1 mek kinase 2 binds and activates protein kinase c-related
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MEK Kinase 2 Binds and Activates Protein Kinase C-related Kinase 2: Bifurcation of Kinase Regulatory Pathways at the Level of a MAPK Kinase Kinase
Weiyong Sun1, Sylvie Vincent2, Jeffrey Settleman2 and Gary L. Johnson1
1Department of Pharmacology, University of Colorado Health Sciences Center and University of Colorado Cancer Center, Denver, CO 80262, 2Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, MA 02129 Address correspondence to: Gary L. Johnson, Ph.D. Department of Pharmacology, C-236 University of Colorado Health Sciences Center 4200 East Ninth Avenue Denver, CO 80262 Phone: 303-315-1009 FAX: 303-315-1022 E-mail: [email protected] Supported by NIH grants DK37871, GM30324, DK48845 and CA58187 Running title: MEKK2 activates PRK2
Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on May 18, 2000 as Manuscript M003148200 by guest on M
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Abstract
MEK kinase 2 (MEKK2) is a 70 kDa protein serine/threonine kinase that has been shown to
function as a mitogen activated protein kinase (MAPK) kinase kinase. MEKK2 has its kinase
domain in the COOH-terminal moiety of the protein. The NH2-terminal moiety of MEKK2 has
no signature motif that would suggest a defined regulatory function. Yeast two-hybrid screening
was performed to identify proteins that bind MEKK2. Protein kinase C-related kinase 2 (PRK2)
was found to bind MEKK2, PRK2 has been previously shown to bind RhoA and the SH3
domain of Nck. PRK2 did not bind MEKK3, which closely related to MEKK2. The MEKK2
binding site maps to amino acids 637-660 in PRK2 that is distinct from the binding sites for
RhoA and Nck. This sequence is divergent in the closely related kinase PRK1 (PKN) that did not
bind MEKK2. In cells, MEKK2 and PRK2 are co-immunoprecipitated and PRK2 is activated by
MEKK2. Similarly, purified recombinant MEKK2 activated PRK2 in vitro. MEKK2 activation
of PRK2 is independent of MEKK2 regulation of the c-Jun NH2-terminal kinase (JNK) pathway.
MEKK2 activation of PRK2 results in a bifurcation of signaling for the dual control of MAPK
pathways and PRK2 regulated responses.
Introduction
Mitogen activated protein kinases (MAPKs) are components of a three kinase module that also
includes a MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK)(1). MEKK2 is a
MAPKKK that we have shown is activated in response to several extracellular stimuli including
antigen receptors in T cells and mast cells and growth factors such as EGF and Kit ligand (stem
cell factor) (2,3). We recently demonstrated that MEKK2 translocates to the cytoplasmic face of
the contact site of T cells interacting with an antigen loaded presenting cell (3). MEKK2 but not
MEKK1 or MEKK3 is translocated to the T cell interface with the antigen presenting cell.
MEKK2 is translocated and activated within seconds of exposure of T cells to antigen
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presentation. MEKK2 activation was required for its translocation because kinase-inactive
MEKK2 was not recruited to the contact between the T cell and antigen presenting cell. In this
system, MEKK2 signaling was found to be required for maintenance of the conjugate formation
between T cell and antigen presenting cell. Although poorly defined, the activation of MEKK2
seems to involve more than one pathway. For example antigen receptor activation of MEKK2 is
inhibted by wortmannin indicating the activation of phosphatidylinositol 3-kinase (PI3K) is
required. In contrast, EGF stimulation of MEKK2 in COS-7 cells is insensitive to wortmannin.
MEKK2 is a 70 kDa serine-threonine kinase that has its kinase domain in the COOH-terminal
half of the protein (4). Analysis of the NH2-terminal moiety of MEKK2 does not reveal an
identifiable motif that has been defined in other proteins that are known to regulate protein-
protein (i.e., SH3, proline-rich, etc.) or protein-lipid (i.e., PH domains) interactions. Thus, the
sequence of MEKK2 does not readily allow predictions of its regulation and interactions with
other molecules in the cell. In an attempt to define the regulation of MEKK2 in antigen and
growth factor responses we have performed two-hybrid analysis to identify proteins that bind
MEKK2. Several binding partners were identified in this screen. As we detail in this report, one
binding partner that was identified in this screen was the protein kinase C-related kinase 2
(PRK2).
Protein kinase C (PKC)-related kinases (PRKs) constitute a subclass of lipid and proteolysis-
activated serine/threonine kinases that are highly homologous to PKCs in their catalytic domains
(5-7). Human PRK1 (also known as PKN, for protein kinase N) and PRK2 share structurally
very similar kinase domains (87% identity), but their regulatory N-termini are less conserved
(48% identity) (6). PKN and PRK2 have been demonstrated to be an effector of the small
GTPase, Rho (8-12). Binding of Rho.GTP activates the kinase activity of PKN and PRK2 (12).
