Interaction of a mitogen-activated protein kinase signaling module with the neuronal protein JIP3

Abstract

The c-Jun NH(2)-terminal kinase (JNK) group of mitogen-activated protein kinases (MAPKs) is activated in response to the treatment of cells with inflammatory cytokines and by exposure to environmental stress. JNK activation is mediated by a protein kinase cascade composed of a MAPK kinase and a MAPK kinase kinase. Here we describe the molecular cloning of a putative molecular scaffold protein, JIP3, that binds the protein kinase components of a JNK signaling module and facilitates JNK activation in cultured cells. JIP3 is expressed in the brain and at lower levels in the heart and other tissues. Immunofluorescence analysis demonstrated that JIP3 was present in the cytoplasm and accumulated in the growth cones of developing neurites. JIP3 is a member of a novel class of putative MAPK scaffold proteins that may regulate signal transduction by the JNK pathway.

FIG. 1

Structure and expression of JIP3. (A) Structure of JIP3 illustrated schematically. (B) Primary sequence of mouse JIP3a and JIP3b deduced from the sequence of cDNA clones, presented in single-letter code. Numbering is based on the sequence of JIP3b. Residues identical to those in JIP3a (.), deletions (-), and termination codons (#) are indicated. (C) Expression of JIP3 mRNA, examined by Northern blot analysis of 2 μg of poly(A)⁺ mRNA isolated from different murine tissues (Clontech Inc.), using a 1.3-kb EcoRI/XbaI fragment of the JIP3a cDNA as a probe (inset). RNA size markers (in kilobases) are indicated on the left. Expression of JIP3 mRNA in different mouse tissues was also examined by dot blot analysis of 5 μg of total RNA hybridized to the JIP3a probe and was quantitated with a PhosphorImager (Molecular Dynamics Inc.). The data are presented graphically as the amount of expression relative to whole brain (100%). Sk, skeletal; Sm, smooth; Submax., submaxillary. (D) Expression of JIP3 protein, examined by Western blot analysis of 75 μg of total protein isolated from different murine tissues and brain subregions (Geno Technology Inc.), using a polyclonal antibody to JIP3. Protein size markers are indicated on the left.

FIG. 1

Structure and expression of JIP3. (A) Structure of JIP3 illustrated schematically. (B) Primary sequence of mouse JIP3a and JIP3b deduced from the sequence of cDNA clones, presented in single-letter code. Numbering is based on the sequence of JIP3b. Residues identical to those in JIP3a (.), deletions (-), and termination codons (#) are indicated. (C) Expression of JIP3 mRNA, examined by Northern blot analysis of 2 μg of poly(A)⁺ mRNA isolated from different murine tissues (Clontech Inc.), using a 1.3-kb EcoRI/XbaI fragment of the JIP3a cDNA as a probe (inset). RNA size markers (in kilobases) are indicated on the left. Expression of JIP3 mRNA in different mouse tissues was also examined by dot blot analysis of 5 μg of total RNA hybridized to the JIP3a probe and was quantitated with a PhosphorImager (Molecular Dynamics Inc.). The data are presented graphically as the amount of expression relative to whole brain (100%). Sk, skeletal; Sm, smooth; Submax., submaxillary. (D) Expression of JIP3 protein, examined by Western blot analysis of 75 μg of total protein isolated from different murine tissues and brain subregions (Geno Technology Inc.), using a polyclonal antibody to JIP3. Protein size markers are indicated on the left.

FIG. 1

Structure and expression of JIP3. (A) Structure of JIP3 illustrated schematically. (B) Primary sequence of mouse JIP3a and JIP3b deduced from the sequence of cDNA clones, presented in single-letter code. Numbering is based on the sequence of JIP3b. Residues identical to those in JIP3a (.), deletions (-), and termination codons (#) are indicated. (C) Expression of JIP3 mRNA, examined by Northern blot analysis of 2 μg of poly(A)⁺ mRNA isolated from different murine tissues (Clontech Inc.), using a 1.3-kb EcoRI/XbaI fragment of the JIP3a cDNA as a probe (inset). RNA size markers (in kilobases) are indicated on the left. Expression of JIP3 mRNA in different mouse tissues was also examined by dot blot analysis of 5 μg of total RNA hybridized to the JIP3a probe and was quantitated with a PhosphorImager (Molecular Dynamics Inc.). The data are presented graphically as the amount of expression relative to whole brain (100%). Sk, skeletal; Sm, smooth; Submax., submaxillary. (D) Expression of JIP3 protein, examined by Western blot analysis of 75 μg of total protein isolated from different murine tissues and brain subregions (Geno Technology Inc.), using a polyclonal antibody to JIP3. Protein size markers are indicated on the left.

