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Originally published In Press as doi:10.1074/jbc.M304212200 on May 19, 2003

J. Biol. Chem., Vol. 278, Issue 31, 28694-28702, August 1, 2003
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Recruitment of JNK to JIP1 and JNK-dependent JIP1 Phosphorylation Regulates JNK Module Dynamics and Activation*

Deepak Nihalani, Hetty N. Wong and Lawrence B. Holzman {ddagger}

From the Division of Nephrology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0676

Received for publication, April 22, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
JIP1 is a scaffold protein that assembles and facilitates the activation of the mixed lineage kinase-dependent JNK module. Results of earlier work led us to propose a model for JIP1-JNK complex regulation that predicts that under basal conditions, JIP1 maintains DLK in a monomeric, unphosphorylated, and catalytically inactive state. Upon appropriate module stimulation, JNK-JIP1 binding affinity increases and DLK-JIP1 affinity decreases. Dissociation of DLK from JIP1 results in subsequent DLK oligomerization, autophosphorylation, and ultimately module activation. Our previous published results suggested the hypothesis that recruitment of JNK to JIP1 and phosphorylation of JIP1 by JNK is prerequisite for activation of the JNK module (Nihalani, D., Meyer, D., Pajni, S., and Holzman, L. B. (2001) EMBO J. 20, 3447–3458). The present study corroborated this hypothesis by demonstrating that JNK binding to JIP1 is necessary for stimulus-induced dissociation of DLK from JIP1, for DLK oligomerization, and for JNK activation. After mapping JNK-dependent JIP1 phosphorylation sites and testing their functional significance, it was observed that phosphorylation by JNK of JIP1 on Thr-103 and not other phosphorylated JIP1 residues is necessary for the regulation of DLK association with JIP1, DLK activation, and subsequent module activation. A refined model of JIP1-JNK module regulation is presented in which JNK phosphorylation of JIP1 is necessary prior to module activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitogen-activated protein kinases (MAPKs)1 link a variety of extracellular signals to a diverse range of cellular responses such as proliferation, differentiation, and apoptosis. Three groups of mammalian MAPKs and the upstream kinases and stimuli that activate them have been studied most extensively. These include the p42/p44MAPK (extracellular signal-regulated kinases, ERK1 and -2), that are generally activated by mitogens and differentiation-inducing stimuli, the stress-activated protein kinases (p46/p54SAPK or JNK1, 2, or 3 and their splice isoforms) and the p38mapk (1–6).

The stress-activated protein kinases have also been termed JNK protein kinases because they were identified as the principal c-Jun N-terminal kinases. The JNK family kinases are activated by cell stress-inducing stimuli such as heat shock, UV irradiation, hyperosmolarity, and ischemia/reperfusion injury, and by activation of specific cell surface receptors (7–9). Studies performed in cell culture systems and investigation of genetic mutants in a number of model organisms has begun to establish a variety of distinct physiological roles for individual stress-activated protein kinases and their associated pathways. Indeed, distinct JNK family kinases have been implicated in multiple specific biological processes that include but are not limited to embryonic morphogenesis, regulation of the apoptotic response to cellular injury, regulation of thymocyte development and activation, and regulation of proliferation (10–17).

Experiments in yeast have provided evidence that MAP kinase pathways are assembled from a unique combination of protein kinases into distinct protein complexes or modules (18, 19). The minimal MAPK module uniformly contains a MAPKKK, a MAPKK, and a MAPK. The components of these modules interact via direct protein-protein interactions and/or are tethered to scaffolding proteins (20–25). Importantly, assembly of MAPK modules appears to allow segregation of MAPK signaling components into units that are responsive to independent stimuli, that obtain appropriate subcellular targeting, that are insulated from similar modules, and that can regulate functionally distinct substrates (26–28). Identification of the JIP family of JNK scaffolding proteins first established that mammalian cells organize JNK pathways into modules in a fashion similar to yeast (29). Three JIP family genes and several splice isoforms have been identified (30–32). JIP proteins can form homo- and hetero-oligomers and are phosphoproteins. Whereas JIP3 is structurally distant from JIP1 and JIP2, each has been demonstrated to associate directly with a mixed lineage kinase, with MKK7 and with JNK (29, 31, 33). It has been proposed that JIP proteins facilitate mixed lineage kinase-dependent signal transduction to JNK possibly by aggregating the three components of a JNK module (30).

Although a considerable amount of work has been done to establish the role of JIP1 in regulating JNK signaling, less is known about the mechanisms that govern this regulation. We previously demonstrated that JNK kinase module components DLK and JNK interact with JIP1 in a dynamic manner to regulate the activation of the associated mixed lineage kinase and ultimately JNK module activity (34). The results of that study suggested a model in which JIP1 maintains DLK in a monomeric, unphosphorylated, and catalytically inactive state. Stimuli resulting in recruitment of JNK to JIP1 also result in release of DLK from JIP1, DLK dimerization, autophosphorylation, and activation, and ultimately induction of JNK catalytic activity. Our previous results implied but did not prove that recruitment of JNK to JIP1 is prerequisite to activating of upstream kinases that are components of a pre-assembled inactive JIP1 complex.

In the present study we have investigated the significance of JNK recruitment to JIP1 and have investigated the mechanism by which JNK recruitment regulates module activation. The data presented corroborates our previous observations using endogenous proteins, demonstrates that JNK binding to JIP1 is necessary for module activation, and shows that activation of JIP1-JNK module dynamics requires phosphorylation of JIP1 on Thr-103 by JNK. A refined model of JNK module regulation is proposed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Polyclonal antibodies to the C-terminal 223 amino acids of DLK was described previously (35, 36). Anti-FLAG epitope monoclonal antibody (M2, Sigma), anti-HA antibody (Sigma), anti-JNK antibody (Santa Cruz Biotechnology, Inc.), and the anti-Myc epitope monoclonal antibody (9E10, Oncogene Science) were obtained commercially. Antibody to JIP1 protein was gift of Dr. Benjamin Margolis (37). Cell culture grade chemical okadaic acid was purchased form Sigma. HN33 cells, an immortalized rat hippocampal neuronal cell line was gift from Dr. Y.-F. Liu (8). All chemical reagents and solvents were purchased from Sigma.

