![]()
|
|
||||||||
J Biol Chem, Vol. 275, Issue 19, 14231-14241, May 12, 2000
§,
¶,
, andFrom the Departments of § Physiology and ¶ Biochemistry, Michigan State University, East Lansing, Michigan 48824
| |
ABSTRACT |
|---|
|
|
|---|
Src homology 3 domain (SH3)-containing
proline-rich protein kinase (SPRK)/mixed-lineage kinase (MLK)-3 is a
serine/threonine kinase that upon overexpression in mammalian cells
activates the c-Jun NH2-terminal kinase pathway. The
mechanisms by which SPRK activity is regulated are not well understood.
The small Rho family GTPases, Rac and Cdc42, have been shown to bind
and modulate the activities of signaling proteins, including SPRK,
which contain Cdc42/Rac interactive binding motifs. Coexpression of
SPRK and activated Cdc42 increases SPRKs activity. SPRKs Cdc42/Rac
interactive binding-like motif contains six of the eight consensus
residues. Using a site-directed mutagenesis approach, we show that SPRK contains a functional Cdc42/Rac interactive binding motif that is
required for SPRKs association with and activation by Cdc42. However,
experiments using a SPRK variant that lacks the COOH-terminal zipper
region/basic stretch suggest that this region may also contribute to
Cdc42 binding. Unlike the PAK family of protein kinases, we find that
the activation of SPRK by Cdc42 cannot be recapitulated in an in
vitro system using purified, recombinant proteins. Comparative
phosphopeptide mapping demonstrates that coexpression of activated
Cdc42 with SPRK alters the in vivo serine/threonine phosphorylation pattern of SPRK suggesting that the mechanism by which
Cdc42 increases SPRKs catalytic activity involves a change in the
in vivo phosphorylation of SPRK. This is, to the best of our knowledge, the first demonstrated example of a Cdc42-mediated change in the in vivo phosphorylation of a protein kinase.
These studies suggest an additional component or cellular environment is required for SPRK activation by Cdc42.
The vast majority of mammalian protein kinases catalyze the
transfer of the Small GTPases regulate certain protein kinases. For instance, by
binding and recruiting the serine/threonine kinase Raf to the plasma
membrane, GTP-bound, farnesylated Ras contributes to the activation of
Raf, and consequently activates the extracellular-regulated protein
kinase
(ERK)1-mitogen-activated
protein kinase (MAPK) pathway (1). Rho family GTPases, which include
Rho, Rac, and Cdc42, play crucial roles in diverse cellular processes
(2, 3), including cytoskeletal rearrangements (4-6), cell cycle
progression (7), cellular transformation (8-14), and nuclear signaling
(15-20). They can also function as protein kinase activators. One well
characterized target of Cdc42 and Rac is the p21-activated kinase (PAK)
(21-23). The interaction between Cdc42 and the serine/threonine kinase PAK requires the CRIB (Cdc42/Rac interactive binding) motif (26), a
14-16-amino acid sequence containing eight consensus amino acids. The
structural determinants required for GTPase binding and the mechanism
of activation of multiple PAK isoforms have been extensively investigated (21, 24, 25, 32-34). CRIB-dependent
interaction of PAK with GTP-bound Rac/Cdc42 induces PAK
autophosphorylation and activation both in vitro and
in vivo.
Diverse proteins, including the tyrosine kinase, activated
Cdc42HS-associated kinase (ACK) (27), and the non-kinase,
Wiskott-Aldrich Syndrome protein (WASP) (28, 29), also contain CRIB
motifs, suggesting that mechanistically diverse regulatory pathways may share this common structural motif. Likewise, not all protein kinases
which interact with Cdc42 and Rac do so through a CRIB motif. For
instance, both the 70-kDa ribosomal S6 kinase (30) and MAPK kinase
kinase-1 (31), which lack CRIB motifs, have been shown to interact with
GTP-bound Cdc42 and Rac. Furthermore, MAPK kinase kinase-4 contains a
modified CRIB motif whose deletion only partially diminishes binding to
Cdc42 and Rac, indicative of a CRIB-independent GTPase binding
determinant (31). Thus the role of CRIB motifs and the mechanisms by
which many protein kinases are activated by small GTPases remains
largely unexplored.
Src homology 3 domain (SH3)-containing proline-rich protein kinase
(SPRK) (35), also called mixed-lineage kinase-3 (MLK-3) (36), or
protein-tyrosine kinase-1 (37), is a member of the so-called
"mixed-lineage" kinases. SPRK contains a CRIB motif bearing six of
the eight consensus amino acids, as well as other domains that may
mediate protein-protein interactions including an
NH2-terminal SH3 domain, a leucine/isoleucine zipper motif, and a large COOH-terminal region that is rich in serine, threonine, and
proline residues (Fig. 1).
![]()
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phosphate of ATP to serine, threonine, or tyrosine residues of their target proteins. Phosphorylation is rendered reversible in vivo by the action of protein phosphatases.
Since phosphorylation is highly regulated in virtually all
physiological processes, it follows that the activities of the protein
kinases, themselves, should be highly regulated. Binding of activating or inhibitory molecules, including lipids, cyclic nucleotides, or
proteins, can modulate the activity of protein kinases. In addition,
post-translational modifications, such as phosphorylation and
proteolysis, can regulate protein kinase activity. These regulatory events may alter the specific activity of a protein kinase or may
change its stability. Finally, access to physiological substrates may
be limited by restricted subcellular localization or translocation of a
protein kinase.

View larger version (18K):
[in a new window]
Fig. 1.
Schematic of SPRK. The
numbers in the diagram represent amino acid number. The
glycine-rich region (amino acids 1-42) is denoted by Gly.
The region containing the zipper motif and the polybasic stretch of
amino acids includes amino acids 400-486. The amino acid sequence
corresponding to this region is shown below with the basic
stretch of amino acids printed in bold letters and the
nonaromatic hydrophobic residues predicted to occupy the d
position in the zipper motifs underlined. The sequence deleted in
SPRK
zip (amino acids 430-486) is boxed.
