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Department of Immunology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030,1 Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, Texas 752352
Received 19 December 2004/ Returned for modification 31 January 2005/ Accepted 26 April 2005
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ABSTRACT |
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INTRODUCTION |
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Despite extensive efforts in the past, it is still largely unknown how each MAP3K is activated and which upstream molecules activate each MAP3K at the molecular level. One major hurdle preventing us from understanding these processes is that when expressed in transient transfection, most of the MAP3Ks become constitutively activated in the absence of any agonist stimuli toward their downstream MAPKs, such as the JNKs, ERKs, and p38. To complicate the matter further, overexpression of many MAP3Ks could also activate the I
B kinase-NF-B pathway in addition to the MAPK cascades (20, 42). These transfection studies suggest a likely possibility that these protein kinases are negatively regulated by inhibitors or through intracellular compartmentalization under normal conditions to maintain their inactive status. Yet, direct evidence supporting this hypothesis remains to be shown. In addition, it is still unclear whether these kinases require an activating kinase (an MAP3K kinase) for their activation in a mammalian system. If not, how are these MAP3Ks activated once the cells are stimulated?
MEKK2 is a Ser/Thr protein kinase belonging to the MEKK/STE11 subgroup of the MAP3K family (1, 30). MEKK2 is expressed in multiple tissues (1, 30), but its physiological functions in different tissues are largely unstudied. Ample in vitro studies show that MEKK2 activates the JNK1/2, ERK1/2, p38, and ERK5 MAPKs (1, 5, 9, 14, 26). The strong and specific JNK activation by MEKK2 is mediated by JNK-activating kinase 2 (JNKK2) through the formation of a tripartite molecular complex consisting of MEKK2, JNKK2, and JNK1 (5). Activation of the ERK1/2, p38, and ERK5 MAPKs by MEKK2 is mediated by MEK1/2, MKK3/6, and MEK5, respectively (1, 4, 9). MEKK2 has been suggested to play a role in T-cell receptor signaling and may participate in immune synapse formation during antigenic stimulation (26, 30). MEKK2-deficient T cells were shown to hyperproliferate suggesting a negative role for MEKK2 in T-cell receptor signaling (16). Consistent with this finding, T cells deficient in JNKK2/MKK7, a major target MAPKK for MEKK2, also displayed hyperproliferative phenotypes (5, 25). MEKK2 has also been shown to regulate cytokine gene expression in mast cells (4, 14), mediate epidermal growth factor receptor (EGFR) and fibroblast growth factor 2 receptor signals (21, 34), and play a role in rheumatoid arthritis (17).
In the study reported here, we investigated the molecular mechanisms of MEKK2 activation and regulation. We identified and cloned an MEKK2 interacting protein (Mip), Mip1, that interacts preferentially with the nonphosphorylated and inactive MEKK2. Mip1 expression led to decreased MEKK2 activity, which inhibited JNKK2, JNK1, ERK5, and AP-1 activation by MEKK2. Stimulation of cells with epidermal growth factor, a known MEKK2 agonist that activates the MEKK2-dependent JNK-AP-1 pathway (34), led to a transient dissociation of MEKK2 from Mip1. In contrast, the knockdown of Mip1 expression by small interfering RNA (siRNA) resulted in the augmented activation of the MEKK2-regulated JNK/AP-1 pathway.
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MATERIALS AND METHODS |
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Plasmids, proteins, and antibodies. Flag-tagged JNK1; HA-tagged JNK1, JNKK2(DD), MEKK2 (1-342), MEKK2 (342-619), MEKK2 (342-424), MEKK2 (1-619), MEKK2(342-619)KM, MEKK2(1-619)KM, and MEKK1; and glutathione transferase (GST)-tagged MEKK2 and MEKK2 (342-619) mammalian expression plasmids, Gal4-luc, Gal4-c-Jun, and GST-c-Jun have been previously described (5, 30, 31, 41, 43). Standard cloning procedures were used to construct hemagglutinin (HA)-tagged Mip1 and Mip1-GFP expression vectors; GST-tagged Mip1 mammalian expression plasmids; and GST-fused Mip1 (1-184), Mip1 (1-457), Mip1 (1-486), Mip1 (152-486), and Mip1 (313-486) bacterial expression plasmids. Anti-HA antibody 12CA5 was prepared from a 12CA5 hybridoma and further purified by using a protein A-Sepharose column. Anti-Flag antibody M2 was purchased from IBI-Kodak (New Haven, CT). MEKK2-specific antibody 1128 was described before (5), and 8384 was prepared by immunizing rabbits with peptide RPALSLQETRKAKSSSPKKQN, and further-affinity-purified. Mip1-specific antibody K87 was prepared by immunizing rabbits with peptide CKNIQWKERSKQSA and further affinity purified.