Bacterial Fusion Protein Construction and Expression—GST-MKK7 and GST— c-Jun-(1–79) fusion protein was prepared as described previously (35, 36). GST-SAP kinase 1b/JNK3 (pre-activated and catalytically competent) were obtained from Upstate Biotechnology. Hexahistidine-tagged full-length JIP1-(1–711) was created by a standard PCR technique using Myc-JIP1 as a template. Briefly, PCR was performed using the following synthetic oligonucleotides, a 5' JIP1 oligonucleotide 5'-GAGCTCGAGGCTGAGCGTGAGTCCGGT-3' and a 3' JIP1 antisense oligonucleotide 5'-GATCAAGCTTCGCTACTCCAGGTAGATATC-3'. The XhoI and HindIII fragments of the resultant amplification product were subcloned into the XhoI and HindIII restriction digested and prepared pRSET-A vector (Invitrogen). The construct was fully DNA sequenced. Full-length HisJIP1 was expressed and purified using the His-Bind purification kit from Novagen (catalog number 70159) according to manufacturers protocol.

Eukaryotic Expression Constructs—Construction and characterization of mammalian expression constructs encoding FLAG-DLK and FLAG-DLK(K185A), were described previously (36). HA-DLK mammalian construct was prepared by a standard PCR cloning technique using FLAG-DLK as a template. The pCDNA3 expression construct expressing the Myc epitope-tagged JIP-1 was a gift of Dr. Benjamin Margolis (37). Mammalian expression constructs encoding various JIP1 mutants were prepared by standard PCR cloning techniques using Myc-JIP1 plasmid as a template. Restriction digestion and DNA sequencing were used to validate all constructs.

Cell Culture—Transient transfections in mammalian cells were carried out in COS7 and HN33 cells. The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen) and 200 units/ml penicillin and streptomycin (Roche Diagnostics). The transfection was performed using FuGENE-6 (Roche Diagnostics) according to the manufacturer's protocols using a total of 2 µg of DNA. Where indicated cells were treated by the addition of indicated concentrations of either okadaic acid or kainic acid 24 h after transfection.

In Vivo Orthophosphate Labeling of Cells—COS7 cells were transfected with the indicated constructs. After 24 h, cells were washed three times with phosphate-free Dulbecco's modified Eagle's medium then incubated at 37 °C in the same medium for 30 min. The cells were then incubated in phosphate-free Dulbecco's modified Eagle's medium containing 0.5% dialyzed fetal bovine serum and 1 mCi/ml [{gamma}-32P]ATP orthophosphate (Amersham Biosciences) for 4 h at 37 °C.

Immunoprecipitations and Immunoblotting—Immunoprecipitations were performed using the indicated antibodies as described previously (38).

JNK Activation Assays—Cells were transfected with plasmid constructs as indicated in the figure legends. Cell lysates were prepared 24 h after transfection using 1 ml of lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM sodium vanadate, 50 mM sodium fluoride, 20 mM {beta}-glycerophosphate, 10% glycerol, 1% Triton X-100, and a mixture of protease inhibitors (Roche Diagnostics, catalogue number 1836170). In vitro reconstituted kinase catalytic activity assays (Figs. 4 and 9A) were performed as described previously (38). Briefly, the immunoprecipitated complexes from the lysates were incubated at 30 °C for 10 min in 50 µl of kinase buffer (25 mM Hepes, pH 7.2, 10% glycerol, 100 mM NaCl, 20 mM MgCl2, 0.1 mM sodium vanadate, and a mixture of protease inhibitors) containing 25 µM ATP, 5 µCi of [{gamma}-32P]ATP (3000 Ci/mmol), and the indicated quantities of GST-JNK or GST-MKK7. The reactions were terminated by the addition of sample buffer, boiled, resolved by SDS-PAGE, transferred to nitrocellulose, and autoradiographed. Expression of DLK or JIP1 constructs in each sample was assessed by immunoblotting.



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  		FIG. 4.
JNK association with JIP1 is necessary for JNK activation. Myc-JIP1 (0.2 µg) or its mutants JIP1(R160G/P161G) (0.2 µg) or JIP1(S197A/T205A/T284A) (0.2 µg) were co-transfected with either FLAG-DLK or inactive FLAG-DLK(K185A) plasmid in COS7 cells. JIP1 was immunoprecipitated from the cell lysates using JIP1 specific antibody. Immunoprecipitates were then assayed in vitro for their ability to activate GST-JNK. Kinase buffer containing recombinant GST-MKK7, GST-JNK, GST-c-Jun, and [{gamma}-32P]ATP was added to immunoprecipitated complexes and incubated at 30 °C for 15 min. Immunoprecipitates were separated on SDS-PAGE, transferred to nitrocellulose, and autoradiographed. Immunoblots from corresponding cell lysates were used to evaluate the relative expression of DLK and JIP in each reaction. Similar results were obtained in three independent experiments.