Upon overexpression in mammalian cell lines SPRK activates c-Jun
NH2-terminal kinase (JNK)/stress-activating protein kinase through phosphorylation and activation of the dual specific kinase, MAPK kinase-4 (MKK-4/SEK1) (38) or MKK-7 (39), and binds the JNK
scaffold proteins, JNK interacting protein (JIP)-1 and JIP-2 (39, 43,
44). In some cell types, SPRK has been reported to activate the MAPK
p38/reactivating kinase via MKK-3/6 (40) as well as ERK (41). SPRK
associates with Rac and Cdc42 in filter binding assays (26) and
coexpression of SPRK, activated Cdc42, and JNK increases SPRK and JNK
activity (42). Whether activation by Cdc42 of the distantly related PAK
and SPRK involves mechanistically analogous processes is unknown. SPRK
is functionally homologous to Raf, and thus mechanistic aspects of
Cdc42-SPRK and Ras-Raf activation may be shared. Here we show that SPRK
contains a functional CRIB motif that is absolutely required for Cdc42
binding to and activation of SPRK. The zipper region and adjacent basic
sequences of SPRK may also contribute to Cdc42 binding. Binding of
Cdc42 to SPRK does not require SPRK catalytic activity. Interestingly, GTP-bound Cdc42 has no effect on the activity of purified,
catalytically active SPRK, suggesting that an additional cellular
component is required for kinase activation. These studies point to an
important distinction between PAK and SPRK in the mode of
GTPase-induced kinase activation. Comparative phosphopeptide mapping
revealed that coexpression of activated Cdc42 with SPRK alters the
in vivo phosphorylation sites on SPRK. This change in
serine/threonine phosphorylation correlates with increased SPRK
activity. These studies represent the first case where a Cdc42-mediated
change in the in vivo phosphorylation sites of a protein
kinase has been documented and provide evidence for the involvement of
in vivo phosphorylation of SPRK for Cdc42-induced
activation. Thus, the Cdc42-mediated activation of SPRK is clearly
distinct from the mechanism previously described for Cdc42-induced
activation of PAK.
| |
EXPERIMENTAL PROCEDURES |
|---|
|
|
|---|
Construction of Mammalian Expression Vectors and Mutagenesis-- Construction of the cytomegalovirus-based expression vectors carrying the cDNAs for wild type SPRK (pRK5-sprk), and for the kinase-defective variant of SPRK (pRK5-sprk K144A) has been described elsewhere (35). Expression plasmids encoding NH2-terminal Flag epitope-tagged wild type Cdc42 (pRK5-Nflag.cdc42) and the constitutively active variant (pRK5-Nflag.cdc42Val-12) of Cdc42 were kindly provided by Avi Ashkenazi (Genentech, Inc.).
Variants of SPRK containing point mutations in the CRIB motif were constructed by a modified recombinant polymerase chain reaction (PCR) method described in detail elsewhere (45). For each mutation two different PCRs were performed, one containing a left mutagenesis primer and a left outside primer, and one containing a right mutagenesis primer and a right outside primer. The left and right outside primers used for all mutations were 5'-GATGAGTCATCTGAATCCAGG-3' and 5'-CTGTGGCCTATGGCGTAGCTG-3', respectively. To obtain the three different CRIB mutants the following left and right mutagenesis primers were used, respectively: SPRK F498A, 5'-GGTGCTTGGCGTCGAGTGGCAT-3' and 5'-ACTCGACGCCAAGCACCGCATC-3'; SPRK H500A, 5'-CTTCAAGGCCCGCATCACCGT-3' and 5'-GGTGATGCGGGCCTTGAAGTCG-3'; SPRK I492A/S493A, 5'-GAAGTCGAGTGGCATGGCGGCACGCTCG-3' and 5'-CGAGCGTGCCGCCATGCCACTCGAC-3'. The presence of the desired mutation was confirmed by DNA sequencing using the Sanger method, and the absence of PCR-introduced errors was verified by automated sequencing.
Deletion of amino acids 430 through 486 of SPRK to yield SPRK
zip was
accomplished by digestion of the expression vector pRK5-sprk with BssHII, followed by ligation with T4 DNA ligase. DNA
modifying enzymes were purchased from New England Biolabs or Life
Technologies, Inc.
Expression and Purification of Recombinant SPRK-- An NcoI-HindIII fragment, containing the full-length SPRK cDNA, was subcloned from pRK5-sprk into the pFastBac HTb baculovirus expression vector (Life Technologies, Inc.) which contains a hexahistidine tag. Recombinant histidine-tagged SPRK was expressed in Sf21 cells and purified by nickel affinity chromatography according to the manufacturer's protocol. Fractions containing histidine-tagged SPRK, as determined by SDS-PAGE followed by Coomassie Blue staining, were pooled and concentrated using a Centriprep concentrator (Amicon).
Cell Lines and Transfections-- Human fetal kidney 293 cells were maintained in Ham's F-12/low glucose Dulbecco's modified Eagle's media (1:1) (Life Technologies, Inc.) supplemented with 8% fetal bovine serum (Life Technologies, Inc.), 2 mM glutamine, and penicillin/streptomycin (Life Technologies, Inc.). Plasmids (5 µg each for 60-mm dishes; 10 µg each for 100-mm dishes) were used to transfect 293 cells using the calcium phosphate technique (46). Cell monolayers were incubated with the DNA precipitate for 4 h, then washed once with PBS (phosphate-buffered saline), and cultured in the medium described above. After 18 h cells were harvested.
Cell Lysis and Immunoprecipitation--
Cells were washed with
ice-cold PBS and lysed for 5 min on ice by the addition of 1 ml of
lysis buffer (50 mM HEPES (pH 7.5), 150 mM
NaCl, 1.5 mM MgCl2, 2 mM EGTA, 1%
Triton X-100, 10% glycerol, 10 mM sodium fluoride, 1 mM Na4PPi, 100 µM
-glycerophosphate, 1 mM Na3VO4,
2 mM phenylmethylsulfonyl fluoride, and 0.15 units/ml aprotinin). The lysate was clarified by centrifugation for 20 min at
14,000 rpm in an Eppendorf centrifuge at 4 °C. Rabbit polyclonal antiserum was raised against a peptide corresponding to the
COOH-terminal 8 amino acids of SPRK and was purified by Protein
A-Sepharose chromatography. Antibodies against the proteins of interest
were prebound to Protein A-agarose beads (SPRK antiserum (0.25 µg/µl slurry), M2 monoclonal antibody (Kodak IBI) directed against
the Flag epitope (0.45 µg/µl slurry), and JNK C-17 antibody (Santa Cruz Biotechnology) (50 ng/µl slurry)) as described previously (35).
Clarified lysate (300 µl) was incubated with 20 µl of antibody-bound Protein A-agarose for 90 min at 4 °C.
Immunoprecipitates were washed with HNTG buffer (20 mM
HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 10%
glycerol). Immunoprecipitates used for kinase assays were washed three
times with HNTG buffer containing 1 M LiCl, three times
with HNTG buffer, and twice with kinase assay buffer (50 mM
Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM
MnCl2, 10 mM MgCl2, 0.1 mM Na3VO4).