PRK2 may also bind another small GTPase, Rac, bound to GTP (12). Rho and Rac are involved
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in the regulation of cytoskeletal organization as well as many other cellular processes including
membrane trafficking, activation of JNK and p38 MAPK pathways, transcription, cell growth
and development (13-16). Consistent with the finding that PKN and PRK2 are effectors for Rho
is the observations that PKN and PRK2 can enhance or mediate changes in the actin cytoskeleton
and gene transcription (9,12,17-20). RhoB has also been reported to mediate PKN association
with endosomes (21).
In addition to its binding to Rho and Rac, PRK2 binds the middle SH3 domain of the SH2-SH3
adaptor protein, Nck (9). PRK2 is, therefore, predicted to be recruited to tyrosine phosphorylated
proteins that bind the Nck SH2 domain. Nck could also bind to proteins having the proline-
directed SH3 binding motif for the first SH3 domain of Nck. Thus, PRK2 activation may
coordinate receptor protein tyrosine kinase signaling with Rho-activated pathways (9,22).
Consistent with this hypothesis is reports that Nck also binds the Wiskott-Aldrich syndrome
protein (WASP) and the p21-activated protein kinase (PAK) 1 and 3 (9,23). WASP and PAK are
effectors for Rac and Cdc42 (24,25). Both WASP and PAKs are involved in regulating the
cytoskeleton. This suggests that like PRK2, Nck could localize Rho-related GTP binding
proteins in the cell for control of the actin cytoskeleton and tyrosine kinase signaling.
Finally, a recent report indicated that the C-terminal 77 residues of PRK2 (termed PIF, the PDK1
interacting fragment) bind the kinase domain of the 3-phosphoinositide-dependent protein
kinase-1 (PDK1) (26). Thus, PRK2 appears to participate in a diverse set of inter-related
signaling programs suggesting it may have a scaffolding function to organize specific signal
transduction responses in the cell. Consistent with this prediction is the observation that PRK2
was rapidly cleaved by caspase3-like proteases during Fas- and staurosporine-induced apoptosis
(27,28). This cleavage event resulted in increased kinase activity but also releases the kinase
domain from tethering to Rho and Nck which would effectively disrupt the signaling complex.
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In the present study we show that MEKK2 and PRK2 are binding partners. MEKK2 interaction
activates PRK2 kinase activity.
Materials and Methods
Antibodies: Mouse monoclonal anti-HA (12CA5), anti-Flag (M5) and anti-V5 antibodies were
purchased from Boehringer Mannheim, Sigma and Invitrogen, respectively. Rabbit polyclonal
anti-MEKK2 antibody has been described elsewhere (2).
Yeast two-hybrid screening of cDNA library and interaction analysis: Full-length mouse
MEKK2 was fused in-frame to the C-terminus of the bacterial DNA binding protein LexA in
vector BTM116 (29). It was used to transform the yeast reporter strain L40 (30) together with
mouse T-cell lymphoma library cDNA (Clontech) cloned C-terminal to the activation domain of
Gal4 (GAD) in plasmid pACT (31). A total of 2.4 x 106 transformants were plated on synthetic
complete plates lacking trytophan, leucine, histidine, lysine and uracil (SC-His) but
supplemented with 10 mM 3-aminotriazole (3-AT). After 3.5 days of incubation at 30 oC, 57 of
the fastest growing clones were picked and streaked on SC-His +15mM 3-AT media. The largest
colony from each of the original 57 clones was then streaked on SC+His plates and tested for β-
galactosidase (β-gal) production using a filter-lift assay (32). Yeast total DNAs were isolated
from LacZ+ clones and the library plasmids were rescued into E. coli strain HB101 (Promega).
After re-transformation into L40 with pLexA-MEKK2, 24 clones were confirmed to be His+
LacZ+.
For two-hybrid interaction analysis, L40 cells were spread directly on SC-His + 3-AT media
after transformation, or streaked on SC-His + 3-AT plates after first growth on plates with
histidine. A second two-hybrid system was also used in the study; in this case yeast CG1945
(Clontech) was used as the host strain, and peptides were fused to the Gal4 DNA binding domain
(GBD) in vector pAS2-1 (Clontech) as “baits”. In both systems, “prey” peptides were fused
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either to the GAD in plasmid pACT (31) or its derivative pACT2 (33), or to the activation
domain of VP16 (VAD) in plasmid pVP16 (30). Quantitation of β-gal activity was assayed on
liquid cultures using o-nitrophenyl-β-D-galactopyranoside (ONPG) as substrate (34).
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Mammalian cell transfection and co-immunoprecipitation: HEK293 cells were grown to 50-
80% confluence in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) and penicillin-streptomycin, and after washing with DMEM were
transfected with various combinations of expression plasmids in the presence of Lipofectamine
(Gibco). Transfected cells were grown in DMEM + 10% FBS for 36-48 hours and harvested in
lysis buffer I (50 mM TrisHCl pH 7.5, 100 mM NaCl, 2 mM MgCl2, 1 mM Na3VO4, 0.5% Triton
X-100, 2 mM PMSF, 10 µg/ml pepstatin A, 50 µM leupeptin, 2 µg/ml aprotinin and 1 mM
DTT). Lysates were cleared by brief centrifugation and 400-800 µg lysates were incubated with
M5 or 12CA5 antibody (as indicated in the figure legends) in a total volume of 400 µl for 2 hr at
4oC with rocking. Thirty µl of 1:1 rec-protein G Sepharose 4B slurry (Zymed) was added to the
mixture and the incubation continued for 1 hr. Beads were washed 3 times with 400 µl lysis
buffer, heat denatured in 1x SDS-PAGE loading buffer and resolved on a 10% SDS-PAGE gel.