 


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  		FIG. 9.
Phosphorylation of JIP1 by JNK on Thr-103 is necessary for JNK activation. A, COS7 cells were co-transfected with the indicated plasmids. JIP1 was immunoprecipitated from the cell lysates using anti-FLAG antibody. Immune complexes were combined in vitro with recombinant GST-MKK7, GST-JNK, and GST-c-Jun in a kinase buffer containing [{gamma}-32P]ATP and incubated at 30 °C for 15 min. Immune complexes were separated on SDS-PAGE, transferred to nitrocellulose, and autoradiographed. Immunoblots from corresponding cell lysates were used to evaluate the relative expression of DLK and JIP1 in each reaction. Similar results were obtained in three independent experiments. B, JIP1 mutants bind JNK with similar affinities. COS7 cells were co-transfected with the indicated plasmids. Cell lysates were immunoprecipitated using anti-FLAG antibody and were analyzed for the presence of JNK using anti-JNK antibody. Cell lysates from the corresponding experiments were also analyzed for the equivalent expression of JIP1 and JNK.

 

Phosphopeptide Mapping—Two-dimensional phosphopeptide mapping was performed as described by Boyle et al. (39). Following an in vitro kinase assay or immunoprecipitation of phosphate-labeled proteins, the substrate was resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and the protein band was identified by autoradiography and excised from the membrane. The nitrocellulose membrane pieces containing the 32P-labeled proteins (wild-type JIP1 and mutant JIP1 proteins) were excised, soaked in 0.5% polyvinylpyrrolidone (PVP-360) in 100 mM acetic acid for 30 min at 37 °C, and washed extensively with water and freshly made 0.05 M NH4HCO3. Tryptic digestion was performed by incubating samples with sequencing grade trypsin (20 µg in 200 µl of freshly prepared 50 mM NH4HCO3 overnight at 37 °C). The supernatants were transferred and dried in a Speed-vac and the samples were solubilized in pH 1.9 buffer (containing 2.5% (v/v) formic acid (88%) and 7.8% (v/v) acetic acid). Aliquots (2–5 µl) of each sample (containing 20,000 cpm) were spotted onto 20 x 20-cm thin-layer cellulose plates and separated in the first dimension by electrophoresis for 45 min at 1000 V using buffer containing 2.5% formic acid and 7.8% acetic acid at pH 1.9. After drying, peptides were further separated in the second dimension by ascending chromatography for 8 h using buffer containing 37.5% (v/v) 1-butanol, 25% (v/v) pyridine, and 5% (v/v) acetic acid. The pattern of tryptic phosphopeptides was visualized by exposing the dried cellulose plates to a PhosphorImager plate (Amersham Biosciences model Storm-860).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Stimulation-induced Dissociation of Endogenous JIP1 and DLK in Cultured Neurons—Our previous experiments suggested that there exists a regulated dynamic relationship between DLK, JIP1, and JNK (34). However, these conclusions were drawn from studies performed by overexpression of module components in COS7 cells. To determine whether a similar relationship between DLK, JIP1, and JNK exists among endogenous module components, the interaction of endogenous DLK and JIP1 and endogenous JNK and JIP1 was studied in HN33 neuronal cell culture. HN33 cells were stimulated with or without okadaic acid and the association of endogenous DLK with JIP1 or JNK with JIP1 was examined in co-immunoprecipitation assays (Fig. 1A). Consistent with our previously published results, treatment of HN33 cells with okadaic acid resulted in recruitment of endogenous JNK to JIP1 and dissociation of endogenous DLK from the JIP1 complex (34). Because JIP1 was expressed at difficult to detect levels within HN33 cells, similar additional experiments were repeated by transfecting these cells with plasmid encoding JIP1 (Fig. 1, B and C). Treatment of neuronal cells in culture with either okadaic acid or kainic acid results in JNK activation and induction of an apoptotic response that might be in part mediated by mixed lineage kinases (8, 40, 41). In a manner similar to that observed when HN33 cells were treated with okadaic acid, kainic acid induced the dissociation of endogenous DLK from JIP1 within 30 min of treatment (Fig. 1B). Moreover, kainic acid treatment of HN33 cells also resulted in JNK recruitment to JIP1 and increased JIP1 phosphorylation (Fig. 1C). These results provide initial evidence that endogenous DLK, JIP1, and JNK interact in a dynamic fashion and that this process is associated with JIP1 phosphorylation.



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  		FIG. 1.
Dynamic nature of endogenous DLK, JIP1, and JNK interaction in stimulated HN33 cells. A, extracts were prepared from HN33 cells (1 x 107) treated with or without 400 nM okadaic acid (Ok. Acid) for 3 h. JIP1 complexes were immunoprecipitated from the cell lysates using JIP1-specific antibody, separated by SDS-PAGE, and analyzed by immunoblotting with anti-DLK and anti-JNK antibody. Immunoprecipitation without JIP1 antibody was used as control. Corresponding lysates were immunoblotted as indicated to determine the endogenous expression of DLK, JIP1, and JNK. B, HN33 cells were transfected with FLAG-JIP1. Cells were treated with either kainic acid or okadaic acid as indicated. JIP1 was immunoprecipitated using JIP1 antibody and immune complexes were analyzed for the presence of DLK. C, HN33 cells were transfected with FLAG-JIP1, metabolically labeled with orthophosphate, and then treated with or without kainic acid for 60 min. JIP1 was immunoprecipitated using JIP1 antibody and immune complexes were analyzed after SDS-PAGE by autoradiography for incorporation of phosphate into JIP1 or by immunoblotting for the presence of DLK or JNK. All experiments were repeated 3 times with similar results.

 

JNK Binding to JIP1 Is Necessary for DLK Dissociation from JIP1 and Its Dimerization in the Presence of Okadaic Acid— Our previous published results suggested the hypothesis that recruitment of JNK to JIP1 is necessary for the activation of the JIP1-dependent JNK module (34). To test this hypothesis, a JIP1 mutant was prepared that does not associate with JNK. Whitmarsh et al. (33) showed that the JIP1 JNK binding domain localized between residues 127 and 282. Kelkar et al. (31) mapped specific residues in JIP3 necessary for the interaction of JIP3 with JNK. Sequence comparison revealed that these residues were highly conserved within the JNK binding domain among all JIP isoforms (31). Therefore, mutations were introduced in JIP1 at residues 160 (arginine to glycine) and 161 (proline to glycine). As shown if Fig. 2, when co-expressed with FLAG-JNK, JNK did not co-immunoprecipitate with this JIP1(R60G/P161G) mutant.