Gel Electrophoresis and Western Blot Analysis-- Lysates and immunoprecipitates of SPRK and Cdc42 were resolved by SDS-PAGE according to Laemmli (47). Proteins were transferred to nitrocellulose and immunoblotted using either SPRK antiserum (1 µg/ml) or M2 Flag monoclonal antibody (9 µg/ml), followed by the appropriate horseradish peroxidase-conjugated secondary antibody (Life Technologies, Inc.). Western blots were developed by chemiluminescence. Multiple exposures of the Western blots were developed, and densitometry (NIH Image) of unsaturated films was used to determine relative expression levels. Statistics were compiled using an unpaired Student's t test. A p value smaller than 0.05 was considered statistically significant.
In Vitro Kinase Assays--
Kinase assays were performed in 20 µl of kinase assay buffer containing 50 µM ATP and 5 µCi of [
-32P]ATP (3000 Ci/mmol) (NEN Life Science
Products). For the SPRK kinase assay 10 µg of mixed histones (Roche
Molecular Biochemicals) was used as the substrate and the reaction was
carried out for 30 min at room temperature. Independent experiments
showed that the reaction was linear within this time range. The
reactions were terminated by the addition of an equal volume of 2 × SDS sample buffer (100 mM Tris (pH 6.8), 4% SDS, 20%
glycerol, 0.2% bromphenol blue, 100 mM dithiothreitol, 1%
-mercaptoethanol) containing 50 mM EDTA (pH 8.0).
For the kinase assays involving purified kinases, recombinant SPRK or
PAK-2 (1 and 2 µg, respectively) was incubated in 50 µl of kinase
assay buffer containing 4 µg of GST (glutathione S-transferase)-Cdc42 that had been preloaded with GTP
S or
GDP and assay was performed as above. GST-Cdc42 (19 µM)
was preloaded with GTP
S or GDP (4 mM) in buffer
containing 50 mM Tris (pH 7.5), 5 mM EDTA, and
1 mM dithiothreitol. The mixture was incubated for 15 min
at 30 °C. The nucleotide loading reaction was quenched by the
addition of 10 mM MgCl2. Purified GST-Cdc42 was
obtained from an Escherichia coli overexpression system; and
purified PAK-2, obtained using the baculovirus expression system, was
kindly provided by Dr. Arie Abo (Onyx Pharmaceuticals) (23).
For the JNK assays, 8 µg of GST-c-Jun was used as the substrate, and the reaction was carried out for 15 min at room temperature. The pGEX-c-Jun (1-115) vector was kindly provided by Dr. Ajay Rana (Massachusetts General Hospital, Harvard Medical School, Boston, MA). GST-c-Jun was expressed in XL-1 Blue E. coli and purified by glutathione-Sepharose chromatography. Following the kinase assay, proteins were separated by SDS-PAGE. Gels were rinsed in PBS, dried, and incorporation of radioactivity into kinase or substrates was determined by PhophorImaging (Molecular Dynamics). To detect JNK expression, proteins were transferred from an SDS-polyacrylamide gel to a polyvinylidene difluoride membrane and immunoblotted using the JNK C-17 antibody (0.5 µg/ml).
MBP Fusion Protein Plasmid Construction-- The predicted leucine zipper coiled coil domain of the SPRK cDNA encoding amino acids 386-477 was generated by PCR amplification of the pRK5-sprk plasmid using the following oligonucleotides: 5'-TCAGAATTCGGATCCGAAATGCCGCGGGACTCCTTC-3' and 5'-ATTGAAGCTTCATGTCCCGCGGCGGCGGCG-3'. The leucine zipper fragment was cloned into the pMAL-c2 vector (New England Biolabs) at the EcoRI and HindIII sites, in-frame with the malE gene, which encodes the monomeric maltose-binding protein (MBP). An MBP control construct was generated using the following linker oligonucleotides encoding a stop codon: 5'-AATTCGGATCCTAATAGCGA-3' and 5'-AGCTTCGCTATTAG GATCCG-3'. The first of these oligonucleotides was phosphorylated with T4 polynucleotide kinase. The two oligonucleotides were annealed and ligated into the pMAL-c2 vector which had been digested with EcoRI and HindIII. Both constructs were confirmed by dideoxy sequencing using Sequenase enzyme (Amersham Pharmacia Biotech).
Expression and Purification of MBP Fusion Proteins--
DH5
derivatives of E. coli containing the recombinant plasmids
were grown at 37 °C in LB medium supplemented with ampicillin (50 µg/ml) and 2% glucose. Protein expression was induced by the addition of 0.3 mM isopropylthiogalactoside for 4 h.
Cells were harvested by centrifugation at 4000 × g for
20 min. Cells were lysed by two passes through a French pressure cell.
MBP proteins were purified by amylose affinity chromatography according
to the manufacturer's protocol. Fractions containing the MBP-zips or
MBP, as determined by SDS-PAGE followed by Coomassie Blue staining, were pooled and concentrated to about 1 mg/ml using a Centriprep concentrator (Amicon).
MBP Pull-down Assay--
One day following transfection of 293 cells with cDNAs encoding SPRK and SPRK
zip, the cells were lysed
in 1 ml of lysis buffer as described above. Amylose resin (30 µl) was
incubated with 10 µg of purified MBP or MBP-zips for 60 min at
4 °C, and then washed twice with column buffer (20 mM
Tris (pH 7.4), 200 mM NaCl, 1 mM EDTA).
Cellular lysate from transfected 293 cells (200 µl) was added to the
amylose resin and incubated at 4 °C with mixing for 90 min. After
removal of the lysate, the resin was washed twice with HNTG buffer. The
binding of SPRK or SPRK
zip to MBP or MBP-zips was analyzed by
SDS-PAGE followed by Western blotting using the SPRK antiserum.
Coomassie Blue staining verified equal amounts of MBP-containing
proteins in the assays.
Phosphopeptide Mapping-- After a 24-h transfection with pRK5-sprk in the presence or absence of pRK5-Nflag.cdc42Val-12, 293 cells were washed five times with phosphate-free medium (Dulbecco's modified Eagle's medium supplemented with 10% dialyzed fetal bovine serum (Summit Biotechnology)), and incubated at 37 °C for 2 h. The cells were then incubated in phosphate-free medium containing 3 mCi/ml [32P]orthophosphate (carrier free; NEN Life Science Products) for 4 h at 37 °C.