Proteins were transferred to a Protran nitrocellulose membrane (Schleicher & Schuell) and
immunoblotted using HRP-coupled goat-anti-mouse secondary antibody and enhanced
chemiluminescence (NEN).
In vitro binding assays: For analyzing binding of PRK2/PKN to endogenous MEKK2, 10 µg
bacterially expressed and purified GST fusion proteins pre-bound to glutathione-Sepharose 4B
beads (Pharmacia) were incubated with 400 µg HEK293 cell lysates at 4oC for 4 or 10 hr with
gentle rocking. After 3 washes beads were subject to SDS-PAGE and Western blotting with anti-
MEKK2 antibody and HRP-conjugated protein A.
Binding of recombinant baculovirus-expressed MEKK2 with transfected PRK2 was performed
by using 800 µg lysate of HEK293 cells that were transfected with either pFlag-hPRK2 or empty
vector. One µg purified MEKK2 was added to the lysate for 2 hr or overnight, followed by
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incubation with M5 antibody and protein G-Sepharose 4B beads for another 2 hr. Binding of
MEKK2 was detected as described above.
In vitro kinase assays: HEK293 cells were transfected with Flag-hPRK2, kinase-inactive Flag-
hPRK2KE or empty vector. Cells were lysed in a higher-strength lysis buffer II (50 mM TrisHCl
pH 7.5, 150 mM NaCl, 50 mM NaF, 5 mM Na4P2O7, 1 mM Na3VO4, 1% Triton X-100, 1 mM
EDTA, 1 mM EGTA, 2 mM PMSF, 10 µg/ml pepstatin A, 50 µM leupeptin, 2 µg/ml aprotinin ,
and 1 mM DTT), and lysates (1500 µg for PRK2KE- and vector-transfected cells) containing
equal amounts of PRK2 or PRK2KE were precipitated with M5 antibody and rec-protein G-
Sepharose 4B beads. Beads were washed twice with lysis buffer II and twice with kinase buffer
(20 mM HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl2 and 5 mM MnCl2) and suspended with 80
µl kinase buffer. Twenty µl of suspension was used in a kinase reaction with 0.5 µg purified
MEKK2 and γ-32P-ATP (90 nM, 4500 Ci/mmol) in a total of 50 µl; in some experiments bovine
serum albumin (BSA) was supplied in reactions without MEKK2 to equalize the amounts of total
protein. Heat-inactivation of MEKK2 was achieved by heating at 1000 C for 15 minutes and
chilling on ice. Kinase reactions were incubated at 30 0C for 20 minutes and terminated by
adding SDS-PAGE loading buffer.
For analysis using myelin basic protein (MBP) as substrate, equal amounts of epitope-tagged
proteins were immunoprecipitated from transfected HEK293 cells (for cells transfected with
empty vector, kinase-inactive PRK2 or MEKK2, 800 µg of lysate was used). Beads were washed
and incubated with 20 µg MBP (Upstate Biotechnology) in a 50 µl kinase reaction.
For analysis of endogenous JNK kinase activity, 200 µg of transfected HEK293 cell lysates were
incubated with 20 µg bacterially expressed and purified GST-cJun1-79 or GST protein pre-bound
to beads in 400 µl lysis buffer I at 4 0C for 2 hr with rocking. Fifty µl of kinase buffer containing
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γ-32P-ATP was then added to the washed beads and the kinase reaction was performed as
described.
Results
MEKK2 interacts with PRK2: In an effort to identify binding partners of MEKK2 the yeast
two-hybrid system (35) was used to screen a mouse T-cell lymphoma cDNA library. Full-length
MEKK2 was fused to the bacterial DNA binding protein LexA and used as “bait” to transform
the yeast reporter strain L40 (30) along with the library cDNA fused to the Gal4 activation
domain (GAD) (36). A total of 2.4 x 106 transformants were plated and 24 clones were strong
positives for both His3 and LacZ reporter constructs. Among them, two clones were identical
isolates encoding the sequence corresponding to residues 479 to 670 of human PRK2 (hPRK2).