Protein purification. The whole-cell lysates of 293T cells transfected with either a GST empty vector or GST-MEKK2 (342-619) were prepared by using lysis buffer (50 mM HEPES, pH 7.6, 300 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1% Triton X-100, and 10% glycerol). After being precleared with protein A-Sepharose beads, the lysates were incubated with reduced glutathione (GSH)-Sepharose beads at 4°C for 4 h on a rotator. The beads were washed six times with lysis buffer, and the precipitates were eluted with sample buffer, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and visualized by silver staining. The putative MEKK2 interacting protein bands were excised from the gel and analyzed by mass spectrometry.
Immunoprecipitation, in vitro kinase assay, and GST pull-down assay. Cell lysate preparation, immunoprecipitation, and in vitro kinase assays were performed as described in our previous reports (5, 31). For the GST pull-down assay, COS-1 cells were transfected with HA-, Flag-, or GST-tagged expression vectors and they were lysed 40 h later using low-salt lysis buffer (50 mM HEPES, pH 7.6, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1% Triton X-100, and 10% glycerol). Nuclear and cellular debris was removed from the lysates by centrifugation for 20 min at 4°C. GST fusion proteins were precipitated from the clarified lysates with GSH-Sepharose at 4°C during a 4-h incubation in a rotator. The beads were washed four times with low-salt lysis buffer, the precipitates were eluted with a sample buffer and resolved by SDS-PAGE, and the interacting proteins were analyzed by immunoblotting with appropriate antibodies.
RNA interference studies. Oligonucleotides 5'-ACCGATTCATCCTCCTTCAATGTTCAAGAGACATTGAAGGAGGATGAATCTTTTTC-3' and 3'-TAAGTAGGAGGAAGTTACAAGTTCTCTGTAACTTCCTCCTACTTAGAAAAAGAGCT-5' (mip1-siRNA1) and 5'-ACCGATTAGAACGACTCCGAAATTCAAGAGATTTCGGAGTCGTTCTAATCTTTTTC-3' and 3'-TAATCTTGCTGAGGCTTTAAGTTCTCTAAAGCCTCAGCAAGATTAGAAAAAGAGCT-5' (mip1- siRNA2) containing 19-nucleotide sequences matching two independent mip1 cDNA sequences (underlined, corresponding to amino acids 40 to 46 and 89 to 95, respectively) in reverse orientation were synthesized (Sigma Chemical Company, St. Louis, MO). The mip1 and nonspecific siRNA oligonucleotides (matching the lacZ sequence) (a gift from X. Qin) were inserted into a pBS-U6 vector (24). 293T cells grown in a six-well plate were transfected with siRNA expression plasmids using Lipofectamine (Invitrogen, La Jolla, CA). Knockdown of the endogenous Mip1 expression was analyzed by immunoblotting with K87 antibody.
Nucleotide sequence accession numbers.
The sequences of the genes encoding Mip1
, Mip1ß, and Mip1
have been assigned GenBank accession numbers AY633624, AY633625, and AY633626, respectively.
