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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: Gary.Johnson@uchsc.edu 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.

References

1. Widmann, C., Gibson, S., Jarpe, M. B., and Johnson, G. L. (1999) Physiol Rev 79(1),

143-80

2. Fanger, G. R., Johnson, N. L., and Johnson, G. L. (1997) Embo J 16(16), 4961-72

3. Schaefer, B. C., Ware, M. F., Marrack, P., Fanger, G. R., Kappler, J. W., Johnson, G. L.,

and Monks, C. R. (1999) Immunity 11(4), 411-21

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

19

4. Blank, J. L., Gerwins, P., Elliott, E. M., Sather, S., and Johnson, G. L. (1996) J Biol

Chem 271(10), 5361-8

5. Morrice, N. A., Gabrielli, B., Kemp, B. E., and Wettenhall, R. E. (1994) J Biol Chem

269(31), 20040-6

6. Palmer, R. H., Ridden, J., and Parker, P. J. (1995) Eur J Biochem 227(1-2), 344-51

7. Yu, W., Liu, J., Morrice, N. A., and Wettenhall, R. E. (1997) J Biol Chem 272(15),

10030-4

8. Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K.,

Iwamatsu, A., and Kaibuchi, K. (1996) Science 271(5249), 648-50

9. Quilliam, L. A., Lambert, Q. T., Mickelson-Young, L. A., Westwick, J. K., Sparks, A. B.,

Kay, B. K., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and Der, C. J. (1996) J Biol Chem

271(46), 28772-6

10. Reid, T., Furuyashiki, T., Ishizaki, T., Watanabe, G., Watanabe, N., Fujisawa, K., Morii,

N., Madaule, P., and Narumiya, S. (1996) J Biol Chem 271(23), 13556-60

11. Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H.,

Ono, Y., Kakizuka, A., and Narumiya, S. (1996) Science 271(5249), 645-8

12. Vincent, S., and Settleman, J. (1997) Mol Cell Biol 17(4), 2247-56

13. Zohar, M., Teramoto, H., Katz, B. Z., Yamada, K. M., and Gutkind, J. S. (1998)

Oncogene 17(8), 991-8

14. Teramoto, H., Coso, O. A., Miyata, H., Igishi, T., Miki, T., and Gutkind, J. S. (1996) J

Biol Chem 271(44), 27225-8

15. Hall, A. (1998) Science 279(5350), 509-14

16. Tapon, N., Nagata, K., Lamarche, N., and Hall, A. (1998) Embo J 17(5), 1395-404

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

20

17. Matsuzawa, K., Kosako, H., Inagaki, N., Shibata, H., Mukai, H., Ono, Y., Amano, M.,

Kaibuchi, K., Matsuura, Y., Azuma, I., and Inagaki, M. (1997) Biochem Biophys Res Commun