An L40 strain expressing both LexA-MEKK2 and GAD-mPRK2aa479-670 was prototrophic for
histidine (Fig. 1A) and exhibited a β-galactosidase (β-gal) activity at least 20-fold higher than
the control transformants (Fig. 1B). To further demonstrate specific interaction between MEKK2
and PRK2, the bait and prey constructs were switched and their binding was tested in a different
two-hybrid system. The mPRK2 aa479-670 was fused to the Gal4 DNA binding domain (GBD)
and MEKK2 was fused to the activation domain of the herpes simplex virus protein VP16
(VAD) (30). Yeast strain CG1945 (from Clontech) transformed with GBD-mPRK2aa479-670
and VAD-MEKK2 could grow into colonies on synthetic complete plates lacking histidine (SC-
His) and supplemented with 15 mM 3-aminotriazole (3-AT). In contrast, cells transformed with
control plasmids plus either GBD-mPRK2aa479-670 or VAD-MEKK2 were not able to grow
even at 1 mM 3-AT (data not shown). We also tested binding of MEKK2 to full-length PRK2
using the two-hybrid analysis using the full length hPRK2. MEKK2 and hPRK2 exhibited a
weaker but still positive interaction in the histidine prototrophy assay compared to PRK2aa479-
670 (data not shown). The weaker interaction of the full length protein relative to the binding
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domain for the partner protein is a common finding in yeast two-hybrid analysis. The findings
suggested that PRK2 is capable of binding MEKK2.
[Figure 1]
To substantiate MEKK2-PRK2 interaction in mammalian cells, HEK293 cells were transfected
with HA-epitope-tagged MEKK2, together with Flag-tagged full-length hPRK2 or control empty
vector plasmid. Cell lysates were immunoprecipitated with anti-Flag antibody and Western
blotted with anti-HA antibody after separation by SDS-PAGE. Fig. 1C shows that MEKK2 was
co-immunoprecipitated with PRK2 but HA-MEKK2 was not detected in immunoprecipitates
from control transfections. Reciprocally, association of PRK2 with MEKK2 was readily detected
when cell lysates were precipitated with anti-HA antibody to immunoprecipitate MEKK2 and
subsequently immunoblotted with the anti-FLAG antibody to detect PRK2 (Fig. 1D). These
findings demonstrate that MEKK2 and PRK2 associate with each other by two independent
experimental techniques: yeast two-hybrid analysis and co-immunoprecipitation.
Refinement of the PRK2 binding region for MEKK2: The PRK2 region required for MEKK2
binding was mapped using the yeast two-hybrid method. Serial truncations of the mPRK2aa479-
670 fragment were fused to the activation domain of Gal4 and tested for their ability to retain
binding of MEKK2 in the yeast L40 strain (Fig. 2A). This approach identified a region of 55
amino acids (corresponding to residues 616 to 670 of hPRK2) that showed binding for MEKK2,
as measured by growth of transformants, comparable to the 479-670 fragment of PRK2.
[Figure 2]
A similar approach was taken in an attempt to define the region of MEKK2 capable of binding
mPRK2aa479-670 (Fig. 2B). However, neither the N-terminal regulatory region nor the C-
terminal kinase domain of MEKK2 alone showed significant interaction with PRK2. One
interpretation of these findings is that PRK2 may interact with more than one site in MEKK2,
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requiring both its NH2- and COOH-termini. Alternatively, the MEKK2 NH2-terminal sequences
expressed may not fold properly in the absence of the COOH-terminal kinase domain. Attempts
at expressing NH2-terminal MEKK2 and MEKK3 sequences in E. coli have proven extremely
difficult with extreme sensitivity to proteases (unpublished observations). This finding is
consistent with the notion that the MEKK2 NH2 terminal constructs do not fold properly and
therefore are not a reliable reagent for mapping the MEKK2 interaction domain for PRK2. Most
likely, a more detailed mutagenesis strategy of full length MEKK2 will probably have to be
undertaken to map the PRK2 interaction sequences.
Specificity of the MEKK2-PRK2 interaction: MEKK2 and MEKK3 are closely related to each
other. The kinase domains of MEKK2 and MEKK3 are 96% conserved in amino acid sequence.
The NH2-terminal moieties of MEKK2 and MEKK3 are approximately 55% conserved in
primary sequence. Therefore, we tested the possible binding of MEKK3 to the PRK2aa479-670
sequence that binds MEKK2 using the yeast two-hybrid system. Yeast L40 cells were
transformed with LexA-MEKK3 plus GAD-mPRK2aa479-670 and plated on SC+His media.
Transformant colonies were then streaked on SC-His + 3 mM 3-AT plates to test for the
transactivation of the His3 reporter gene (Fig. 3A), or grown in liquid media and analyzed for
LacZ expression (Fig. 3B). As shown, L40 cells carrying plasmids LexA-MEKK3 and GAD-
mPRK2aa479-670 were not able to grow on SC-His + 3 mM 3-AT plates, even after incubation
for over one week (not shown). In comparison, cells with LexA-MEKK3 plus VAD-14-3-3ε
showed robust growth on the same minimal plates and strong β-gal activity, consistent with our
previous report that MEKK3 binds 14-3-3ε protein (37). This indicates that LexA-MEKK3 was
expressed and nucleus-localized, and the failure of GAD-mPRK2aa479-670 to transactivate the
reporter genes was due to its inability to bind MEKK3. In support of this conclusion, in a
different two-hybrid system, we found that yeast CG1945 cells transformed with GBD-
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mPRK2aa479-670 plus VAD-MEKK3 were negative for histidine prototrophy (data not shown);
in contrast, as mentioned above, GBD-mPRK2aa479-670 bound VAD-MEKK2 in CG1945 cells.