Cells were washed five times with ice-cold PBS and then lysed in 1 ml of lysis buffer. Lysates were clarified by centrifugation for 15 min at 14,000 rpm in an Eppendorf centrifuge at 4 °C. SPRK was immunoprecipitated with SPRK antiserum as described above. Immunoprecipitated proteins were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Radiolabeled bands that comigrated with SPRK, as judged by Western blotting, were excised from the polyvinylidene difluoride membrane. After washing three times with methanol and three times with water, the radioactive piece of membrane was blocked with 1 ml of 0.5% polyvinylpyrrolidine-360 (Sigma) containing 100 mM acetic acid for 30 min at 37 °C, and then washed five times with water. Tryptic digestion was performed with 10 µg of sequencing grade trypsin (Roche Molecular Biochemicals) for 2 h in 200 µl of 50 mM NH4HCO3 (pH 8.3) at 37 °C. An additional 10 µg of trypsin was added and the digestion mixture was incubated for an additional 2 h at 37 °C. The membrane was then sonicated for 3 min in 300 µl of water to remove additional tryptic peptides. The solution containing the released tryptic peptides was concentrated in a SpeedVac (Savant Instruments).
The peptides were separated on cellulose thin layer chromatography (TLC) plates (Kodak, 20 × 20 cm) by thin layer electrophoresis (TLE) in the first dimension in pH 1.9 buffer (formic acid (88% w/v)/glacial acetic acid/water, 25:78:897, v/v) at 0 °C and 1000 V for 30 min, and separated in the second dimension by TLC in phosphochromatography buffer (n-butanol/pyridine/glacial acetic acid/water, 15:10:3:12, v/v). The radiolabeled phosphopeptides were visualized and quantitated using a PhosphorImager.
Phosphoamino Acid Analysis--
The tryptic peptides were
hydrolyzed in 200 µl of 6 M HCl for 1 h at
100 °C. The phosphoamino acids were concentrated in a SpeedVac.
Unlabeled phosphoamino acid standards (Sigma) and xylene cyanol FF
marker dye (Sigma) were added to each sample. The phosphoamino acids
were separated by one-dimensional TLE in pH 2.5 buffer (66.7% pH 3.5 buffer (glacial acetic acid/pyridine/water, 50:5:945, v/v) and 33.3%
pH 1.9 buffer on cellulose TLC plates at 0 °C and 500 V for 1.5 h. Unlabeled phosphoamino acid standards were visualized by ninhydrin
staining and the 32P-labeled phosphoamino acids derived
from the SPRK peptides were visualized by PhosphorImaging.
| |
RESULTS |
|---|
|
|
|---|
Association of Activated Cdc42 with SPRK Does Not Require SPRK
Kinase Activity--
Recently Hall and co-workers have shown that
several proteins, including SPRK, associate with GTP-bound Cdc42 and
Rac in filter binding assays (26). All of these proteins contain a
14-16-amino acid sequence that includes eight consensus residues,
which has been coined the CRIB motif. The CRIB motif of SPRK contains
six of the eight consensus amino acids (Fig.
2). In this study we examined the
structural requirements and mechanism of Cdc42 binding and activation
of SPRK.
|
SPRK and Flag epitope-tagged Cdc42 expression vectors were transiently
transfected in 293 cells. To mimic the GTP-bound state of Cdc42 we used
a constitutively active mutant of the GTPase, i.e.
Cdc42Val-12. Cellular lysates from 293 cells were
immunoprecipitated with the Flag antibody and the presence of
associated SPRK was assessed by Western blot analysis with a SPRK
antibody. In these co-immunoprecipitation experiments (Fig.
3A), the constitutively active
form of Cdc42, but not wild type Cdc42, associates with SPRK
(first panel). We have not observed association of SPRK with
the dominant negative variant Cdc42Asn-17, but, in our
hands, expression of this Cdc42 variant has been low. Expression levels
of SPRK and Cdc42 in cellular lysates were determined by Western blot
analysis (Fig. 3A, lower panels). These data indicate that
SPRK preferentially associates with the GTP-bound form of Cdc42.
|
SPRK can autophosphorylate in vitro (35). Although SPRKs postulated site of interaction with Cdc42 is not within the catalytic domain, it is plausible that either SPRK autophosphorylation or SPRK phosphorylation of another interacting molecule might modulate the Cdc42-SPRK interaction. To examine whether SPRKs catalytic activity effects its ability to bind to Cdc42, the kinase-defective SPRK variant (SPRK K144A) was tested for its ability to associate with Cdc42Val-12. The SPRK K144A variant shows no autophosphorylation in an in vitro kinase assay (35).
Co-transfection and co-immunoprecipitation experiments with active and inactive SPRK, and Cdc42Val-12, demonstrate that constitutively active Cdc42 associates equally well with wild type SPRK and the kinase-defective variant SPRK K144A (Fig. 3B, upper panel). This indicates that SPRKs catalytic activity is not required for its association with Cdc42. The retarded mobility of wild type SPRK, as compared with that of inactive SPRK, suggests a more highly phosphorylated form of SPRK that is likely due, at least in part, to autophosphorylation (Fig. 3B, upper and middle panels).
To determine whether association with Cdc42 effects SPRKs catalytic activity, an in vitro kinase assay for SPRK was developed. While its kinase domain shares overall sequence similarity with both serine/threonine and tyrosine kinases, SPRK shows high amino acid sequence identity with the serine/threonine kinase B-Raf in a small region of subdomain VIb and VIII that is important for substrate specificity and catalytic activity (48). In addition, both Raf and SPRK appear to function as MKKKs leading primarily to the activation of ERK and JNK, respectively. Since histones are commonly employed as exogenous substrates for Raf in in vitro kinase assays (49, 50), a mixture of histones was tested as an exogenous substrate for SPRK. SPRK, immunoprecipitated from cellular lysates of transiently transfected 293 cells, exhibits basal autophosphorylation as well as phosphorylation of the histones H3 and H4 (Fig. 3B). In contrast, the kinase-defective SPRK shows no autophosphorylation or histone phosphorylation, indicating that the phosphorylation events observed in these assays are attributable to SPRK, rather than to contaminating kinases.
The expression of Cdc42Val-12 in transiently transfected 293 cells increases the autophosphorylation activity as well as the substrate phosphorylation activity of SPRK about 3-5-fold (Fig. 3C). No background phosphorylation is observed with the kinase-defective SPRK variant in the presence or absence of Cdc42Val-12. Interestingly, under these in vitro kinase assay conditions, we do not detect Cdc42 associated with SPRK by Western blotting (data not shown).
SPRKs CRIB Motif Is Necessary for Association with Cdc42 and for
Cdc42-induced Activation of SPRK--
To test whether SPRKs potential
CRIB motif actually functions in the binding of Cdc42 we took a
site-directed mutagenesis approach. Three different SPRK variants were
generated by mutating conserved amino acids in the CRIB motif to
alanine residues: SPRK F498A, SPRK H500A, and SPRK I492A/S493A (Fig.
