel mammalian MEKKs (MEKK 2 and 3).
MEKK 2 and 3 encode proteins of 69.7 and 71 kDa, respectively. The
kinase domains encoded in the COOH-terminal moiety are 94% conserved;
the NH-terminal moieties are approximately 65% homologous,
suggesting this region may encode sequences conferring differential
regulation of the two kinases. Expression of MEKK 2 or 3 in HEK293
cells results in activation of p42/44
and JNK but not of
p38/Hog1 kinase. Immunoprecipitated MEKK 2 phosphorylated the MAP
kinase kinases, MEK 1, and JNK kinase. Titration of MEKK 2 and 3
expression in transfection assays indicated that MEKK 2 preferentially
activated JNK while MEKK 3 preferentially activated
p42/44
. These findings define a family of MEKK proteins
capable of regulating sequential protein kinase pathways involving MAPK
members.
A variety of extracellular signals including growth factors,
hormones, cytokines, antigens, and stresses such as heat shock and
osmotic imbalance activate members of the mitogen-activated protein
kinase (MAPK) ()family(1, 2, 3, 4, 5) .
MAPKs are characterized as serine/threonine-protein kinases activated
by dual phosphorylation on both a tyrosine and a threonine(6) .
The MAPK family includes p42/44
(also referred to as
ERK2 and -1)(7) , the c-Jun kinases (JNKs which are also
referred to as stress-activated protein kinases)(8) , and p38,
the osmotic imbalance responsive kinase similar to the yeast Hog1
enzyme(5) . The regulation of different MAPKs including
p42/44
, JNKs, and p38 involves sequential protein kinase
pathways whose upstream activators include the MEKs (MAPK/ERK kinases)
and the JNK kinases (also referred to as SEKs or stress/ERK
kinases)(9, 10, 11, 12, 13) .
MEKs and JNK kinases (JNKKs) phosphorylate specific MAPK family members
on both a tyrosine and threonine resulting in MAPK activation.
Raf-1 and B-Raf are serine/threonine-protein kinases that selectively phosphorylate and activate MEK 1 and MEK 2(14, 15, 16, 17) . Recently, we isolated the cDNA for a novel serine/threonine-protein kinase referred to as MEK kinase (MEKK 1) that phosphorylates and activates MEK 1 and 2 and JNKKs(13, 18, 19, 20) . The catalytic domain of MEKK 1 is homologous to the kinase domains of the Ste11 and Byr2 serine/threonine-protein kinases, involved in the control of mating in Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively(21, 22, 23) . In this report, we have isolated and expressed the cDNAs for two new MEKK proteins. MEKK 2 and 3, when expressed in cells, are similar to MEKK 1 in that they are capable of regulating MEK and JNKK activities. Thus, there exists a family of MEKKs controlling sequential protein kinase systems involving MAPK members in addition to the Raf family of protein kinases.
Figure 5:
Selective regulation of p42/44 and JNK by MEKK 2 and 3. HEK293 cells were transfected with 5, 1,
0.25, 0.05, and 0.01 µg of plasmid DNA encoding either MEKK 2 or 3.
Cells were harvested 48 h post-transfection and assayed for both JNK
and p42/44
activity. The results are representative of
two independent experiments for both MEKK 2 and 3. B, MEKK 2
phosphorylation of JNKK stimulates JNK kinase activity. MEKK 2
immunoprecipitates were incubated with the indicated combinations of
wild-type or kinase-inactive JNKK and JNK as described under
``Materials and Methods. Gst-c-Jun
phosphorylation by JNK was used as a measure for activation of
the JNKK/JNK pathway.
To demonstrate MEKK activation of JNKK activity, the in vitro kinase reactions were performed with different combinations of
recombinant wild type or kinase-inactive JNKK (lysine 116 mutated to
methionine) and wild type or kinase-inactive JNK. Kinase-inactive JNK
was made by mutating the active site lysine 55 to methionine (provided
by Dr. Matt Jarpe). Incubations were for 30 min at 30 °C in the
presence of 50 µM ATP.
GST-c-Jun-Sepharose beads were then added, and
the mixture was rotated at 4 °C for 30 min. The beads were washed,
suspended in 40 µl of c-Jun kinase assay buffer containing 20
µCi of [
-
P]ATP, and incubated for 15
min at 30 °C. Reaction mixtures were added to Laemmli sample
buffer, boiled, and phosphorylated proteins were resolved on SDS-10%
polyacrylamide gels.
Figure 1: DNA and deduced amino acid sequences for MEKK 2 and 3. A, MEKK 2; B, MEKK 3. In-frame stop codons 5` to the predicted start methionine are underlined.
The 5` ends of both MEKK 2 and 3 are highly G/C-rich making DNA sequencing difficult. To verify the presence of stop codons in all three possible reading frames 5` to the predicted start site methionine, the MEKK 2 and 3 cDNAs were inserted in pRSET A, B, and C (Invitrogen) and expressed in Escherichia coli (not shown). Each construct gave a truncated RSET fragment confirming that the MEKK 2 and 3 cDNAs encoded 5` stop sites and that the isolated cDNAs encode full-length proteins.