These findings indicated that the PRK2 interaction was specific for MEKK2 because a closely
related kinase, namely MEKK3, did not bind PRK2aa479-670. The results also argue that the
binding of PRK2aa479-670 requires specific sequences in the NH2-terminus of MEKK2 that are
not conserved in MEKK3 because the kinase domains are nearly identical.
[Figure 3]
To experimentally confirm that MEKK2 but not MEKK3 binds PRK2 in mammalian cells, we
transfected HEK293 cells with Flag-hPRK2 and either C-terminally V5-epitope-tagged MEKK3
(MEKK3-V5) or MEKK2-V5. Cell lysates were immuno-precipitated with anti-Flag antibody
and immunoblotted with anti-V5 antibody. As predicted, MEKK2-V5 co-immunoprecipitated
with Flag-hPRK2 but MEKK3-V5 did not (Fig. 3C). MEKK3-V5 was expressed at similar
protein levels as MEKK2-V5 and Flag-hPRK2 was expressed at similar levels in the presence of
either MEKK2-V5 or MEKK3-V5 (Fig. 3C). Therefore MEKK2 but not MEKK3 binds PRK2.
Obviously, it was also important to determine if MEKK2 could bind PKN (PRK1), a kinase
sharing 87% homology in the kinase domain and 48% homolgy in the NH2-terminal regulatory
domain to PRK2. Thus, a fragment of human PKN (residues 477 to 628) that is homologous to
PRK2aa479-670 was fused to the activation domain of Gal4 and tested for its ability to interact
with MEKK2 in yeast L40. By the criteria of histidine prototrophy and β-gal activity, no
significant binding could be demonstrated. The corresponding regions from the human and the
mouse PRK2 protein interacted strongly with MEKK2 (Fig. 4A & B). The yeast two-hybrid data
indicates MEKK2 binds PRK2 but not PKN.
[Figure 4]
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To gain direct evidence for the selectivity of MEKK2-PRK2 interaction, an in vitro binding
assay was employed. Initially, we generated glutathione S-transferase (GST) fusion constructs of
hPRK2aa479-670 and hPKNaa477-628. Unfortunately, despite a variety of expression
conditions we tried, neither protein was expressed in bacteria. As shown in Figure 2A, we had
mapped the binding site for MEKK2 to a 55-residue region (aa616-670) of mPRK2. Therefore,
we decided to fuse GST to hPRK2aa616-670 and the corresponding region (aa585-628) of
hPKN. Purified GST, GST-hPKNaa585-628 or GST-hPRK2aa616-670, pre-bound to
glutathione-Sepharose 4B beads, was incubated with HEK293 cell lysates. After extensive
washing, bead bound proteins were resolved by SDS-PAGE and immunoblotted with anti-
MEKK2 antibody (Fig. 4C). The results definitively show hPRK2aa616-670 binds MEKK2 at
endogenous expression levels of MEKK2 in HEK 293 cells. MEKK2 does not bind hPKNaa585-
628.
Fig. 4D shows the alignment of the MEKK2 binding region for hPRK2 and mPRK2 with PKN.
Within the start of the kinase domain residues 619-628 of hPKN are identical to the
corresponding sequences of PRK2 (aa661-670). We also demonstrated that PRK2aa479-622
does not bind MEKK2 indicating that amino acids 623-660 encode the PRK2 binding sequence
for MEKK2. The sequence corresponding to hPRK2aa637-660 where the kinase domain begins
is particularly conserved between the mouse and human PRK2 sequences and either absent or
divergent in PKN consistent with this sequence contributing to the PRK2 domain that binds
MEKK2.
MEKK2 activates PRK2: In vivo activation of PRK2 was characterized by analyzing the kinase
activity of PRK2 immunoprecipitated from transfected HEK293 cells (Fig. 5). Since no
physiological PRK2 substrates have been identified, we used myelin basic protein (MBP) as a
substrate in the PRK2 kinase assay (7,12). In the absence of transfected MEKK2, expressed
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Flag-tagged hPRK2 had little measureable kinase activity (Figure 5A, lane 2). Strikingly, when
Flag-hPRK2 was co-transfected with MEKK2, a dramatic increase in PRK2 activity was
observed in anti-Flag immunoprecipitations from cell lysates (lane 3). Surprisingly, cell
expression of kinase inactive MEKK2 also activated PRK2 (lanes 4 &5). This result suggested
that an interaction independent of MEKK2 kinase activity resulted in the activation of PRK2.