2). While we cannot absolutely rule out the possibility that
introduction of these mutations might alter SPRKs conformation, the
expression levels of the SPRK CRIB mutants in transient transfections
of 293 cells are at least as high as that of wild type SPRK (Fig.
4A), suggesting that these
variants are stable. The CRIB variants were coexpressed with
Cdc42Val-12 in 293 cells and the cellular lysates were
subjected to co-immunoprecipitation experiments. While wild type SPRK
associates with Cdc42Val-12, none of the SPRK CRIB mutants
detectably associates with the activated GTPase (Fig.
4A).
|
If Cdc42-induced activation of SPRK is mediated through its interaction with SPRKs CRIB motif, one would expect that the SPRK CRIB mutants should exhibit a defect in Cdc42-induced activation. Accordingly, cells were transiently transfected with cDNAs encoding wild type SPRK or SPRK I492A/S493A, in the presence or absence of Cdc42Val-12. The activity of the immunoprecipitated SPRK or SPRK CRIB mutant was measured in an in vitro kinase assay. In the absence of Cdc42Val-12, both wild type SPRK and the SPRK CRIB variant show similar levels of autophosphorylation and substrate phosphorylation (Fig. 4, B and C). In the absence of Cdc42Val-12, the differences in the activities of SPRK and the SPRK CRIB variants were not statistically significant. This further supports the idea that the mutations in the CRIB motif do not grossly perturb SPRKs structure or inherent catalytic activity. However, both autophosphorylation and substrate phosphorylation of the CRIB variant is markedly lower (3-fold) than that of wild type SPRK, when each is coexpressed with the activated GTPase (Fig. 4, B and C). The small increase in the catalytic activity of the SPRK CRIB mutant when activated Cdc42 is coexpressed may be due to Cdc42-activated endogenous SPRK. Alternatively there may be residual binding of the CRIB variant to activated Cdc42 in vivo, which we do not detect in our in vitro co-immunoprecipitation assay. Taken together these results demonstrate that SPRK does contain a functional CRIB motif, and that Cdc42-induced activation of SPRK is mediated via association with this CRIB motif.
Effects of Deleting the COOH-terminal Portion of SPRKs Zipper
Domain--
In WASP (51), PAK (32, 52), and ACK (27) the CRIB motif is
necessary but not sufficient for GTPase binding. Outside of the CRIB
motif SPRK shares no sequence similarity with the minimal
GTPase-binding domains that have been defined for these proteins.
Instead, SPRK contains two closely spaced leucine/isoleucine zipper
motifs spanning amino acids 400-462, COOH-terminal to the CRIB motif
(Fig. 1). Considering the close vicinity in linear sequence of the
zippers and the CRIB motif we asked whether the zipper motif might
contribute to the binding of SPRK to Cdc42Val-12.
Accordingly, a variant of SPRK, SPRK
zip, which lacks amino acids
430-486, as shown in Fig. 1, was constructed. This deletion removes
the second half of the zipper region as well as 22 COOH-terminal amino
acids which includes a short basic region, but leaves the entire CRIB
motif intact.
SPRK
zip is expressed in transiently transfected 293 cells at levels
comparable to that of wild type SPRK (Fig.
5A). The ability of
Cdc42Val-12 to associate with SPRK
zip was tested in
co-immunoprecipitation experiments with cellular lysates harvested from
transiently transfected 293 cells. The deletion of the second
zipper/basic stretch greatly diminishes the ability of SPRK to bind to
Cdc42Val-12, despite the presence of the complete CRIB
motif (Fig. 5A). Based on these and our previous results,
both the CRIB motif and the second half of the zipper region and a
stretch of basic amino acids may contribute to Cdc42 binding.
Alternatively, the COOH-terminal zipper/basic stretch may not directly
interact with Cdc42, but may be required for the proper presentation
and binding of the CRIB motif to the GTPase.
|
SPRK
zip has approximately 70% of the basal autophosphorylation
activity of wild type SPRK (Fig. 5, B and C).
However, in contrast to wild type SPRK, there is no Cdc42-induced
increase in autophosphorylation of SPRK
zip, consistent with the
finding that SPRK
zip binds Cdc42Val-12 only very weakly.
In an in vitro kinase assay, SPRK
zip expressed with or
without Cdc42Val-12 (Fig. 5, B and D)
lacks the ability to phosphorylate histones. To address whether the
lack of histone phosphorylation might be due to some unique feature of
histones we performed the same experiment with myelin basic protein as
a substrate and obtained the analogous results (data not shown). These
data suggest that the zipper domain/basic stretch may be fundamentally
required for substrate phosphorylation.
SPRK
zip Fails to Activate JNK--
SPRK has been identified as
an upstream activator of the JNK/stress-activating protein kinase
pathway (38, 40, 42). JNK activity was measured after
immunoprecipitation of endogenous JNK from cellular lysates of
transiently transfected 293 cells in an immune complex in
vitro kinase assay using GST-c-Jun as the substrate.
Overexpression of SPRK in 293 cells leads to a 5-fold increase in JNK
activity over the basal activity in vector control-transfected cells
(Fig. 6, A and B).
Transient expression of Cdc42Val-12 increases JNK activity
some 2-3-fold. Although coexpression of SPRK and
Cdc42Val-12 increases SPRK catalytic activity, as measured
in an in vitro kinase assay, the observed SPRK-induced JNK
activation with coexpressed Cdc42Val-12 over that of SPRK
alone does not reach statistical significance. A likely explanation for
this finding is that the activity of overexpressed SPRK alone is
sufficient to maximally activate the endogenous JNK in 293 cells.
SPRK
zip is unable to phosphorylate an exogenous substrate in an
in vitro kinase assay, and, indeed, we have found that it
completely lacks the ability to activate the JNK pathway (Fig. 6,
A and B). Thus, it appears that the zipper motif
is critical for substrate phosphorylation by SPRK both in vitro and in vivo.
|
The Leucine Zipper Domain Is Sufficient for SPRK
Oligomerization--
Despite its substantial autophosphorylation
activity, SPRK
zip fails to activate JNK. To characterize the
oligomerization properties of SPRK
zip, we engineered a cDNA
construct encoding amino acids 386-477 of SPRK fused to the coding
sequence of the monomeric MBP of E. coli, designated
MBP-zips, and tested purified MBP and MBP-zips for their ability to
associate with full-length SPRK and SPRK
zip. As shown in Fig.