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  		FIG. 2.
Point mutations in the JNK binding domain of JIP1 abolish JIP-JNK interaction. COS 7 cells were co-transfected with plasmids encoding Myc-JIP1 (0.5 µg) or JIP1(R160G/P161G) (0.5 µg) and FLAG-JNK (0.5 µg) as indicated. Cell lysates were analyzed by immunoprecipitation using JIP1 antibody. Immune complexes were analyzed for the presence of JNK using anti-FLAG antibody. Cell lysates from corresponding experiments were analyzed for equivalent expression of JIP1 and JNK.

 

Using this mutant, we tested whether preventing recruitment of JNK to JIP1 inhibited dissociation of DLK from JIP1 as predicted by our model. FLAG-DLK was co-expressed with either JIP1 or JIP1(R160G/P161G). Cells were treated with okadaic acid or control buffer for 3 h. JIP1 or its mutant was immunoprecipitated from cell lysates using JIP1-specific antibodies and immunoprecipitated complexes were analyzed for the presence of DLK (Fig. 3A). As shown in Fig. 3A, both wild type JIP1 and JIP1(R160G/P161G) bound DLK under control conditions. As reported previously, okadaic acid treatment resulted in dissociation of DLK from JIP1. However, under the same conditions, DLK did not dissociate from JIP1(R160G/P161G).



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  		FIG. 3.
JNK-JIP1 association is necessary for DLK dissociation from JIP1 and DLK oligomerization. A, COS7 cells were co-transfected with plasmids encoding FLAG-DLK (0.5 µg) and either M-JIP1 (0.5 µg), JIP1(R160G/P161G) (0.5 µg) or JIP1(S197A/T205A/T284A) (0.5 µg). Cells were treated for 3 h with 400 nM okadaic acid. Cell lysates were immunoprecipitated with anti-JIP1 antibody, separated on SDS-PAGE, and immunoblotted with anti-DLK antibody. Cell lysates from corresponding experiments were immunoblotted with the indicated antibodies to evaluate the expression of JIP and DLK. B, COS7 cells were co-transfected as indicated with HA-DLK (0.05 µg), FLAG-DLK (0.05 µg), and either M-JIP1 (0.9 µg) or JIP1(R160G/P161G) (0.9 µg). Cells were treated for 3 h with 400 nM okadaic acid. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with HA antibody. Corresponding cell lysates were immunoblotted with the indicated antibodies to evaluate the expression of HA-DLK, FLAG-DLK, M-JIP1, and JIP1(R160G/P161G). These experiments were repeated three times with similar results.

 

Treatment of cells with okadaic acid also results in DLK oligomerization in the presence of JIP1 (34). Because DLK dissociation from JIP1 is thought to precede DLK oligomerization (34), it was anticipated that preventing JNK binding to JIP1 after treatment with okadaic acid would also inhibit DLK oligomerization. To test this hypothesis, FLAG-DLK and HA-DLK were co-expressed with either JIP1 or JIP1(R160G/P161G) in COS7 cells (Fig. 3B). Cells were treated with okadaic acid or control buffer for 3 h. FLAG-DLK was immunoprecipitated using anti-FLAG-agarose beads and immune complexes were analyzed for the presence of HA-DLK. As previously observed, JIP1 inhibited DLK oligomerization under basal conditions (Fig. 3B, lane 2) and DLK oligomerization in the presence of JIP1 was promoted upon treatment of cells with okadaic acid (Fig. 3B, lane 3). However, while JIP1(R160G/P161G) prevented DLK oligomerization, okadaic acid failed to promote DLK oligomerization in the presence of this JIP1 mutant. Combined with observations described above, these results provide evidence that JNK binding to JIP1 is necessary for dissociation of DLK from JIP1 and the subsequent oligomerization of DLK.

JNK Association with JIP1 Is Necessary for JIP1-dependent JNK Activation—Because DLK oligomerization is required for its activation (34) the above results suggested that JNK recruitment to JIP1 should also be necessary for DLK-mediated JNK activation. To test whether JNK binding to JIP1 is necessary for DLK-mediated JNK activation, the module was reconstituted in vitro and assayed for JNK activation as described previously (34) (Fig. 4). DLK or catalytically inactive DLK(K185A) was co-expressed with JIP1, JIP1(R160G/P161G), or JIP1(S197A/T205A/T284A) in COS7 cells. The various JIP1-DLK complexes were obtained after immunoprecipitation of JIP1(X) from cell lysates using anti-JIP1 antibodies. GST-JNK activation was determined by incubating the immunoprecipitated complexes with a mixture of bacterially expressed and purified recombinant GST-MKK7, GST-JNK, and GST-c-Jun in a kinase buffer containing radiolabeled ATP. In this system, GST-JNK was activated when incubated in the presence of DLK and JIP1. In contrast, GST-JNK was not activated in the absence of DLK or in the presence of catalytically inactive DLK(K185A) and JIP1 (Fig. 4, compare lanes 1 and 2; see also Fig. 9, lanes 1 and 2). GST-JNK was activated in a similar manner when the JIP1(S197A/T205A/T284A)-DLK complex was substituted for wild type JIP1-DLK in the reaction mixture (Fig. 4, lanes 4–6). However, GST-JNK activation was substantially attenuated when JIP1(R160G/P161G) was substituted for wild type JIP1 (Fig. 4, lanes 7–9). In conclusion, these results refine the previously published model and suggest that JNK recruitment to JIP1 is necessary for DLK dissociation, DLK oligomerization, and DLK activation, and ultimately for induction of JNK activity.