However, the level of PRK2 activation was less with kinase inactive MEKK2 than that observed
with the wild type kinase indicating that wild type MEKK2 was more active than the kinase
inactive mutant in stimulating PRK2 activity. Immunoblotting of cell lysates (Panel B) revealed
that even when the kinase-inactive form of MEKK2 was expressed at a higher level than the wild
type MEKK2, it was less effective than the wild type kinase in activating PRK2 (compare lanes
3 and 5). PRK2 is clearly the kinase assayed in the immunoprecipitates because expression of
Flag-tagged kinase inactive PRK2 (Flag-PRK2KE) in the presence of MEKK2 does not result in
MBP phosphorylation in the in vitro kinase assay (lane 6). The immunoblots in panel B also
show that the expressed wild type MEKK2 migrates as a slower band in SDS-PAGE than the
kinase inactive MEKK2 due to autophoshorylation of expressed MEKK2. The kinase inactive
MEKK2 does not show this gel shift. The lower bands in Panel B seen most clearly in lanes 1
and 2 are non-specific bands recognized by the anti-HA antibody and are unrelated to MEKK2.
[Figure 5]
The ability of MEKK2 to activate PRK2 was verified using purified recombinant MEKK2 (Fig.
6). Immunoprecipitated PRK2 was incubated with MEKK2 purified from Sf9 cells infected with
baculovirus encoding the MEKK2 cDNA. Immunoprecipitations demonstrated that purified
recombinant MEKK2 bound to Flag-PRK2 similar to endogenous cellular MEKK2 (not shown).
Immunoprecipitated PRK2 by itself had little kinase activity (lane 2). Addition of MEKK2 to the
Flag-PRK2 immunoprecipitate dramatically increased the phosphorylation of PRK2 (compare
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lanes 2 and 3). Interestingly, incubation of PRK2 with heat-inactivated MEKK2 to inhibit
MEKK2 kinase activity also stimulated the phosphorylation of PRK2 (lane 1). The increased
phosphorylation of PRK2 required the kinase activity of PRK2, demonstrating the MEKK2
dependent stimulation of PRK2 autophosphorylation (compare lanes 1, 3 and 5).
Co-incubation of hPRK2 and MEKK2 led to an increased phosphorylation of MEKK2 (lane 3).
Clearly, kinase inactive PRK2 (PRK2KE) does not enhance MEKK2 phosphorylation (lane 5)
and heat-inactivated MEKK2 is not a substrate for activated PRK2 (lane 1). Thus, PRK2 may
phosphorylate MEKK2 or increase the autophosphorylation activity of MEKK2 resulting from
their interaction.
[Figure 6]
PRK2 does not influence MEKK2 activation of the JNK pathway: HEK293 cells were
transfected with MEKK2, hPRK2, or a combination of both, and cell lysates assayed for JNK
activity (Fig. 7). Expression of MEKK2 gave a pronounced stimulation of JNK activity (compare
lanes 1, 2 and 3). Cells transfected with hPRK2 showed basal JNK activity (compare lanes 3 and
4). Furthermore, cells co-transfected with hPRK2 plus MEKK2 showed the same JNK activity as
cells transfected with MEKK2 alone (compare lanes 1 and 2), indicating that PRK2 does not
regulate the JNK pathway under conditions where MEKK2 activates both the JNK pathway and
PRK2.
[Figure 7]
Discussion
PRK2 apparently has a complex regulation responding to several different regulatory pathways
in cells (6,38). It has been shown to be activated by RhoA and to bind Rac (12,39). In addition,
PRK2 can be activated by cardiolipins and is a substrate for caspase-dependent cleavage and
activation (7,27,28,40). Activation of PRK2 by RhoA, cardiolipins and protease-catalyzed
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cleavage appears to involve the release of pseudo-substrate inhibition of PRK2 activity (7,41).
These findings indicate that the primary mechanism defined for PRK2 activation involves its
interaction with regulatory molecules that release the internal inhibition of the kinase encoded
within the PRK2 sequence.
The results we have presented demonstrate that MEKK2 and PRK2 are binding partners. The
region of PRK2 that binds MEKK2 is divergent in the closely related PKN protein and PKN
does not interact with MEKK2. This finding demonstrates a unique regulatory function for the
control of PRK2 activity relative to PKN by MEKK2. Importantly, the interaction of MEKK2
and PRK2 activates PRK2 kinase activity. MEKK2 regulation of PRK2 was demonstrated in
cells and in vitro with purified recombinant MEKK2. As mentioned above, previously described
regulatory mechanisms for the activation of PRK2 have involved the release of a pseudo-
substrate inhibition of its kinase activity (7,41). Our findings with MEKK2 activation of PRK2
are consistent with a protein-protein interaction induced release of a pseudo-substrate inhibition
of PRK2 kinase activity, with the kinase activity of MEKK2 not being required for its activation
of PRK2.
The activation of one kinase by the interaction with a second kinase, independent of the second
kinase’s phosphorylation activity has been described previously. The kinase suppressor of Ras
(KSR) has been proposed to regulate the activity of Raf independent of its kinase activity (42).