FIG. 2

JIP3 binds to the JNK group of MAPKs. (A) Interaction of JIP3 with MAPKs, examined by expression of GST-JIP3a and HA epitope-tagged MAPKs in COS7 cells. GST-JIP3a was isolated from cell lysates by incubation with glutathione-agarose beads and was detected by immunoblot analysis using an antibody that binds GST. The binding to MAPKs was examined by immunoblot analysis using an antibody that binds the HA epitope tag. The interaction of JIP3 with JNK1α1, JNK2α2, JNK3α2, p38α, and ERK2 was investigated. Control experiments were performed by transfection of an empty expression vector instead of the MAPK expression vectors. (B) Epitope-tagged T7-JIP3a and HA-JNK1 were expressed in COS7 cells. Lysates were prepared, and the amount of JIP3 and JNK1 was examined by immunoblot analysis using monoclonal antibodies to the T7 and HA epitopes. HA-JNK1 was immunoprecipitated with the HA antibody, and T7-JIP3a in the immunoprecipitates was detected by immunoblot analysis with an antibody to the T7 epitope tag. (C) Comparison of the binding of 10 JNK isoforms to JIP3. GST and GST-JIP3a were expressed in COS7 cells and immobilized on glutathione-agarose beads. The JNK MAPKs were prepared by in vitro translation in the presence of [³⁵S]methionine and incubated with immobilized GST and GST-JIP3a. No interaction of JNK with GST was detected. However, the JNK protein kinases bound to GST-JIP3a. The bound JNK was detected by SDS-PAGE and autoradiography. The radioactivity was quantitated by PhosphorImager analysis (Molecular Dynamics) and is presented graphically as relative binding. (D) Deletion analysis of JIP3. A series of GST-JIP3b fragments were expressed in bacteria, purified, and immobilized on glutathione-agarose beads. The interaction of these JIP3b fragments with JNK was examined by incubation of the immobilized JIP3b proteins with lysates prepared from COS7 cells expressing Flag epitope-tagged JNK1α1. Bound JNK was detected by immunoblot analysis using antibody M2, which binds the Flag epitope tag. (E) Mutational analysis of the JNK binding domain of JIP3. The function of the JNK binding domain was examined in binding assays using immobilized GST (lane 1) or GST-JIP3b (residues 141 to 241) (lanes 2 to 13) and Flag epitope-tagged JNK1α1 (lane 14). Competition assays were performed by including in the binding assay a synthetic peptide corresponding to the JNK binding domain (10 μg/ml) (lane 3). The effect of replacement of residues in the JNK binding domain with Gly was examined (lanes 4 to 13). The binding of JNK to the immobilized GST-JIP3b is presented. (F) Primary sequences of the JNK binding domains of JIP3 and JIP1 and the consensus sequence.