234(3), 621-5

18. Mukai, H., Toshimori, M., Shibata, H., Kitagawa, M., Shimakawa, M., Miyahara, M.,

Sunakawa, H., and Ono, Y. (1996) J Biol Chem 271(16), 9816-22

19. Mukai, H., Toshimori, M., Shibata, H., Takanaga, H., Kitagawa, M., Miyahara, M.,

Shimakawa, M., and Ono, Y. (1997) J Biol Chem 272(8), 4740-6

20. Sahai, E., Alberts, A. S., and Treisman, R. (1998) Embo J 17(5), 1350-61

21. Mellor, H., Flynn, P., Nobes, C. D., Hall, A., and Parker, P. J. (1998) J Biol Chem 273(9),

4811-4

22. Braverman, L. E., and Quilliam, L. A. (1999) J Biol Chem 274(9), 5542-9

23. Bokoch, G. M., Wang, Y., Bohl, B. P., Sells, M. A., Quilliam, L. A., and Knaus, U. G.

(1996) J Biol Chem 271(42), 25746-9

24. Knaus, U. G., and Bokoch, G. M. (1998) Int J Biochem Cell Biol 30(8), 857-62

25. Symons, M., Derry, J. M., Karlak, B., Jiang, S., Lemahieu, V., McCormick, F., Francke,

U., and Abo, A. (1996) Cell 84(5), 723-34

26. Balendran, A., Casamayor, A., Deak, M., Paterson, A., Gaffney, P., Currie, R., Downes,

C. P., and Alessi, D. R. (1999) Curr Biol 9(8), 393-404

27. Cryns, V. L., Byun, Y., Rana, A., Mellor, H., Lustig, K. D., Ghanem, L., Parker, P. J.,

Kirschner, M. W., and Yuan, J. (1997) J Biol Chem 272(47), 29449-53

28. Takahashi, M., Mukai, H., Toshimori, M., Miyamoto, M., and Ono, Y. (1998) Proc Natl

Acad Sci U S A 95(20), 11566-71

29. Bartel, P. L., and Fields, S. (1995) Methods Enzymol 254, 241-63

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

21

30. Hollenberg, S. M., Sternglanz, R., Cheng, P. F., and Weintraub, H. (1995) Mol Cell Biol

15(7), 3813-22

31. Durfee, T., Becherer, K., Chen, P. L., Yeh, S. H., Yang, Y., Kilburn, A. E., Lee, W. H.,

and Elledge, S. J. (1993) Genes Dev 7(4), 555-69

32. Breeden, L., and Nasmyth, K. (1985) Cold Spring Harb Symp Quant Biol 50, 643-50

33. Li, L., Elledge, S. J., Peterson, C. A., Bales, E. S., and Legerski, R. J. (1994) Proc Natl

Acad Sci U S A 91(11), 5012-6

34. Guarente, L. (1983) Methods Enzymol 101, 181-91

35. Fields, S., and Song, O. (1989) Nature 340(6230), 245-6

36. Ma, J., and Ptashne, M. (1987) Cell 48(5), 847-53

37. Fanger, G. R., Widmann, C., Porter, A. C., Sather, S., Johnson, G. L., and Vaillancourt,

R. R. (1998) J Biol Chem 273(6), 3476-83

38. Mellor, H., and Parker, P. J. (1998) Biochem J 332(Pt 2), 281-92

39. Zong, H., Raman, N., Mickelson-Young, L. A., Atkinson, S. J., and Quilliam, L. A.

(1999) J Biol Chem 274(8), 4551-60

40. Mukai, H., Kitagawa, M., Shibata, H., Takanaga, H., Mori, K., Shimakawa, M.,

Miyahara, M., Hirao, K., and Ono, Y. (1994) Biochem Biophys Res Commun 204(1), 348-56

41. Yoshinaga, C., Mukai, H., Toshimori, M., Miyamoto, M., and Ono, Y. (1999) J Biochem

(Tokyo) 126(3), 475-84

42. Michaud, N. R., Therrien, M., Cacace, A., Edsall, L. C., Spiegel, S., Rubin, G. M., and

Morrison, D. K. (1997) Proc Natl Acad Sci U S A 94(24), 12792-6

43. Joneson, T., Fulton, J. A., Volle, D. J., Chaika, O. V., Bar-Sagi, D., and Lewis, R. E.

(1998) J Biol Chem 273(13), 7743-8

by guest on March 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

22

44. Denouel-Galy, A., Douville, E. M., Warne, P. H., Papin, C., Laugier, D., Calothy, G.,

Downward, J., and Eychene, A. (1998) Curr Biol 8(1), 46-55

45. Cacace, A. M., Michaud, N. R., Therrien, M., Mathes, K., Copeland, T., Rubin, G. M.,

and Morrison, D. K. (1999) Mol Cell Biol 19(1), 229-40

46. Therrien, M., Chang, H. C., Solomon, N. M., Karim, F. D., Wassarman, D. A., and

Rubin, G. M. (1995) Cell 83(6), 879-88

47. Therrien, M., Michaud, N. R., Rubin, G. M., and Morrison, D. K. (1996) Genes Dev

10(21), 2684-95

48. Yu, W., Fantl, W. J., Harrowe, G., and Williams, L. T. (1998) Curr Biol 8(1), 56-64

49. Singer, W. D., Brown, H. A., Jiang, X., and Sternweis, P. C. (1996) J Biol Chem 271(8),

4504-10

50. Pombo, C. M., Kehrl, J. H., Sanchez, I., Katz, P., Avruch, J., Zon, L. I., Woodgett, J. R.,

Force, T., and Kyriakis, J. M. (1995) Nature 377(6551), 750-4

51. Schaeffer, H. J., Catling, A. D., Eblen, S. T., Collier, L. S., Krauss, A., and Weber, M. J.

(1998) Science 281(5383), 1668-71

52. Dickens, M., Rogers, J. S., Cavanagh, J., Raitano, A., Xia, Z., Halpern, J. R., Greenberg,

M. E., Sawyers, C. L., and Davis, R. J. (1997) Science 277(5326), 693-6

53. Garrington, T. P., and Johnson, G. L. (1999) Curr Opin Cell Biol 11(2), 211-8

54. Stapleton, G., Nguyen, C. P., Lease, K. A., and Hille, M. B. (1998) Dev Biol 193(1), 36-

46

55. Henikoff, S., and Henikoff, J. G. (1992) Proc Natl Acad Sci U S A 89(22), 10915-9

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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FIGURE 1

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FIGURE 2

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FIGURE 3

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FIGURE 4

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FIGURE 5

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FIGURE 6

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FIGURE 7

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FIGURE 8

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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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