Expression experiments indicate that modest expression of KSR can activate Raf and high
expression inhibits Raf activation (42,43). The function of KSR for Raf activation has been
proposed to be that of a scaffold for organization of the Raf-MEK-ERK signaling module
independent of its kinase function (44-48). PKCα has been shown to interact with and activate
phospholipase D (PLD) independent of PKCα kinase activity (49). Finally, the Ste20-like
germinal centre kinase (GCK) was shown to activate the JNK pathway independent of its kinase
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activity (50). The mechanism for kinase-inactive GCK activation of the JNK pathway was not
defined but was predicted to be due to protein-protein interactions reminiscent of KSR regulation
of the Raf-MEK-ERK pathway (47). These examples indicate that scaffolding and possibly
oligomerization of hetero-kinases can regulate the kinase activity of specific binding partners in
the macromolecular complex. MEKK2 regulation of PRK2 is another example of such a
regulatory mechanism.
Figure 8 shows a model of the potential organization of the PRK2-MEKK2 signaling complex in
cells. PRK2 has been shown to bind the middle SH3 domain of Nck (9), to bind PDK1 (26) and
to be activated by RhoA (12). These findings would suggest that multiple mechanisms exist for
the activation of PRK2 involving tyrosine kinase binding of Nck, phosphatidylinosotol 3,4,5
trisphosphate (PtdIns(3,4,5)P3) activation of PDK1, and stimulation of RhoA GTP binding, in
addition to PRK2 interaction with MEKK2. Based on these findings, our hypothesis is that
PRK2-MEKK2 interactions function to co-localize two protein kinases that regulate different
and divergent signaling pathways in the cell. The requirement for the organization of signaling
complexes for MAPK pathways in yeast and mammalian cells is becoming increasingly
apparent. In yeast it is clear that Ste5 and PBS2 have scaffolding functions for regulating the
mating response and activation of Hog-1 in response to high osmolarity, respectively. In
mammalian cells, MP-1 has been shown to function as a scaffold for MEK1 and ERK1 (51) and
KSR appears to be a scaffold for Raf, MEK and ERK (47). Similarly, JIP-1 was shown to
function as a scaffold for HPK1, MLK3, MKK7 and JNK1/2 (52). MEKK1 has also been
proposed to be a scaffold for MKK4 and JNK1/2 (53). Our findings may be most like that for
KSR and Raf in that the two kinases are not in the same pathway.
[Figure 8]
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Both MEKK2 and PRK2 have been shown to re-distribute in the cell following specific
stimulation of cells (3,54). MEKK2 has been shown to localize to the T cell receptor signaling
complex in response to antigen presentation. PRK2 has been shown to re-distribute from the
cytoplasm to the germinal vesicle soon after hormone treatment of starfish oocytes. RhoA is
widely distributed in the cytoplasmic compartment of the cell and signals from tyrosine kinases
and other GTP binding proteins can activate RhoA. Thus, it is likely that PRK2-MEKK2
signaling complexes could be formed by several different mechanisms in different locations in
the cell. At present the function of PRK2 is unclear. Findings from different laboratories have
suggested that PRK2 may be involved in the regulation of the cytoskeleton and specific gene
expression (9,12). MEKK2 is clearly involved in the regulation of the JNK (4) and ERK5 (W.
Sun and G. L. Johnson, in preparation) pathways. The association of PRK2 and MEKK2 would
allow for the coordinate regulation of their respective functions. In this sense, PRK2 and
MEKK2 would be components of a macromolecular signaling module. This module would
regulate multiple pathways in response to specific stimulatory inputs. Such a signaling module
need not be necessarily preformed but could be brought together, for example, by the activation
of RhoA, PI-3,4,5 kinase or a phosphotyrosine motif that binds the SH2 domain of Nck. It will
be important to define the structure and organization of modules such as that of PRK2- MEKK2
to understand the modular nature of signal transduction.
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Abbreviations: DMEM, Dulbecco modified Eagle medium; ERK, extracellular signal-
regulated kinase; β-gal, β-galactosidase; GAD, Gal4 activation domain; GBD, Gal4 DNA
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binding domain; GST; glutathione S-transferase; JNK, c-Jun N-terminal kinase; MAPK,
mitogen-activated protein kinase; MBP, myelin basic protein; MEK, MAPK/ERK kinase;
MEKK, MEK kinase; ONPG, o-nitrophenyl-β-D-galactopyranoside; PAGE, polyacrylamide gel
electrophoresis; PDK, 3-phosphoinositide dependent protein kinase; PI3K, phosphatidylinositol
3-kinase; PKC, protein kinase C; PKN, protein kinase N; PRK, PKC-related kinase; VAD, VP16
activation domain.
Figure Legends
Fig. 1: MEKK2 interacts with PRK2. (A and B) MEKK2 binds PRK2 (residues 479-670) in the
yeast two-hybrid system. (A) Yeast L40 cells were transformed with the indicated combinations
of plasmids and plated on SC+His medium. Transformant colonies were then streaked on SC-His
+ 15 mM 3-AT plates and incubated at 30 oC for 2.5 days. (B) Liquid cultures of yeast
transformant cells were analyzed for β-galactosidase (β-gal) activity using ONPG as substrate.