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RESULTS |
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As shown in Fig. 1A, many unique protein bands (putative Mips) were precipitated specifically by GST-MEKK2 (342-619) but not by GST alone. Because of potential problems with the degradation products, we isolated only those Mips larger than GST-MEKK2 (342-619) for peptide sequencing by mass spectrometry. In comparing the sequence data with the gene bank database, we found that the Mips included hsp70, hsp90, kinesin-like protein-1, PARP1, and several novel proteins (Fig. 1A and data not shown). One of the Mips, which we identified by mass spectrometry and named Mip1 (Fig. 1B), with an apparent molecular mass of 65 kDa, was particularly interesting to us because it shared sequence homology with a conserved gene called JC310, which encodes a truncated protein that was characterized as a Ras-inhibitory factor (8) (a full-length cDNA for JC310 was recently deposited in the National Center for Biotechnology Information gene bank during the course of this study). Although the function of the JC310 clone has not been studied in mammalian cells, it was shown that a yeast gene encoding a protein called Sty-1-interacting protein 1 (Sin1) that is involved in the yeast MAPK pathway shared considerable homology with the JC310 clone (38). In addition, the loss of function of the yeast Sin1 (ySin1) could be compensated for by a chicken homolog gene isolated from chicken hindbrain with unknown function (38). These results strongly suggest that Mip1 is a critical regulator of the MAPK cascades in mammalian cells.
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Comparing the sequence of human Mip1 with the sequences of chicken Sin1 and JC310 revealed a stretch of 36 amino acids that was missing from the human Mip1, indicating that additional forms of human Mip1 may exist. To obtain these different forms, we designed primers to flank this region and carried out a reverse transcriptase PCR in which human Jurkat cDNA was used as a template. We identified three different isoforms of human Mip1, which we named , ß, and ; the corresponding genes encode 486, 522, and 323 amino acids, respectively (accession numbers AY633624, AY633625, and AY633626, respectively) (data not shown). A searching of the human genomic DNA database revealed that these isoforms are derived from the alternative splicing of different exons on chromosome 9q34.12, locus ID79109. Although we still do not know the functional differences between these isoforms, the present study was performed with Mip1. Northern blot analysis showed that Mip1 is ubiquitously expressed; however, it is expressed at the highest levels in the heart and skeletal muscle (Fig. 1C). During the course of this study, Schroder and colleagues also reported the cloning and identification of these differential spliced forms of human Mip1, which they named human Sin1ß, Sin1, and Sin1 (29). They also found that Mip1/hSin is ubiquitously expressed with highest expression in heart and skeletal muscle. Interestingly, high MEKK2 expression in heart and skeletal muscle compared to other tissues was also observed (30).
Transiently expressed and endogenous Mip1 interacts with MEKK2. To confirm that the human cDNA indeed encodes the Mip1 that interacts with MEKK2, we constructed expression vectors with HA and GST tags with this cDNA and carried out GST pull-down and coimmunoprecipitation assays. As shown in Fig. 2A, the transiently expressed Mip1 interacted with both the catalytic domain (the bait) and the full-length MEKK2. This interaction appeared to be specific, as Mip1 did not interact with MEKK1, another member of the MEKK/Ste11 family, in the same assay (Fig. 2B). Furthermore, we prepared a Mip1-specific peptide antibody (K87), and using this antibody, we detected the transiently transfected Mip1 (Fig. 2C, left panel) and the endogenous Mip1 protein with an apparent molecular mass of 65 kDa (Fig. 2C, middle panel). The interaction of MEKK2 and Mip1 was further confirmed by using the K87 antibody to detect the endogenous Mip1 protein that was pulled down by GST-MEKK2 (342-619) (Fig. 2C, right panel), thereby confirming that the cDNA is indeed the Mip1 gene.
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Thus the above results suggested that Mip1 negatively regulated MEKK2 activation by preventing MEKK2 from forming dimers. If this were the case, we would expect that Mip1 expression should disrupt MEKK2 dimer formation. To further confirm that Mip1 interferes with MEKK2 dimer formation, we performed a GST pull-down assay to examine the dimer formation between GST-MEKK2 (342-619) and either MEKK2 (342-619) or MEKK2 (342-619)(KM) in the presence or absence of Mip1. As shown in Fig. 4C, Mip1 expression significantly inhibited MEKK2 dimer formation with either MEKK2 (342-619) or MEKK2 (342-619)(KM). Concomitantly, GST-MEKK2 (342-619) formed a complex with Mip1 (Fig. 4C). We also observed similar results when we used GST-MEKK2 (342-619) to pull down the full-length MEKK2 protein (data not shown). To further confirm that Mip1 is able to directly disrupt MEKK2 dimer formation, we purified GST-MEKK2, HA-MEKK2 (342-619), and GST-Mip1 and then examined if Mip1 could disrupt MEKK2 dimer formation. As shown in Fig. 4D, addition of GST-Mip1 but not control GST recombinant proteins significantly blocked MEKK2 dimer formation.