JIP1 Is Phosphorylated by JNK on Multiple Sites—Because JIP1 phosphorylation was observed to correlate with alterations in the affinity of JIP1 for JNK and DLK (Fig. 1), and because JNK-JIP1 interaction negatively influenced DLK and JIP1 binding affinity, we hypothesized that JIP1 module dynamics are dependent upon JNK-mediated phosphorylation of JIP1. Wild type JIP1 or the JIP1(R160G/P161G) mutant obtained by immunoprecipitation from transfected COS7 cells were combined with preactivated recombinant GST-JNK in a kinase buffer containing radiolabeled ATP. JIP1 phosphorylation required binding of JNK to JIP1 and occurred only in the presence of JNK (Fig. 5, lanes 1–3).



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  		FIG. 5.
JIP1 phosphorylation by recombinant activated GST-JNK is dependent on the JNK binding domain. A, schematic of JIP1 structure illustrating the potential proline-directed Ser/Thr phosphorylation sites. B, JIP1 is phosphorylated by JNK in vitro. Myc-JIP1 or its various mutants were expressed in COS7 cells. Following immunoprecipitation, JIP1(X) complexes were incubated at 30 °C for 30 min in vitro in a kinase buffer containing recombinant pre-activated GST-JNK and [{gamma}-32P]ATP. Immune complexes were separated by SDS-PAGE, transferred to nitrocellulose, and autoradiographed. In addition to the mutants shown, similar analysis was performed on JIP1 mutations at Ser-234, Ser-421, and Thr-447 with similar results. Immunoblots from corresponding cell lysates were used to evaluate the relative expression of JIP1 proteins in each reaction. This experiment was repeated at three times with similar results.

 

Primary sequence analysis indicated that JIP1 has 11 potential proline-directed Ser/Thr phosphorylation sites (Fig. 5B). To examine this possibility, radiolabeled JIP1 obtained from COS7 cells was phosphorylated in vitro by recombinant JNK. Phosphorylated JIP1 was isolated and trypsinized, and JIP1 tryptic peptides were resolved by two-dimensional chromatography. Multiple radiolabeled phosphopeptides were observed on this two-dimensional map of JNK-phosphorylated JIP1 tryptic peptides (Fig. 6A). In an effort to further map JNK-dependent JIP1 phosphorylation sites a series of JIP1 deletion mutants as well as JIP1 point mutants were created at the sites indicated in Fig. 5B and were phosphorylated in vitro in the presence of recombinant JNK. As shown in Fig. 5B, none of these individual mutations resulted in a significant decrease in JIP1 phosphorylation after normalization for protein abundance. For this reason, two-dimensional phosphopeptide mapping using multiple JIP1 mutants was performed to identify specific tryptic phosphopeptides. Collective analysis allowed the identification of several phosphorylated residues on JIP1 (Fig. 6A). Alternative efforts were made to separate JIP1 tryptic phosphopeptides using a standard microbore high performance liquid chromatography system with subsequent analysis of individual peptides by mass spectrometry. Although a number of peaks containing phosphorylated peptides were observed, success in confirming the identity of phosphopeptides was technically limited. However, peptides containing strongly phosphorylated sites including Ser-197 and Ser-11/15/29 were confirmed by this method (data not shown).



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  		FIG. 6.
Identification of JIP1 phosphorylation sites by phosphopeptide mapping. A, for in vitro phosphorylation of JIP1 by recombinant JNK cells, JIP1 or various JIP1 point mutants described in Fig. 5 were obtained from COS7 cells by immunoprecipitation and phosphorylated in vitro by recombinant GST-JNK in a kinase buffer containing radiolabeled ATP. For in vivo phosphorylation of JIP1, COS7 cells were transfected with plasmids encoding Myc-JIP1 or JIP1 mutants, metabolically labeled with radiolabeled orthophosphate, and treated with 400 nM okadaic acid for 3 h. JIP1 was obtained by immunoprecipitation from cell lysates. Phosphorylated bands representing JIP1 protein or its mutants were isolated by SDS-PAGE and processed for two-dimensional peptide mapping. Representative peptide maps for radiophosphate-labeled wild type JIP1 and JIP1(S197A) obtained by both in vivo or in vitro phosphorylation methods are shown for comparison. The absence of spot in the peptide map of JIP1(S197A) is indicated with an arrow. Enlargements of two-dimensional maps obtained after both in vivo or in vitro phosphorylation are shown for comparison. Identified tryptic phosphopeptides are labeled and a tabulated summary of peptide characteristics is presented at the left. In this table, the down arrow represents decreased phosphorylation and the up arrow represents increased phosphorylation. Peptides 2 and 7 are grouped to indicate phosphorylation state interdependence. R/P, JIP1(R160G/P161G). B, representative examples of two-dimensional maps obtained after substituting JIP1(R160G/P161G) for wild type JIP1 and demonstrating loss of peptides 2A and 2B after mutation of JIP1(T103A). Or, origin; JIP1(RP/G), JIP1(R160G/P161G).

 

Phosphorylation of JIP1 was also investigated in vivo using JIP1-transfected okadaic acid-treated COS7 cells that were metabolically labeled with radiolabeled orthophosphate. Okadaic acid treatment was employed here because preliminary experiments indicated that JIP1 phosphorylation increased following okadaic acid treatment (data not shown) and because experiments described above indicated that okadaic acid induced JIP1 module activation. By this approach, two-dimensional phosphopeptide mapping showed that phosphorylation of JIP1 occurred on most of the same peptides as that observed under the in vitro conditions discussed above (Fig. 6A). Four differentially phosphorylated tryptic peptides were identified and are tabulated in Fig. 6A.