Results were calculated in Miller units and represented as average ± standard deviation (SD). (C
and D) MEKK2 associates with full-length PRK2 in mammalian cells. (C) HEK293 cells were
transfected with HA-MEKK2 plus either Flag-hPRK2 or empty pCMV5 vector. After lysis cell
lysates were immunoprecipitated with anti-Flag M5 antibody and blotted with anti-HA 12CA5
antibody. (D) Reciprocally, cells were transfected with Flag-hPRK2 plus HA-MEKK2 or
pCMV5. Cell lysates were precipitated with 12CA5 and blotted with M5 antibody.
Fig. 2: Mapping the MEKK2-PRK2 binding sites. (A) Various truncations of the mouse PRK2
(amino acids 479-670) were fused to the activation domain of Gal4 (GAD) in plasmid pACT or
its derivative pACT2 and transformed into yeast L40 together with LexA-MEKK2 cloned in
pBTM116. Interaction was determined by the ability of the transformant cells to grow on SC-His
+ 15 mM 3-AT. (B) Different fragments of MEKK2 was fused to LexA in pBTM116 and
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transformed along with GAD-mPRK2aa479-670 into L40. Binding was examined by the
histidine prototrophy assay.
Fig. 3: MEKK3 does not bind PRK2. (A and B) Interaction of MEKK3 with PRK2 was
examined in the yeast two-hybrid system. (A) Yeast L40 was transformed with the indicated
combinations of plasmids and grown on SC+His medium. Yeast colonies were then streaked on
SC-His + 3 mM 3-AT plates and incubated at 30 oC for 3 days. (B) β-gal assay of liquid cultures
using ONPG as substrate. (C) MEKK3 does not bind PRK2 in mammalian cells. Top panel:
HEK293 cells were transfected with the indicated expression plasmids. Cells lysates were then
immunoprecipitated with anti-Flag M5 antibody and blotted with anti-V5 antibody. Middle
panel: 20 µg lysates were blotted with anti-V5 antibody. Bottom panel: 20 µg lysates were
blotted with M5 antibody.
Fig. 4: PKN does not bind MEKK2. (A and B) Yeast two-hybrid analyses of PKN-MEKK2
interaction. (A) The region of hPKN (residues 477-628) that is homologous to PRK2aa479-670
was tested for its interaction with MEKK2 in yeast L40. After growth on SC+His medium cells
were streaked on SC-His + 15 mM 3-AT plates and incubated at 30 oC for 3 days. (B) β-gal
activities of liquid cultures using ONPG as substrate. (C) PKN does not bind MEKK2 in vitro.
Human PRK2 (residues 616-670) and the corresponding region from hPKN were expressed as
GST fusions in bacteria and purified by binding to glutathion Sepharose beads. Beads were
incubated with HEK293 cell lysates and binding of endogenous MEKK2 was detected by
Western blotting with anti-MEKK2 antibody. (D) Alignment of the MEKK2-binding region of
hPRK2 (residues 616-670) with the mouse sequence and the related region from human PKN.
Residues identical to hPRK2 are highlighted in black, and conservative substitutions [to which
the BLOSUM-62 matrix (55) gives a positive score] are shaded in gray. The start of the kinase
domains (7) is indicated by an arrow.
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Fig. 5: PRK2 is activated by MEKK2 in vivo. (A) HEK293 cells were transfected with the
indicated combinations of expression plasmids. Equal amount of Flag-hPRK2 or Flag-hPRK2KE
was immunoprecipitated from cell lysates and incubated in a kinase reaction using myelin basic
protein (MBP) as substrate. (B) Equal amounts of cell lysates were Western blotted with anti-
MEKK2 antibody.
Fig. 6: MEKK2 binding is sufficient for activation of PRK2 in vitro, but the kinase activity of
PRK2 is required for phosphorylation of MEKK2. Flag-tagged human PRK2 or kinase inactive
PRK2KE was immunoprecipitated from transfected HEK293 cells and incubated with
recombinant MEKK2 purified from baculovirus-infected insect cells in a kinase reaction. The
reactions were resolved on an SDS-PAGE gel and exposed to a Kodak X-Omat film.
Fig. 7: PRK2 does not contribute to JNK activation. HEK293 cells were transfected with the
indicated plasmids and equal amounts of cell lysates were incubated with purified GST-cJun1-79
or GST bound to Sepharose beads. Endogenous JNK activity was subject to analysis in a kinase
reaction followed by subsequent SDS-PAGE and autoradiography. (A) 5 min. exposure to a film.
(B) 10 sec. exposure.
Fig. 8: Model depicting the different association complexes possible for PRK2 and MEKK2 with
Nck, RhoA and PDK1 (see text for details).
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Weiyong Sun, Sylvie Vincent, Jeffrey Settleman and Gary L Johnsonkinase regulatory pathways at the level of a MAPK kinase Kinase
MEK kinase 2 binds and activates protein kinase C-related kinase 2: bifurcation of
published online May 18, 2000J. Biol. Chem.
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