7A, full-length SPRK binds MBP-zips but not MBP, as judged by immunoblotting. Furthermore, SPRK
zip fails to bind either MBP-zips or MBP. Equal amounts of MBP
or MBP-zips in the assays were verified by Coomassie Blue staining of a duplicate gel (Fig. 7B). Western blotting of
cellular lysates using a SPRK antibody shown in Fig. 7C,
reveals that full-length SPRK and SPRK
zip were expressed at similar
levels. These data provide direct evidence that the leucine
zipper/basic stretch of SPRK is capable of protein-protein interactions
and is sufficient to mediate SPRK homo-oligomerization.
|
Activated Cdc42 Fails to Activate SPRK in Vitro--
The small
GTPases Rac and Cdc42 can stimulate the autophosphorylation activity of
the CRIB-containing serine/threonine kinase PAK-2 in vitro
(23). To determine whether Cdc42-induced activation of SPRK can be
recapitulated in a completely in vitro system, hexahistidine
NH2-terminal tagged SPRK was expressed using the baculovirus system, and purified by metal-chelate chromatography. GST-Cdc42 was expressed in and purified from E. coli. The
purified SPRK is catalytically active as judged by its basal
autophosphorylation activity. GTP
S- or GDP-loaded GST-Cdc42 was
incubated with purified SPRK or PAK-2 in an in vitro kinase
assay (Fig. 8B). While
GTP
S-loaded Cdc42 activates purified PAK-2, it fails to activate
purified SPRK. Likewise, SPRK immunoprecipitated from transfected 293 cells cannot be activated in vitro by the addition of
GTP
S-loaded Cdc42 (Fig. 8A). These data support the
requirement of a cellular context or coactivator for SPRK activation by
Cdc42.
|
Cdc42 Alters the in Vivo Phosphorylation Pattern of SPRK-- As described above, co-immunoprecipitation experiments and in vitro kinase assays show that Cdc42Val-12 when coexpressed with SPRK can associate with SPRK and modulate its catalytic activity. However, purified, activated Cdc42 cannot stimulate the autophosphorylation of SPRK in vitro. In order to determine if the presence of activated Cdc42 alters SPRK phosphorylation in vivo, two-dimensional phosphopeptide analysis of SPRK labeled in vivo, in the absence and presence of Cdc42Val-12, was performed.
The net incorporation of radiolabel into SPRK increased approximately
3-fold when SPRK was coexpressed with Cdc42Val-12.
Two-dimensional TLE/TLC revealed that while the basic pattern of
phosphopeptides from the two samples is similar (Fig.
9A), there are notable
differences. The major changes are observed in the triangular cluster
of phosphopeptides b, c, and d.
Phosphopeptide a predominates in both samples.
Phosphopeptides b and c are detected in the
triangular cluster of the SPRK map, but are low in abundance relative
to peptide a. In the corresponding map of SPRK that had been
expressed in the presence of Cdc42Val-12, however,
phosphopeptide b is not detected. Instead, phosphopeptide c is the prominent phosphopeptide in the triangular cluster,
with 70% of the radioactivity of phosphopeptide a (Fig.
9B). Furthermore, phosphopeptide d, nearly
undetectable in the map of SPRK, appears at high levels in the map of
SPRK expressed with Cdc42Val-12. For comparison, the level
of another peptide (x) relative to peptide a is
essentially constant in both maps. The labeling and mapping from three
independent experiments yielded the same results. These data indicate
that the presence of activated Cdc42 changes the in vivo
phosphorylation state of SPRK, which correlates with an increase in
SPRK catalytic activity. Phosphoamino acid analysis of the pooled
tryptic phosphopeptides (Fig. 9C) reveals predominantly serine phosphorylation, some threonine phosphorylation, and no tyrosine
phosphorylation. The ratios of phosphoserine to phosphothreonine in the
two samples are not substantially different.
|
| |
DISCUSSION |
|---|
|
|
|---|
Small GTPases of the Ras superfamily have been shown to regulate protein kinases. PAK has emerged as the paradigm CRIB-containing serine/threonine kinase that is activated by GTP-bound Cdc42 and/or Rac. The PAKs play roles in diverse processes, including apoptosis, modulation of actin cytoskeleton, gene transcription, and cell cycle (53). SPRK is a member of the so-called mixed-lineage kinases. Except for the presence of a loosely conserved CRIB motif, SPRK differs dramatically from the PAKs, both structurally and functionally. While the mammalian PAK-1, -2, and -3 share 95% sequence similarity in their catalytic domains, SPRKs catalytic domain is just 20% similar to those of the mammalian PAKs. The CRIB motif of the PAKs is found NH2-terminal to the catalytic domain, whereas SPRKs CRIB motif is COOH-terminal to the catalytic domain. Flanking SPRKs catalytic domain is an NH2-terminal SH3 domain and a COOH-terminal leucine zipper motif, both lacking in the PAKs. The only well established function thus far ascribed to SPRK is as an MKKK in the activation of the JNK pathway. Because the MLKs are so different from the PAKS, it is important to determine whether the structural features of their binding to and the mechanisms of activation by Cdc42 and/or Rac also differ from that of the PAKs.
Whereas the three mammalian PAK isoforms contain perfect consensus CRIB motifs, as defined by Burbelo et al. (26), SPRKs CRIB motif contains only six of the eight consensus residues (Fig. 2). We show that mutations in conserved residues of SPRKs CRIB motif disrupt the ability of the Cdc42 to bind to and activate SPRK, indicating that SPRK does indeed contain a functional CRIB motif. WASP and ACK, two other proteins whose CRIB-dependent binding to Cdc42 has been well established, also contain less than perfect CRIB motifs (Fig. 2), with WASP (28, 29), and ACK (27) containing 7 of the 8 consensus residues, and 6 of the 8 consensus residues, respectively. It may well be that the CRIB consensus motif is biased toward PAK, due to the large number of PAK isoforms that have been identified.
SPRK and the closely related MLK-2 lack the second of the two conserved histidine residues of the consensus CRIB motif (Fig. 2). We show here that mutation of the first conserved histidine in SPRK to an alanine residue (H500A) disrupts binding to Cdc42. The fact that both SPRK (26) and MLK-2 (26, 54) have been demonstrated to bind Cdc42 may indicate that the second of the two conserved histidines in the consensus CRIB motif in other CRIB-containing proteins is not required for binding to Cdc42. Further support for this notion is provided by the finding that the conserved Asp38 in Cdc42 interacts primarily with the first of the two conserved histidine residues (His520) in ACK (55). In addition, mutation of the first of the two conserved histidine residues in N-WASP to aspartate (H208D) decreases the in vitro binding affinity for Cdc42 and Rac, as well as the activity of N-WASP in vivo and in vitro (56). The regions outside of the CRIB motifs of ACK and WASP exhibit low sequence similarity, and, perhaps not surprisingly, low structural similarity when bound to Cdc42 (55, 57). It is likely that the GTPase-binding domain of SPRK, with the exception of the CRIB motif, will differ structurally from those of both WASP and ACK.