Alignment of the deduced amino
acid sequences demonstrated significant homology between the two
proteins (Fig. 2A). Overall, the two proteins are
approximately 77% homologous. The catalytic domain is encoded in the
COOH-terminal moiety of both MEKK 2 and 3. The first consensus kinase
domain (32) comprising the catalytic site of MEKK 2 and 3
begins at residues 361 and 367, respectively. The COOH-terminal
catalytic domains of MEKK 2 and 3 are approximately 94% conserved,
whereas the NH-terminal moieties are only 65% conserved in
amino acid sequence. These findings indicate that the primary sequences
of MEKK 2 and 3 diverge significantly in the NH
-terminal
half of the proteins. The conservation in sequence of the catalytic
domains suggests they may recognize an overlapping set of substrates.
The divergent NH
termini would be consistent with this
region encoding sequences for the differential regulation of the two
proteins.
Figure 2: Comparison of amino acid sequences for MEKK 2 and 3. A, amino acids not having homology are boxed in the alignment of MEKK 2 and 3. B, alignment of the catalytic domains for MEKK 1, 2, and 3. Roman numerals indicate the 11 conserved regions within the protein kinase catalytic domain, with the most highly conserved residues underlined(32) . Lowercase letters represent nonconserved amino acids in one or more of the MEKK sequences.
Fig. 2B shows the alignment of MEKK 1, 2,
and 3 catalytic domains. The 11 conserved subdomains comprising the
protein kinase catalytic domain are designated by Roman numerals. The
COOH terminus of MEKK 1 encoding the catalytic domain is only 50%
homologous to the corresponding regions of MEKK 2 and 3. Thus, the
catalytic domains of MEKK 2 and 3 are very similar to each other but
significantly divergent from MEKK 1. As shown below, MEKK 1, 2, and 3
can all stimulate JNK and p42/44 activities in
transfected cells. The significance of the sequence differences in the
catalytic domains of MEKK 1, 2, and 3 is presently unclear.
Figure 3: Stimulation of JNK activity in MEKK 2 and 3 transfected HEK293 cells. Cells were harvested 48 h post-transfection and assayed for GST-c-Jun phosphorylating activity by mixing of lysates with a slurry of GST-c-Jun-Sepharose (A). Alternatively, lysates were fractionated using a 0-0.5 M NaCl linear gradient on a Mono Q ion exchange column before assay with GST-c-Jun-Sepharose as substrate (B).
Transient expression of MEKK 2 and 3
also stimulated p42/44 activity (Fig. 4).
Immunoblotting of hemagglutinin (HA) epitope-tagged MEKK 2 and 3
indicated that MEKK 2 and 3 were expressed at similar levels in HEK293
cells when 2 µg of plasmid DNA was used per transfection (not
shown). To determine whether MEKK 2 and 3 demonstrated selectivity in
activating the JNK and p42/44
pathways, plasmid DNAs
were titrated over a range of concentrations in the transfections. Fig. 5shows that MEKK 2 has a greater selectivity for
stimulation of the JNK pathway. In contrast, MEKK 3 had a greater
selectivity for activating p42/44
relative to JNK. Thus,
even though the kinase domains are approximately 94% conserved, MEKK 2
and 3 differ in their selectivity for regulation of the JNK and
p42/44
pathways. This was particularly evident for MEKK
3 at low plasmid concentrations where the p42/44
pathway
was preferentially activated.
Figure 4:
MAPK activity in HEK293 cells transfected
with MEKK 2 and 3. Cells were harvested 48 h post-transfection, and
lysates were fractionated using a linear 0-0.5 M NaCl
gradient on a Mono Q ion exchange column. p42/44 activity was assayed as described under ``Materials and
Methods'' using the EGF receptor 662-681 peptide as
substrate.
Figure 6:
Phosphorylation of recombinant MEK 1 and
JNKK by immunoprecipitated MEKK 2. A, HEK293 cells were
transfected with pCMV5 alone(-) or encoding HA epitope-tagged
MEKK 2 or 3. Forty-eight h post-transfection, cells were lysed, and
MEKK 2 and 3 were immunoprecipitated using the 12CA5 monoclonal
antibody. Immunoprecipitates were then assayed for kinase activity
using recombinant kinase-inactive MEK 1 or JNKK as substrate. The JNKK
only lane shows the low level of autophosphorylation of recombinant
JNKK. Results are representative of 5-6 experiments for both MEKK
2 and 3. B, MEKK 2 phosphorylation of JNKK stimulates JNK
kinase activity. MEKK 2 immunoprecipitates were incubated with the
indicated combinations of wild-type or kinase-inactive JNKK and JNK as
described under ''Materials and Methods.
Gst-c-Jun phosphorylation by JNK was used as a
measure for activation of the JNKK/JNK
pathway.