To extend this in vivo analysis, the JIP1(R160G/P161G) mutant was used to identify JNK-JIP interaction-dependent phosphorylation sites on JIP1 after treatment with okadaic acid (Fig. 6B and tabulation in A). In contrast to results obtained in vitro, most JIP1 tryptic phosphopeptides identified on the two-dimensional phosphopeptide map of wild type JIP1 were also observed on the map of JIP1(R160G/P161G). However, comparison of these peptide maps with maps of all other mutants revealed several distinct dissimilarities. In experiments repeated 3 times, there was an absence of spots representing peptides 2A and 2B on the map of JIP1(R160G/P161G), a reproducible decrease in the intensity of phosphorylation on peptides 7A and 7B, and an increase in phosphorylation on peptide 7C (Fig. 6B and tabulation in A). Comparison of maps obtained from JIP1(R160G/P161G) with those obtained from JIP1(T103A) and JIP1-(113–711) revealed that both phosphopeptides 2A and 2B were absent only when residue Thr-103 was mutated to Ala. This suggested that phosphorylation of Thr-103 and possibly another undefined interdependent phosphorylation site was dependent on JNK binding to JIP1. Comparison of maps obtained from JIP1(R160G/P161G) with those obtained from JIP1(T205A), JIP1(T205A/T284A), and JIP1(S197A/T205A/T284A) revealed that phosphopeptides 7A, 7B, and 7C were absent when residue Thr-205 was mutated to Ala. Again, phosphorylation on Thr-205 appeared to be inter-dependent with the phosphorylation on possibly two additional sites. Therefore, phosphorylation on JIP1 at Thr-103 and Thr-205 and several additional interdependent undefined phosphorylation sites was dependent on JNK binding to JIP1 in this system.

To establish that JNK could directly phosphorylate JIP1, recombinant hexahistidine-JIP1 purified from Escherichia coli was combined in vitro with activated recombinant JNK (Fig. 7A). In these experiments, JNK directly phosphorylated recombinant JIP1 and a recombinant GST-c-Jun positive control. JIP1 was not phosphorylated when JNK was not included in the reaction mixture. JIP1 sites that were directly phosphorylated by JNK and not merely dependent on JNK binding to JIP1 were identified by isolating JNK-labeled hexahistidine-JIP1 and by subjecting this radiolabeled JIP1 to two-dimensional phosphopeptide mapping (Fig. 7B). Tryptic phosphopeptides 1, 2A, 2B, 4, 5, 6, 7A, and 7B were identified by this approach. No unidentified phosphopeptides were observed. Therefore, Thr-103 and Thr-205 are phosphorylated directly by JNK after JNK binds to JIP1.



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  		FIG. 7.
JNK directly phosphorylates JIP1 on several residues including Thr-103 and Thr-205. A, full-length recombinant hexahistidine-JIP1 or GST-c-Jun were incubated at 30 °C for 30 min in vitro in a kinase buffer containing or not containing recombinant preactivated GST-JNK and [{gamma}-32P]ATP. Note that purified hexahistidine-JIP1 readily degraded but that the product of least mobility on SDS-PAGE was resolved at the anticipated molecular weight. Reaction mixtures were separated on SDS-PAGE and autoradiographed. B, two-dimensional tryptic phosphopeptide map of JNK-phosphorylated hexahistidine-JIP1 produced as described above and labeled using the protocol established in Fig. 5. Or, origin.

 

Phosphorylation of JIP1 on Site Thr-103 Is Necessary for Okadaic Acid-induced Dissociation and Dimerization of DLK and Activation of JNK Module—If the hypothesis is true that JIP1 module dynamics are dependent upon JNK-mediated phosphorylation of JIP1 then one might expect that mutation of one or more residues identified in mapping JNK-dependent JIP1 phosphorylation sites should prevent both normal module dynamics and module activation. To determine whether phosphorylation of JIP1 at sites Thr-103 and Thr-205 is necessary for okadaic acid-induced DLK dissociation from JIP1 and subsequent DLK dimerization, JIP1 Thr to Ala mutants at these sites were used in DLK dissociation and dimerization assays described previously in this study (Figs. 3, A and B, 8, A and B). Similar to wild type JIP1, JIP(T103A) associated with DLK and inhibited DLK dimerization. However, in contrast to wild type JIP1 but like JIP1(R160G/P161G), okadaic acid treatment did not result in dissociation of DLK from JIP1(T103A) and did not promote DLK dimerization in the presence of JIP1(T103A) (Fig. 8, A and B). Similar results were obtained when JIP1- (113–711) (which lacks site Thr-103) was substituted in these assays. These results suggested that phosphorylation of JIP1 at Thr-103 is required for dissociation of DLK from JIP1 and its dimerization subsequent to JNK binding JIP1. Similar experiments carried out with JIP1(T205A) and JIP1(S197A/T205A/T284A) demonstrated that these mutants behaved in a manner similar to wild type JIP1 (Fig. 3, A and B, and data not shown). Therefore, despite that fact that Thr-205 phosphorylation was altered in okadaic acid-treated JIP1(R160G/P161G) experiments, phosphorylation at this site was not necessary for regulating DLK-JIP affinity. In control experiments, all remaining JIP1 mutants were also tested in DLK-JIP1 dissociation assays (data not shown); mutations at these alternative sites did not result in behavior different from that observed for wild type JIP1. In summary, mutation only at Thr-103, where phosphorylation was dependent on JNK binding to JIP1, disrupted normal JIP1 module dynamics.