Because of the proximity of SPRKs zipper and CRIB motifs in linear
sequence, and because sequences flanking the CRIB motif in other
proteins contribute to Cdc42/Rac binding, we tested whether deletion of
the COOH-terminal half of the zipper motif effects Cdc42 binding. The
binding of SPRK
zip (Fig. 1), which contains an intact CRIB motif, to
activated Cdc42 is reduced more than 10-fold, suggesting that, in
addition to SPRKs CRIB motif, the zipper domain or the following short
basic region of SPRK may contribute to Cdc42 binding. Interestingly,
the basal autophosphorylation activity of SPRK
zip is about 70% that
of wild type SPRK. This may indicate some intramolecular
autophosphorylation activity. Alternatively, SPRK
zip may
homo-oligomerize and undergo intermolecular autophosphorylation. Leung
and Lassam (58) recently reported a very large reduction in
GST-SPRK/MLK-3 autophosphorylation activity in vitro upon
deleting the entire zipper region, but leaving the basic stretch intact.
Recent site-directed mutagenesis studies indicate that the
serine/threonine kinase PAK contains a basic stretch consisting of
three contiguous lysine residues upstream of the CRIB motif, whose
charge is important for binding to Rac1 and Rac2, and whose presence is
required for efficient PAK-1 activation by Rac1, Rac2, and Cdc42 (33).
SPRK contains four contiguous arginine residues between the zipper
domain and the CRIB motif. These basic amino acids are deleted in the
SPRK
zip variant, which exhibits greatly reduced binding to activated
Cdc42. Thus, it is plausible that the arginine tract in SPRK may
contribute to Cdc42 binding or activation of SPRK. Currently we are
defining a minimal Cdc42-binding domain of SPRK and are assessing the
relative contributions of various amino acids within this domain to
Cdc42 binding and Cdc42-induced SPRK activation.
We show that SPRK and activated Cdc42 can be co-immunoprecipitated from cellular lysates. However, under the conditions of our in vitro kinase assay, which clearly show a Cdc42-induced increase in SPRK autophosphorylation and histone phosphorylation, Cdc42 is not detected. Possibly once SPRK is activated by Cdc42, SPRK has a reduced affinity for the GTPase as has been demonstrated with PAK-2 (21). Furthermore, the sites of in vitro autophosphorylation of SPRK expressed with and without Cdc42, and isolated from mammalian cells, are essentially identical as judged by mapping of tryptic phosphopeptides (data not shown). The mechanism by which the highly conserved PAK family members (PAK-1, -2, and -3) are activated by Rac and Cdc42 has been well studied. Increased autophosphorylation activity is observed upon incubation of purified activated Cdc42 with purified PAK. In contrast to PAK, purified, catalytically active SPRK cannot be further activated in vitro by the addition of GTP-bound Cdc42. These data are consistent with a catalytic model in which Cdc42 activates SPRK in vivo, but is not required to maintain SPRK in its activated state. We therefore decided to assess whether Cdc42 induces differential phosphorylation of SPRK in vivo.
Phosphoamino acid analysis of in vivo labeled SPRK revealed serine and threonine, but no tyrosine, phosphorylation. Two-dimensional tryptic phosphopeptide mapping studies of in vivo labeled SPRK, expressed with or without constitutively active Cdc42, showed similar phosphopeptide maps, with major differences observed in a triangular cluster of phosphopeptides b, c, and d (Fig. 9A). In the map of SPRK alone, phosphopeptides b and c are present, but at low levels. When activated Cdc42 is coexpressed with SPRK, phosphopeptide b is not detected and phosphopeptides c and d appear at high levels. Since the change in in vivo phosphorylation of SPRK with Cdc42 correlates with increased SPRK activity, it is likely that phosphopeptides c and d contain activating phosphorylation sites. Phosphopeptides c and d may be distinct phosphopeptides. However, since their chromatographic mobilities are essentially identical, it is also possible that phosphopeptides c and d result from differential trypsin digestion. Phosphopeptides b and c lie on a diagonal which slopes toward the anode, characteristic of peptides that are phosphoisomers. Upon phosphorylation, the negative charge and polar nature of the added phosphate group reduces a peptides mobility in both the electrophoretic and chromatographic dimensions (59). Therefore, phosphopeptide c may differ from phosphopeptide b by the addition of a phosphate group(s). This is consistent with the observation that when activated Cdc42 is coexpressed with SPRK, phosphopeptide c emerges while phosphopeptide b disappears.
Cdc42 may induce differential SPRK autophosphorylation in vivo or, alternatively, another SPRK-activating kinase may be responsible for the in vivo change in SPRK phosphorylation. Because Cdc42 is geranylgeranylated (60) and has been localized to cellular membranes (61, 62, 63) as well as to cytoskeletal elements (64), it is possible that Cdc42 recruits SPRK to the vicinity of an activating kinase. SPRK and the serine/threonine kinase Raf both appear to function as MKKKs, activating the JNK and ERK pathways, respectively. The idea that Cdc42 may recruit SPRK to an activating kinase is reminiscent of the proposed mechanism by which the small GTPase Ras activates Raf. Analogous to our findings with Cdc42 and SPRK, the addition of purified, activated Ras to Raf in vitro is not sufficient for full activation of Raf. However, appending a membrane targeting motif to the COOH terminus of Raf causes Raf translocation and activation in the absence of activated Ras (1).
It has been recently shown that PAK-3 phosphorylates Raf in
vivo and in vitro leading to an increase in Raf
activity (65), although it has yet to be determined whether this event
requires Raf translocation. In yeast, the PAK homolog STE20 functions
as an MKKK in the activation of a yeast MAPK pathway. By analogy, potential SPRK-activating kinases may include PAK-related kinases (53)
such as hematopoietic protein kinase-1. In coexpression studies
hematopoietic protein kinase-1 binds to SPRKs SH3 domain and
phosphorylates SPRK, but the effect on SPRK activity is not reported
(66). Interestingly, unlike PAK-1, -2, and -3, hematopoietic protein
kinase-1 lacks a CRIB motif. Our finding that catalytically active SPRK
cannot be further activated in vitro by GTP-bound Cdc42
suggests that the GTPase activates SPRK differently than the PAKs.