FIG. 3

JIP3 is phosphorylated by JNK in vivo and in vitro. (A) JIP3 is phosphorylated by JNK in vitro. Epitope-tagged JNK1α1, p38α, and ERK2 MAPKs were expressed in COS-7 cells. The JNK and p38 MAPKs were activated by treatment of the cells without (−) and with (+) UV-C radiation (80 J/m²). The ERK MAPK was activated by treatment of the cells without (−) and with (+) 100 nM phorbol myristate acetate. The MAPKs were isolated by immunoprecipitation using a monoclonal antibody to the HA epitope tag, and immunocomplex protein kinase assays were performed with [γ-³²P]ATP and GST-JIP3b (residues 190 to 380) as the substrate. Control experiments were performed with known substrates for JNK (GST-ATF2), p38 (GST-ATF2), and ERK (GST–c-Myc). Phosphorylation of the substrate proteins was detected following SDS-PAGE by autoradiography. Phosphorylation of JIP3b by JNK, but not by p38 or ERK, was observed. (B and C) Mutational analysis of JIP3b phosphorylation by JNK. Three potential JNK phosphorylation sites (Ser/Thr-Pro) were identified by sequence analysis (Thr²⁶⁶, Thr²⁷⁶, and Thr²⁸⁷). These potential phosphorylation sites were replaced with Ala residues, and the wild-type and mutated JIP3b proteins were examined as substrates for JNK in vitro. The phosphorylation of these JIP3b proteins was detected following SDS-PAGE by autoradiography (B) and was also examined by phosphoamino acid analysis (C). (D) JNK activation in vivo decreases the electrophoretic mobility of JIP3. Flag epitope-tagged JIP3b was expressed in COS7 cells and was detected by immunoblot analysis using antibody M2. The cells were treated without (−) and with (+) UV-C radiation (80 J/m²) to activate JNK. The effect of replacement of the JNK phosphorylation sites Thr²⁶⁶, Thr²⁷⁶, and Thr²⁸⁷ with Ala (Thr/Ala) was examined. (E) COS7 cells expressing epitope-tagged wild-type and mutated [Ala²⁶⁶, Ala²⁷⁶, Ala²⁸⁷] JIP3b were metabolically labeled with [³²P]phosphate. The phosphorylated JIP3b proteins were detected following immunoprecipitation and SDS-PAGE by autoradiography (left) and were examined by phosphoamino acid analysis (right). (F) Analysis of JIP3 phosphorylation by phosphopeptide mapping. Wild-type and mutated (Thr/Ala) [Ala²⁶⁶, Ala²⁷⁶, Ala²⁸⁷] JIP3b phosphorylated in vivo were investigated by phosphopeptide mapping. Maps of JIP3 phosphorylated by JNK1 in vitro were also examined. Comparative phosphopeptide maps were prepared by mixing equal amounts of radioactivity derived from wild-type JIP3 phosphorylated in vivo and in vitro. The origin (o) is indicated on the right. The horizontal and vertical dimensions are electrophoresis and chromatography, respectively. The major [³²P]phosphopeptide present in maps of in vivo phosphorylated wild-type JIP3b and absent in maps of mutated (Thr/Ala) JIP3b is indicated with an arrow.

FIG. 4

JIP3 binds to the MAPKK MKK7. (A) JIP3a was expressed as a GST fusion protein in COS7 cells together with epitope-tagged MEK1, MKK3, MKK4, MKK6, and MKK7. Control experiments were performed with an empty vector instead of the MAPKK expression vector. The expression of JIP3a and MAPKK was examined by immunoblot analysis of cell lysates. GST-JIP3a was isolated on glutathione-agarose beads, and the bound MAPKKs were detected by immunoblot analysis. (B) Epitope-tagged T7-JIP3a and Flag-MKK7 were expressed in COS-7 cells. Lysates were prepared, and the amount of JIP3 and MKK7 was examined by immunoblot analysis using monoclonal antibodies to the T7 and Flag epitopes. The Flag-MKK7 was immunoprecipitated with antibody M2, and T7-JIP3a in the immunoprecipitates was detected by immunoblot analysis with an antibody to the T7 epitope tag. (C) Purified recombinant GST and GST-JIP3a were immobilized on glutathione-agarose and incubated with purified recombinant Flag-tagged MKK7. Bound MKK7 was detected by immunoblot analysis using an antibody that binds the Flag epitope tag. (D) Deletion analysis of JIP3. To define the MKK7 binding region of JIP3, in vitro-translated fragments of JIP3a (residues 1 to 442, 410 to 815, and 800 to 1337) were prepared in the presence of [³⁵S]methionine. Control experiments were performed with in vitro-translated luciferase. These proteins were incubated with GST or GST-MKK7 immobilized on glutathione-agarose. The binding of JIP3 was detected following SDS-PAGE by autoradiography. Binding of in vitro-translated JIP3 to GST-MKK7, but not to GST, was detected.