Figure 7: Measurement of p38 kinase activation in MEKK 2 and 3 transfected cells. A, stimulation of p38 kinase activity in response to sorbitol. HEK293 cells were incubated for 20 min with 0.4 M sorbitol. Cells were then lysed, and p38 was immunoprecipitated using a rabbit antisera raised against a COOH-terminal peptide sequence of p38. Recombinant ATF 2 was used in an in vitro kinase assay as a substrate for p38 as described under ``Materials and Methods.'' The results are representative of two independent experiments. B, HEK293 cells were transfected with pCMV5 plasmids encoding no cDNA or MEKK 2 or 3. Lysates were prepared 48 h post-transfection and fractionated using a 0-0.5 M NaCl gradient on a Mono Q ion exchange column. Fractions were assayed for kinase activity using recombinant ATF 2 as substrate. JNK and p38 were identified in the column fractions by immunoblotting using specific antibodies for JNK and p38 (not shown).
The cloning and characterization of MEKK 2 and 3 define a
family of MEKK proteins. MEKK 1, 2, and 3 are all capable of regulating
both p42/44 and JNK activities. MEKK 1 and 2 appear to
preferentially regulate the JNK pathway, whereas MEKK 3 shows a
preference for activation of the p42/44
pathway in
vivo. Cumulatively, our current and previous results (19) indicate that the different MEKKs when transiently
expressed do not display a high selectivity for the p42/44
or JNK regulatory pathways. At more modest levels of expression,
a greater degree of selectivity is observed for MEKK regulation of
sequential protein kinase pathways. In MEKK 1-inducible clones of
NIH3T3 (34) and Swiss 3T3 (35) cells, JNK is
preferentially activated relative to p42/44
. MEKK 1, 2,
and 3 do not measurably activate the p38 kinase pathway in these cell
types.
Based on the biochemical characterization of MEKK proteins,
it is evident that their activities are quite distinct from those of
Raf-1 and B-Raf kinases. The Raf kinases selectively regulate MEK 1 and
2 and do not recognize the JNKK
proteins(1, 14, 20, 33, 34, 35) .
Thus, Raf proteins which evolved in metazoan organisms appear to be
highly selective for the regulation of p42/44 pathways(1, 8) . At present, no distinct
pathways or substrates other than MEK and p42/44
have
been defined for Raf kinases, although it is probable that additional
MEK-like kinases will be identified in the future that serve as Raf
substrates. MEKK proteins, in contrast, are capable of regulating both
JNK and p42/44
pathways.
The ability of MEKKs to
regulate multiple sequential protein kinase pathways in the cell
suggests that a different mechanism exists for their regulation
compared to the Raf kinases. The simplest prediction would be that MEKK
proteins are selectively organized in ``signalsome''
complexes much like that for the mating pathway in S.
cerevisiae. In this pathway, the protein kinases Ste11
(MEKK-like), Ste7 (MEK-like), and Fus3 (MAPK-like) are held together in
a high affinity complex by Ste5(36, 37, 38) .
Ste5 functions as a scaffold to keep these proteins in an organized
complex. Expression of gain-of-function Ste11 mutants can result in
overcoming a threshold where Ste11 crosses over to activate another
sequential protein kinase pathway such as that involved in
morphogenesis(21, 22, 23) . No Ste5
equivalent has yet been reported for mammalian cells and MEKKs. If such
scaffold-like proteins do exist in mammalian cells and their expression
is limiting, it would explain the ability of transiently expressed
MEKKs to regulate both JNK and p42/44 pathways. It will
obviously be necessary to define the organization of potential MEKK
signalsome complexes in the cell and the constituent kinases in each
complex.
Finally, the MEKK/JNK pathways can be activated by a diverse set of stimuli. These include cytokines such as TNF and IL-1(39) , low molecular weight GTP-binding regulatory proteins including Ras, Rac, and Cdc42(40, 41) , high intracellular calcium(42) , and stresses such as ultraviolet light, heat shock, osmotic imbalance, sphingomyelinase activity, protein synthesis inhibitors, etc.(1, 8, 13, 14, 20) . Based on this array of stimuli capable of activating JNK, it is likely that several independent pathways converge to regulate the JNK sequential protein kinase pathway. It is possible that MEKK 1, 2, and 3 may all regulate the JNK pathway and each functions to respond to different upstream inputs. Alternatively, it is possible that MEKK 1, 2 and 3 are not only capable of regulating the JNK pathway but also other sequential protein kinase pathways as well. Such a mechanism would allow co-ordinate regulation of both the JNK pathway and additional parallel sequential protein kinase pathways in the cell. In support of this hypothesis, we have found that MEKK 1 also selectively regulates a protein kinase pathway leading to the phosphorylation and transactivation of c-Myc that is independent of JNK and c-Jun(35) . The magnitude of a specific MEKK activation in response to a stimulus could selectively regulate different pathways such as those for JNK and c-Myc kinase activity by requiring different thresholds of MEKK activity to be obtained for stimulation of each pathway. The thresholds for MEKK regulation of these pathways might be regulated in part by the relative abundance or cellular localization of specific signalsome complexes. The cloning of MEKK 2 and 3 allows us to now address these potential regulatory mechanisms for the three MEKK proteins using both genetic and biochemical approaches.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U43186 [GenBank](MEKK 2) and U43187 [GenBank](MEKK 3).