Mip1 interacts with nonphosphorylated and inactive MEKK2. Transiently expressed MEKK2 (342-619) was a phosphor protein with a retarded mobility on an SDS-PAGE gel, whereas the kinase-inactive MEKK2 (342-619)(KM) mutant was nonphosphorylated, with only the band of fastest mobility (Fig. 4C). Protein phosphatase treatment of MEKK2 (342-619) led to its dephosphorylation so that it regained the fast mobility on an SDS-PAGE gel like the mutant MEKK2 (342-619)(KM) (Fig. 5A). Thus we were able to distinguish if the transiently expressed MEKK2 (342-619) protein was phosphorylated or not phosphorylated according to its mobility. Interestingly, we found that the interaction between Mip1 and MEKK2 (342-619) was significantly increased when MEKK2 (342-619) had been pretreated with a protein phosphatase compared to the untreated sample (Fig. 5A). In addition, more MEKK2 (342-619)(KM) than MEKK2 (342-619) was coprecipitated by Mip1 (Fig. 5A). Most importantly, we found that almost all the MEKK2 protein pulled down by Mip1 corresponded to the nonphosphorylated MEKK2 as shown by their fastest mobility on the SDS-PAGE gel, suggesting that Mip1 preferentially interacted with the nonphosphorylated, and likely inactive, MEKK2 (Fig. 5A).
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Epidermal growth factor stimulation dissociates the MEKK2-Mip1 complex. If MEKK2 is negatively regulated through its binding to Mip1, Mip1 may need to dissociate from MEKK2 in order for MEKK2 to be activated during cell stimulation. To test this possibility, we examined the association of MEKK2 and Mip1 in nonstimulated cells and in cells treated with EGF for different times since EGF is a potent MEKK2 activator (13). Using an MEKK2-specific antibody, we then immunoprecipitated the endogenous MEKK2 from the unstimulated and stimulated cells and examined the Mip1 that was coprecipitated with MEKK2 by immunoblotting. In the absence of EGF treatment, we were able to detect the endogenous MEKK2 and Mip1 complex (Fig. 5C). However, after 10 min of EGF treatment, there was a significant decrease of the MEKK2-Mip1 complex. Interestingly, the MEKK2-Mip1 complex appeared to reform after 60 min of EGF stimulation, indicating that Mip1 may also be involved in the negative feedback regulation of MEKK2 signaling following cell stimulation (Fig. 5C).
Knockdown Mip1 expression in vivo activates the JNK-AP-1 pathway. Although the above studies strongly suggest that Mip1 is a negative regulator of the MEKK2 signaling pathway, it is still possible that Mip1 might act as an adaptor molecule to positively control the MEKK2 signal transduction. However, if Mip1 could acts as an endogenous inhibitor, it is conceivable that, under normal conditions, most endogenous MEKK2 is either sequestered in specific intracellular compartments or associated with Mip1, thus preventing MEKK2 from forming dimers and activating itself. When MEKK2 is ectopically expressed, there may not be enough Mip1 in the cells to inhibit MEKK2 dimer formation and activation, thus allowing the nonspecific activation of the downstream JNK-AP-1 pathway. If this were the case, we would expect that the knockdown of Mip1 might allow MEKK2 to activate the JNK-AP-1 pathway. To test this possibility, we constructed mip1 siRNA expression vectors that target two independent mip1 coding sequences. As shown in Fig. 5D, the mip1 siRNAs but not the control siRNA specifically knocked down the expression of a cotransfected Mip1-GFP fusion protein. Using these two mip1 siRNAs, we knocked down the endogenous Mip1 protein expression (Fig. 5E) and examined how it would affect the JNK and AP-1 activation by an in vitro kinase assay and an AP-1 reporter assay. As shown in Fig. 5E and 5F, transfection of the mip1-specific siRNA but not control siRNA led to the augmented activation of both JNK and the AP-1 reporter gene. Together, these results confirm that Mip1 is a negative regulator of MEKK2 in vivo.