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  		FIG. 8.
Phosphorylation of JIP1 on Thr-103 is necessary for okadaic acid-induced dissociation of DLK from JIP1 and DLK dimerization in the presence of JIP1. A, COS7 cells were co-transfected with plasmids encoding HA-DLK (0.5 µg) and FLAG-JIP1 (0.5 µg), FLAG-JIP1(R160G/P161G) (0.5 µg), FLAG-JIP1-(113–711) (0.5 µg), or FLAG-JIP1(T103A) (0.5 µg). After 24 h, the indicated samples were treated for 3 h with 400 nM okadaic acid. Cell lysates were immunoprecipitated with anti-FLAG antibody, separated on SDS-PAGE, and immunoblotted with anti-DLK antibody. Cell lysates from corresponding experiments were immunoblotted with the indicated antibodies to evaluate the expression of JIP and DLK. B, COS7 cells were co-transfected as indicated with HA-DLK (0.05 µg), FLAG-DLK (0.05 µg), and Myc-JIP1 (0.9 µg), JIP1(R160G/P161G) (0.9 µg), Myc-JIP1- (113–711) (0.9 µg), or JIP1(T103A) (0.9 µg). Where indicated, cells were treated for 3 h with 400 nM okadaic acid. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with HA antibody. Corresponding cell lysates were immunoblotted with the indicated antibodies to evaluate the expression of HA-DLK, FLAG-DLK, and JIP1. These experiments were repeated three times with similar results.

 

The necessity of phosphorylation at defined residues in JIP1 for module activation was also examined. In vitro JNK activation assays were performed as described above in which JIP1 mutants were substituted for wild type JIP1 (Fig. 9A). DLK or catalytically inactive DLK(K185A)] was co-expressed with JIP1 or its mutants (JIP1(R160G/P161G) or JIP1(T103A)) in COS7 cells. JIP1-DLK complexes were immunoprecipitated from cell lysates using JIP1 antibody and JNK activation was assayed in vitro after reconstituting the JNK module with recombinant GST-MKK7, GST-JNK, and GST-c-Jun. Substitution of JIP1(T103A) for wild type JIP1 in this system reproducibly attenuated JNK activation relative to control in a manner similar to substitution of JIP1(R160G/P161G) (Fig. 9A). Control experiments were performed to assure that the affinity of JIP1(T103A) for JNK was not altered in this system. In these experiments, FLAG-JIP1(T103A), FLAG-JIP1(S197A/T205A/T284A), JIP1(R160G/P161G), or FLAG-JIP1 were co-expressed with Myc-JNK in COS7 cells. JIP1 complexes were immunoprecipitated using FLAG antibody and immune complexes were examined for the presence of JNK by immunoblotting. In these experiments, FLAG-JIP1(T103A) and FLAG-JIP1(S197A/T205A/T284A) bound JNK with affinity similar to wild type JIP1. Taken together, the results presented demonstrate that JNK recruitment to JIP1 and JNK-binding dependent phosphorylation of JIP1 on residue Thr-103 are necessary for DLK-dependent activation of JNK via effects on JIP1 module dynamics.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Increased understanding of cell signaling has highlighted the complexity of biological information processing. Although significant advances have been made in discovering various components of signaling pathways, the mechanisms by which these pathways are integrated remain incompletely understood. Recent studies have focused on understanding the subcellular organization of signaling components that are often governed by the action of signaling complexes that include signaling enzymes, their substrates, and adapter or scaffolding proteins (9). It has been proposed that the JIP group of proteins behave as mammalian scaffolds that may facilitate mixed lineage kinase-dependent JNK activation (42). Although a number of studies have established the role of JIP proteins in JNK module signaling, the mechanisms that govern the assembly and regulation of this complex have required additional investigation.

In a previous study we provided experimental evidence to suggest a mechanism by which the DLK-dependent JIP1-JNK module is regulated (34). In this model, DLK and JNK do not co-exist simultaneously on JIP1. Under basal conditions, DLK associated with JIP1 is held in a monomeric, unphosphorylated and catalytically inactive state. Appropriate cellular stimulation results in recruitment of catalytically competent JNK to the JIP1 scaffold (31, 42). In turn, recruitment of JNK coincides with significantly decreased affinity of JIP1 and DLK. DLK dissociation from JIP1 results in DLK oligomerization, autophosphorylation, activation, and subsequent activation of JNK (34). Here, DLK catalytic activity is necessary for JNK activation. It is reassuring that in experiments in neuronal culture described herein, endogenous DLK, JIP1, and JNK behave in a manner consistent with this proposed model.

These results and the derived model suggested that JNK recruitment to JIP1 initiated DLK dissociation from JIP1 and subsequent module activation. Indeed, the observations presented herein provide novel insight into the regulation of JIP1 function by demonstrating that JNK binding to JIP1 is necessary for DLK dissociation and dimerization and JNK activation. It is possible that association of JNK with JIP1 introduces conformational changes in the pre-assembled inactive DLK-JIP1 complex that results in dissociation of DLK and permits its subsequent activation. The importance of JIP1 complex conformation in governing module activation was recently suggested. In studying the association between Akt and JIP1, Kim et al. (43) demonstrated that Akt binding to JIP1 attenuated the affinity of JIP1 for JNK module components independent of Akt catalytic activity. Whereas the mechanism by which this effect is propagated has not been discerned it could be reasonably argued that this effect was mediated by effecting a conformational change in JIP1.

The concept that the assembly and regulation of MAPK modules is dependent on phosphorylation of associated scaffold proteins has recently emerged. Phosphorylation-dependent regulation of JIP3 (JSAP1) by the MAP3K ASK1 appears to facilitate JIP3-JNK module assembly and activation in response to oxidative stress (44). Similar phosphorylation-induced JIP3-dependent assembly of the integrin-related signaling complex composed of FAK and its associated proteins was also recently reported (45). Additionally, phosphorylation by MAP3K TAK1 on KSR (which scaffolds the Raf-MEK-ERK module) determines the subcellular localization of KSR and its associated proteins and participates in regulating module activation (46). Whereas our previous study established that JIP1 is a phosphoprotein, the functional importance of JIP1 phosphorylation had not been determined.