FIG. 5

Interaction of JIP3 with MAPKKK. (A) Epitope-tagged MAPKKKs and GST-JIP3a were expressed in COS7 cells. Control experiments were performed with empty vector instead of the MAPKKK expression vectors. GST-JIP3a protein was isolated on glutathione-agarose beads. The binding of MAPKKK to JIP3 was examined by immunoblot analysis using an antibody that binds the epitope tag. (B) Epitope-tagged T7-JIP3a and HA-MLK3 were expressed in COS7 cells. Lysates were prepared, and the amount of JIP3a and MLK3 was examined by immunoblot analysis using monoclonal antibodies to the T7 and HA epitopes. HA-MLK3 was immunoprecipitated with the HA antibody, and T7-JIP3a in the immunoprecipitates was detected by immunoblot analysis with an antibody to the T7 epitope tag. (C) Interaction of purified recombinant MLK3 with JIP3a. Epitope-tagged HA-MLK3 was isolated by immunoprecipitation and was eluted by incubation with HA synthetic peptide (20 μg/ml) for 2 h at 4°C. The purified soluble MLK3 was incubated with GST or GST-JIP3a immobilized on glutathione-agarose. The amount of MLK3, GST, and GST-JIP3a was examined by immunoblot analysis using HA or GST antibodies. The agarose beads were washed, and bound MLK3 was detected by immunoblot analysis with an antibody to the HA epitope tag. (D) Deletion analysis of JIP3. To define the MLK3 binding region of JIP3, fragments of JIP3a (residues 1 to 1337, 1 to 815, 1 to 442, 420 to 815, and 800 to 1337) fused to GST were immobilized on glutathione-agarose. Control experiments were performed with immobilized GST. These immobilized proteins were incubated with MLK3 (top) or luciferase (bottom) prepared by in vitro translation in the presence of [³⁵S]methionine. Binding to the immobilized proteins was examined following SDS-PAGE by autoradiography.

FIG. 6

Identification of JIP3 complexes. (A) The interaction of JIP3 with components of the JNK signaling pathway was examined by coimmunoprecipitation analysis. Soluble extracts prepared from mouse brain were immunoprecipitated with a nonimmune antibody (Control) and with antibodies to JNK, MKK7, JIP1, and JIP2. The immunoprecipitates were examined by immunoblot analysis with an antibody to JIP3. (B) Complex formation with JIP3 is increased by JNK activation. COS cells were transfected with an empty expression vector (Control) or with an expression vector for Flag-tagged JIP3b. The effect of replacement of the three JNK phosphorylation sites (Thr²⁶⁶, Thr²⁷⁶, and Thr²⁸⁷) with Ala was examined. The cells were exposed without (−) and with (+) UV-C radiation (80 J/m²) and incubated for 1 h. JIP3b was isolated by immunoprecipitation with monoclonal antibody M2. The presence of JNK in the immunoprecipitates was examined by immunoblot analysis by probing with a rabbit polyclonal antibody to JNK.

FIG. 7

JIP3 potentiates MLK3-stimulated JNK activity. The effect of JIP3 on MLK3-stimulated JNK activity was examined in cotransfection assays using HA epitope-tagged JNK1α1 (A), JNK2α2 (B), and JNK3α2 (C). The effect of expression of MLK3 and JIP3a was examined. Control experiments were performed with the JIP1 and JIP2 scaffold proteins. The expression of MLK3, JIP1, JIP2, JIP3, and JNK was investigated by immunoblot analysis. JNK was immunoprecipitated with an antibody that binds the HA epitope tag, and protein kinase activity was measured with [γ-³²P]ATP and c-Jun as substrates. The phosphorylated c-Jun was detected following SDS-PAGE by autoradiography and was quantitated by PhosphorImager (Molecular Dynamics) analysis. The data presented were derived from one experiment. Similar data were obtained in seven independent experiments.