Whereas the PAKs can be activated in vitro by interaction with unprenylated GTP-bound Cdc42, the activation of SPRK by Cdc42 appears to require prenylation of Cdc42, the cellular environment, or
an as yet unidentified cellular component. Our data supports a model in
which the in vivo CRIB-dependent interaction of
SPRK and Cdc42 either allows SPRK to adopt a conformation that leads to
autophosphorylation or recruits SPRK to the vicinity of a
serine/threonine kinase that phosphorylates and activates SPRK.
Determination of the precise sites of Cdc42-induced phosphorylation of
SPRK, coupled with subcellular localization studies, should shed
further light on the detailed mechanism of SPRK activation by Cdc42.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to Hua Zhang for expression and purification of recombinant GST-Cdc42; Arie Abo (Onyx Pharmaceuticals) for providing purified PAK-2; and Avi Ashkenazi (Genentech, Inc.) for mammalian Cdc42 expression vectors. We thank Donald B. Jump and Walter J. Esselman for valuable discussions and critical reading of the manuscript. We appreciate the support and reagents from Paul Godowski (Genentech, Inc.).
| |
FOOTNOTES |
|---|
* This work was supported in part by National Institutes of Health Grant CA76306.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Contributed equally to the results of this work.
Supported by the National Science Foundation-REU program at
Michigan State University.
** To whom correspondence should be addressed: Depts. of Physiology and Biochemistry, 108 Giltner Hall, Michigan State University, East Lansing, MI 48824. Tel.: 517-355-6475; Fax: 517-355-5125, E-mail: gallo@psl.msu.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
ERK, extracellular
regulated protein kinase;
MAPK, mitogen-activated protein kinase;
PAK, p21-activated kinase;
CRIB, Cdc42/Rac-interactive binding;
ACK, activated Cdc42HS-associated kinase;
WASP, Wiskott-Aldrich Syndrome
protein;
SPRK, SH3 domain-containing proline-rich kinase;
SH3, Src-homology 3;
MLK, mixed-lineage kinase;
JNK, c-Jun
NH2-terminal kinase;
MKK, MAPK kinase;
JIP, JNK interacting
protein;
PCR, polymerase chain reaction;
PBS, phosphate-buffered
saline;
PAGE, polyacrylamide gel electrophoresis;
GST, glutathione
S-transferase;
MBP, maltose-binding protein;
GTP
S, guanosine 5'-3-O-(thio)triphosphate.
| |
REFERENCES |
|---|
|
|
|---|
| 1. |
Stokoe, D.,
Macdonald, S. G.,
Cadwallader, K.,
Symons, M.,
and Hancock, J. F.
(1994)
Science
264,
1463-1467 |
| 2. |
Van Aelst, L.,
and D'Souza-Schorey, C.
(1997)
Genes Dev.
11,
2295-2322 |
| 3. |
Hall, A.
(1998)
Science
279,
509-514 |
| 4. | Nobes, C. D., and Hall, A. (1995) Cell 81, 53-62[CrossRef][Medline] [Order article via Infotrieve] |
| 5. | Kozma, R., Ahmed, S., Best, A., and Lim, L. (1995) Mol. Cell. Biol. 15, 1942-1952[Abstract] |
| 6. | Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. (1992) Cell 70, 401-410[CrossRef][Medline] [Order article via Infotrieve] |
| 7. |
Olson, M. F.,
Ashworth, A.,
and Hall, A.
(1995)
Science
269,
1270-1272 |
| 8. | Qiu, R-G., Chen, J., Kirn, D., McCormick, F., and Symons, M. (1995) Nature 374, 457-459[CrossRef][Medline] [Order article via Infotrieve] |
| 9. | Khosravi-Far, R., Solski, P. A., Clark, G. J., Kinch, M. S., and Der, C. J. (1995) Mol. Cell. Biol. 11, 6643-6453 |
| 10. | Qiu, R.-G., Abo, A., McCormick, F., and Symons, M. (1997) Mol. Cell. Biol. 17, 3449-3458[Abstract] |
| 11. | Tang, Y., Chen, Z., Ambrose, D., Liu, J., Gibbs, J. B., Chernoff, J., and Field, J. (1997) Mol. Cell. Biol. 17, 4454-4464[Abstract] |
| 12. |
Wu, W. J.,
Lin, R.,
Cerione, R. A.,
and Manor, D.
(1998)
J. Biol. Chem.
273,
16655-16658 |
| 13. |
Whitehead, I. P.,
Abe, K.,
Gorski, J. L.,
and Der, C. J.
(1998)
Mol. Cell. Biol.
18,
4689-4697 |
| 14. |
Tang, Y., Yu, J.,
and Field, J.
(1999)
Mol. Cell. Biol.
19,
1881-1891 |
| 15. |
Zhang, S.,
Han, J.,
Sells, M. A.,
Chernoff, J.,
Knaus, U. G.,
Ulevitch, R. J.,
and Bokoch, G. M.
(1995)
J. Biol. Chem.
270,
23934-23936 |
| 16. | Coso, O. A., Chiariello, M., Yu, J.-C., Teramoto, H., Crespo, P., Xu, N., Miki, T., and Gutkind, J. S. (1995) Cell 81, 1137-1146[CrossRef][Medline] [Order article via Infotrieve] |
| 17. |
Bagrodia, S.,
Dérijard, B.,
Davis, R. J.,
and Cerione, R. A.
(1995)
J. Biol. Chem.
270,
27995-27998 |
| 18. | Brown, J. L., Stowers, L., Baer, M., Trejo, J., Coughlin, S., and Chant, J. (1996) Curr. Biol. 6, 598-605[CrossRef][Medline] [Order article via Infotrieve] |
| 19. | Minden, A., Lin, A., Claret, F.-X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157[CrossRef][Medline] [Order article via Infotrieve] |
| 20. | Hill, C. S., Wynne, J., and Treisman, R. (1995) Cell 81, 1159-1170[CrossRef][Medline] [Order article via Infotrieve] |
| 21. | Manser, E., Leung, T., Salihuddin, H., Zhao, Z., and Lim, L. (1994) Nature 367, 40-46[CrossRef][Medline] [Order article via Infotrieve] |
| 22. |
Bagrodia, S.,
Taylor, S. J.,
Creasy, C. L.,
Chernoff, J.,
and Cerione, R. A.
(1995)
J. Biol. Chem.
270,
22731-22737 |
| 23. | Martin, G. A., Bollag, G., McCormick, F., and Abo, A. (1995) EMBO J. 14, 1970-1978[Medline] [Order article via Infotrieve] |
| 24. | Manser, E., Huang, H.-Y., Loo, T.-H |