It was hypothesized that JIP1 module dynamics are dependent upon JNK-mediated phosphorylation of JIP1 because JIP1 phosphorylation correlates with a change in the affinity of JIP1 for JNK and DLK, because JNK-JIP1 interaction negatively influences DLK and JIP1 binding affinity, and because JNK catalytic competency is required for DLK activation. The reported results support this hypothesis by establishing that JNK phosphorylation of JIP1 on Thr-103 is necessary for the dissociation and activation of DLK within the module. It remains to be determined by what mechanism phosphorylation on JIP1 results in DLK dissociation and activation; however, it is likely that alterations in complex conformational structure participate in this process.

Evidence from two-dimensional peptide mapping suggests that JIP1 is phosphorylated on multiple sites. Because many of these sites are not directly phosphorylated by JNK, it is likely that kinases already known to associate with JIP1 such as the mixed lineage kinases and MKK7 participate in JIP1 phosphorylation. However, inputs to JIP1 from other signaling pathways that are mediated by unidentified protein kinases or phosphatases might play a role in JIP1 phosphorylation. The role of phosphorylation on JIP1 on sites other than Thr-103 remains undefined. However, work in other systems suggests that these phosphorylation events might regulate complex assembly, subcellular localization, or activation (44–46). As such, JIP1 might serve as a site that facilitates the integration of signals from multiple pathways.

Comparison of JIP1 phosphorylated in vitro by JNK with in vivo phosphorylation on JIP1(R160G/P161G) in the presence of okadaic acid suggests that several residues that can be phosphorylated by JNK do not necessarily require direct binding of JNK to JIP1. Several explanations are possible. As suggested recently by Park et al. (47) nonspecific tethering of MAPK module components to the MAPK scaffolded complex might be sufficient to allow phosphorylation and activation of module components. Therefore, despite abolition of the JIP1-JNK binding motif, JNK might remain loosely associated with JIP1 via indirect interactions with other module components. Alternatively, in the presence of okadaic acid, other protein kinases might phosphorylate these same sites either in a physiologically meaningful or insignificant manner. Nevertheless, it is reassuring that phosphorylation of Thr-103 is both directed by JNK and requires JNK-binding to JIP1.

Contrary to standard convention, the results reported here imply that JNK recruitment to JIP1 and JNK-mediated phosphorylation of JIP1 precedes the activation of DLK and ultimately increased JNK catalytic activity. However, this result does not necessarily suggest that JNK "activation" is the proximal event and is therefore "upstream" of DLK activation. Rather, it could be speculated that phosphorylation on JIP1 by kinases other than JNK is a proximal event in module assembly and activation. In this scenario, JIP1 phosphorylation could result in alteration in the affinity of JIP1 for JNK, resultant recruitment of catalytically competent JNK to the module, then module activation through the mechanism elucidated in the present work. This model further implicates the existence of a positive feedback mechanism for JNK activation that moves the module in a switch-like manner from a slow to rapid rate of catalysis.

In this report we have shown that both JNK recruitment to JIP1 and phosphorylation of JIP1 by JNK are required for activation of JIP1-based modules. In light of these observations, a refined but speculative model of JIP1 scaffold function is proposed (Fig. 10). According to this model, DLK is complexed to JIP1 in a monomeric, catalytically inactive state under basal conditions. Upon appropriate stimulation (possibly by initial phosphorylation on JIP1), JNK is recruited to JIP1 and JIP1 is phosphorylated by JNK. As a result, DLK is dissociated from JIP1, oligomerizes, and becomes catalytically active. Activated DLK phosphorylates and activates MKK7 that subsequently phosphorylates JNK. Activation of JNK reinforces DLK activation via a positive feedback loop.



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  		FIG. 10.
Interaction of JIP1 with components of the JNK pathway. Schematic illustration of the dynamic relationship between JIP1, DLK, and JNK. Under basal conditions DLK is bound to JIP1 in a monomeric, inactive state. Upon stimulation, JNK is recruited to JIP1. This recruitment leads to JIP1 phosphorylation, DLK dissociation from JIP1, and subsequent dimerization and activation of DLK and ultimately JNK.

 


   FOOTNOTES
 
* This work was supported by NIDDK, National Institutes of Health Public Health Service Grant DO-52788 (to L. B. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: University of Michigan Medical School, 1560 MSRB II, Ann Arbor, MI 48109-0676. Tel.: 734-764-3157; Fax: 734-763-0982; E-mail: lholzman{at}umich.edu.

1 The abbreviations used are: MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; HA, hemagglutinin; GST, glutathione S-transferase; JIP, JNK interacting protein; DLK, dual leucine zipper kinase; MKK7, mitogen activated-protein kinase kinase 7. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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J. J. Song and Y. J. Lee
Dissociation of Akt1 from its negative regulator JIP1 is mediated through the ASK1-MEK-JNK signal transduction pathway during metabolic oxidative stress: a negative feedback loop
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Y. Wei, D. Wang, and M. J. Pagliassotti
Fructose Selectively Modulates c-jun N-Terminal Kinase Activity and Insulin Signaling in Rat Primary Hepatocytes
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Scaffold Proteins in Mammalian MAP Kinase Cascades
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L. M. Mooney and A. J. Whitmarsh
Docking Interactions in the c-Jun N-terminal Kinase Pathway
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N. Allaman-Pillet, J. Storling, A. Oberson, R. Roduit, S. Negri, C. Sauser, P. Nicod, J. S. Beckmann, D. F. Schorderet, T. Mandrup-Poulsen, et al.
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