FIG. 8

JIP3 selectively interacts with JIP scaffold proteins. (A) Epitope (T7)-tagged JIP3a was coexpressed with GST-tagged JIP3a in COS7 cells and incubated with glutathione-agarose. Bound proteins were detected by immunoblot analysis with an antibody to the T7 epitope. Expression of T7-JIP3 and GST-JIP3 proteins in the cell lysates was examined by immunoblot analysis. (B) Epitope (T7)-tagged JIP1 and JIP2 were expressed in COS7 cells. JIP3a was also expressed as a GST fusion protein in COS7 cells and immobilized on glutathione-agarose beads. Bound proteins were detected by immunoblot analysis with an antibody to the T7 epitope. Expression of T7-JIP1, T7-JIP2, and GST-JIP3a proteins in the cell lysates was monitored by immunoblot analysis. (C) Epitope-tagged T7-JIP3a and Flag-JIP2 were expressed in COS-7 cells. Lysates were prepared, and the amount of JIP3 and JIP2 was examined by immunoblot analysis using monoclonal antibodies to the T7 and Flag epitopes. The Flag-JIP2 was immunoprecipitated with antibody M2, and T7-JIP3 in the immunoprecipitates was detected by immunoblot analysis with an antibody to the T7 epitope tag. (D) Deletion analysis of JIP3. To define the JIP2 binding region of JIP3a, fragments of JIP3a (residues 1 to 1337, 1 to 815, 1 to 442, 420 to 815, and 800 to 1337) fused to GST were immobilized on glutathione-agarose. Control experiments were performed with immobilized GST. These immobilized proteins were incubated with JIP2 prepared by in vitro translation in the presence of [³⁵S]methionine. Binding of JIP2 to the immobilized proteins was examined following SDS-PAGE by autoradiography. (E) Deletion analysis of JIP2. To define the JIP3 binding region of JIP2, fragments of JIP2 fused to GST were immobilized on glutathione-agarose. Control experiments were performed with GST. These immobilized proteins were incubated with JIP3a prepared by in vitro translation in the presence of [³⁵S]methionine. The binding of JIP3 to an NH₂-terminal fragment of JIP2 (residues 1 to 229) and a COOH-terminal fragment of JIP2 (residues 557 to 824) was examined following SDS-PAGE by autoradiography.

FIG. 9

NGF regulates the expression of JIP3. (A) JIP3 expression was induced by treatment of PC12 cells with NGF. PC12 cells were differentiated to a neuron-like phenotype by culture in the presence of NGF for 12 days. The expression of JIP3 was examined by immunoblot analysis using preimmune and immune sera prepared from a rabbit immunized with recombinant JIP3. Control experiments were performed with COS7 cells transfected with an empty expression vector or with a JIP3 expression vector. (B) Expression of JIP3 by PC12 cells treated with NGF for various times was examined by immunoblot analysis. Extracts prepared from COS cells transfected with a JIP3b expression vector were examined in control experiments (JIP3). (C) Differentiated PC12 cells (+ NGF) were deprived of NGF (− NGF) for 24 h in the presence and absence of the caspase inhibitor zVAD (0.05 mM). Both the adherent (Adher.) and nonadherent (Non-Adher.) populations of cells were collected. The expression of JIP3 was examined by immunoblot analysis. (D) JIP3b (residues 1 to 781) was prepared by in vitro translation in the presence of [³⁵S]methionine and incubated in the absence (−) or presence (+) of recombinant active caspase 1 (Casp-1) and caspase 3 (Casp-3). The effect of caspase digestion was examined following SDS-PAGE by autoradiography. Control experiments were performed with in vitro-translated PARP, which is a substrate of caspase 3. (E) In vitro-translated JIP3b (residues 1 to 781) was incubated without (−) and with (+) caspase 3 in the presence of the caspase inhibitors zVAD and DEVD (0.1 and 0.01 mM). The effect of caspase digestion was examined following SDS-PAGE by autoradiography. (F) Mutational analysis of the caspase 3 consensus site in JIP3. In vitro-translated JIP3b (residues 1 to 781) was incubated without (−) and with (+) caspase 1 and caspase 3. The effect of the replacement of Asp-344 with Glu in the predicted caspase 3 cleavage site of JIP3 was investigated. The products of caspase digestion were examined following SDS-PAGE by autoradiography. Sizes in panels C to F are indicated in kilodaltons.

FIG. 10

Subcellular localization of JIP3. PC12 cells differentiated in the presence of NGF for 12 days were fixed and processed for dual-label indirect immunofluorescence microscopy. The cells were stained with an antibody to JIP3 (red), neuron-specific enolase (red), and tubulin (green). Competition experiments were performed by including recombinant JIP3 (10 μg/ml) in the incubation with the primary antibody.