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, in which we attempted to elucidate the underlying molecular mechanisms governing the activation of the MAP3K MEKK2, we identified a MEKK2-interacting protein, Mip1, that interacts with the inactive MEKK2 and negatively regulates MEKK2 activation by preventing MEKK2 dimer formation. We recently found that MEKK2 dimer formation is a critical step for MEKK2 activation (6). The MEKK2 and Mip1 interaction essentially blocks the MEKK2 dimerization motif that is required for MEKK2 to form a dimer thus keeping it from being activated. Mip1 expression decreased MEKK2 activity and inhibited JNKK2, JNK1, ERK5, and AP-1 activation by MEKK2. In contrast, EGF stimulation led to a transient dissociation of MEKK2 from Mip1 suggesting that Mip-1 may control the basal activity of MEKK2. Furthermore, we found that the knockdown of Mip1 expression by siRNA resulted in the augmented activation of the MEKK2 downstream target JNK/AP-1 pathway. We have thus identified a novel mechanism for the negative regulation of MEKK2 involving an intracellular inhibitors as depicted in Fig. 6.
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Interestingly, the C terminal of chicken Sin1 was required to complement the loss of function of ySin-1 in yeast MAPK Sty-1 signaling, whereas, in our study, the N-terminal region was involved in regulating MEKK2 activation. Sequence analysis of Mip1 homologues from different species revealed several conserved regions in both the N and C termini of Mip1 suggesting that different regions of Mip1 have distinct functions in regulating the MAPK module (29). It is possible that in addition to its function as a MEKK2 regulator, Mip1 may also act as a scaffold protein to modulate the MAPK cascade similar to that of JIP1 in the MLK-MKK7-JNK module (44). In this regard, we found that Mip1 was also capable of interacting with JNK1/2 although this interaction seems not to facilitate JNK activation by MEKK2 nor does it affect the MEKK2-Mip1 interaction (data not shown). In addition, Mip1 appeared not to affect JNKK2-induced JNK activation. Future investigation on this issue may reveal a distinct function of Mip1 in the MAPK cascade regulation.
We mapped the motifs in MEKK2 that are required for MEKK2 dimerization and for Mip1 binding and discovered that the same motif in MEKK2 confers the ability both for MEKK2 to form dimers and for MEKK2 to interact with Mip1. These data may partially explain how Mip1 regulates MEKK2 activation; that is, an MEKK2-Mip1 complex prevents MEKK2 from being activated by occupying the motif required for MEKK2 dimer formation, an essential step for MEKK2 activation. At the molecular level, the interaction motif in Mip1 contains a unique coiled-coil structure, as predicted by computer modeling. Such a coiled-coil structure has been found in large numbers of proteins and is suggested to form a specific protein-protein interaction surface (2, 3). Thus, Mip1 may use this putative coiled coil structure to interact with MEKK2.
Although our present study established Mip1 as an important regulator of MEKK2 signaling, it is likely that Mip1 is also a critical regulator of MEKK3 since the Mip1 interaction motif in MEKK2 is conserved between MEKK2 and MEKK3. Indeed, a preliminary study confirmed that Mip1 and MEKK3 interact (data not shown). The use of mip1-specific siRNAs to knock down Mip1 in nonstimulated cells led to augmented JNK and AP-1 reporter gene expression, further suggesting that Mip1 is a naturally existing inhibitor of the MEKK2 and perhaps also the MEKK3 signaling pathways. Future studies on how Mip1 regulates the MEKK2 and MEKK3 pathways under normal physiological conditions should prove fruitful.
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ACKNOWLEDGMENTS |
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This study was supported partially by grants AI44016 (NIH), HL070225 (NIH), and ARP (Texas Higher Education) (to B.S.) and Cancer Center Core grant CA16672 (M. D. Anderson Cancer Center).
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FOOTNOTES |
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