Several proteins are encoded by different transcripts derived from the Jip4 gene by alternative splicing. The gene that encodes JIP4 is located on mouse chromosome 11 and consists of a total of 31 exons. The structure of the gene is illustrated schematically. The gene encodes three proteins (JIP4, JLP, and SPAG9) that are encoded by the selective inclusion of different 5′ exons of the Jip4 gene. The JIP4 mRNA is encoded by exons 2, 3, 4, 5, and 7 through 31 and includes an in-frame termination codon in the 5′ untranslated region. The JLP mRNA is encoded by exons 1, 2, 4, 5, and 7 through 31. The SPAG9 mRNA is encoded by exons 6 and 7 through 31. In-frame termination codons in the 5′ untranslated region of JLP and SPAG9 are not present. The GenBank entries for the genes that encode JIP1, JIP2, and JIP3 are designated Mapk8ip1, Mapk8ip2, and Mapk8ip3. This nomenclature suggests that the GenBank entry for the gene that encodes JIP4 should be designated Mapk8ip4.

Subcellular localization of JIP3 and JIP4. (A) COS cells transfected with Flag-tagged JIP3 or JIP4 were fixed and processed for confocal microscopy. Control or nocodazole-treated cells were stained (green) with antibodies to Flag-tagged JIP3, Flag-tagged JIP4, or α-tubulin. The nucleus was identified by staining DNA with 4,6-diamidino-2-phenylindole (blue). (B) The possible colocalization of JIP3 or JIP4 (green) with organelles was examined by staining (red) with mitotracker red (mitochondria) or antibodies to protein disulfide isomerase (endoplasmic reticulum) or Grasp 65 (Golgi).

JIP4 is not a scaffold for the MLK3-MKK7-JNK module. The effect of JIP3 and JIP4 on MLK3-stimulated JNK1 activity was examined in a cotransfection assay using COS cells. The levels of expression of JIP3, JIP4, MLK3, and JNK were examined by immunoblot analysis (upper panel). JNK1 activity was measured by using an immune complex kinase assay with [γ-³²P]ATP and c-Jun as the substrates. The phosphorylated c-Jun was detected following SDS-PAGE by autoradiography (middle panel) and quantitated by PhosphorImager (Molecular Dynamics) analysis (lower panel). The data presented were derived from one experiment. Similar data were obtained in three independent experiments.

JIP4 is a substrate for JNK and p38 MAP kinase but not ERK2. (A) JIP4 is a substrate for JNK and p38α MAPK but not ERK2. UV-stimulated JNK1 (upper panel) and p38α MAPK (middle panel), together with phorbol myristate acetate (PMA)-stimulated ERK2 (lower panel), were employed in immune complex kinase assays using [γ-³²P]ATP and GST-JIP3 or GST-JIP4 as the substrates. Control experiments were performed using known substrates for JNK (c-Jun), p38α MAPK (ATF2), and ERK2 (Elk-1). The phosphorylation of the substrates was examined by SDS-PAGE and autoradiography. (B) Immune complex kinase assays were performed using wild-type JIP3 and a mutated JIP3 [JIP3(mu)] protein in which the three sites of phosphorylation by JNK were replaced with Ala (Thr-266, Thr-276, and Thr-287). These phosphorylation sites are conserved in JIP4 (Thr-52, Thr-61, Thr-96, and Thr-112). These Thr phosphorylation sites were replaced with Ala residues. Wild-type JIP4 and the mutant JIP4 protein [JIP4(mu)] were examined as substrates for JNK and p38α MAP kinase. Control experiments were performed using wild-type and mutant JIP3 proteins for JNK assays (upper panel) and ATF2 for p38α MAP kinase assays (lower panel).

JIP4 requires MKK3 and MKK6 for the potentiation of p38 MAP kinase activation. (A) Total protein extracts were prepared from wild-type MEF, Mkk3^−/− MEF, Mkk6^−/− MEF, and Mkk3^−/− Mkk6^−/− compound mutant MEF. The expression levels of MKK3, MKK6, and tubulin were examined by immunoblot analysis. (B) The effects of JIP3 and JIP4 on ASK1-stimulated p38 MAPK activity in wild-type MEF were examined by cotransfection assays. The activity of HA-tagged p38α MAP kinase was examined in an immune complex kinase assay using the substrate ATF2. (C) The effects of JIP3 and JIP4 on ASK1-stimulated p38α MAPK activity in an Mkk3^−/− Mkk6^−/− compound mutant MEF were examined by cotransfection assays. The activity of HA-tagged p38α MAP kinase was examined in an immune complex kinase assay using the substrate ATF2.

Primary structures and expression levels of JIP3 and JIP4. (A) Schematic representation of the domain structures of JIP3 and JIP4. We have previously reported the primary sequence of JIP3 (GenBank accession numbers AF178636 and AF178637) (13). We now report the primary sequence of JIP4 (GenBank accession number AY823270). TM, transmembrane. (B) Northern blot analysis of 2 μg of poly(A)⁺ RNA isolated from several murine tissues. The blot was probed with double-stranded probes derived from JIP3, JIP4, and β-actin. The migration of RNA standards is indicated on the left.

The cytoplasmic proteins JIP3 and JIP4 interact with kinesin light chain. (A) JIP3 and JIP4 are cytoplasmic proteins. Flag-tagged JIP proteins were expressed in COS cells. Cytosolic (C) and membrane (M) fractions of the cells were prepared and examined by an immunoblot analysis using antibodies to the Flag-tagged JIP3 and JIP4. (B) Nerve growth factor-differentiated PC12 cells were separated into cytosolic (C) and membrane (M) fractions. Endogenously expressed JIP3 and JIP4 were examined by immunoblot analysis using antibodies to JIP3 and JIP4. An immunoblot analysis using an antibody to the microsomal membrane protein STT3 was used as a control for cell fractionation. (C) Flag-tagged JIP3 and JIP4, together with V5 epitope-tagged kinesin light chain 1 (KLC), were expressed in COS cells. The cell lysates were examined by immunoprecipitation (IP) and immunoblot (IB) analyses using the indicated epitope tag antibodies. (D) COS cells expressing epitope-tagged JIP1, JIP2, JIP3, or JIP4 were incubated in serum or were serum starved (24 h). Cell lysates were prepared and incubated with the bacterially expressed TPR domain of KLC fused to GST. Protein complexes were collected on glutathione-agarose, and bound Flag-tagged JIP proteins were detected by immunoblot analysis. (E and F) The leucine zipper regions of JIP3 and JIP4 interact with the TPR domain of KLC. Bacterially expressed GST and GST-TPR domain of KLC (GST-KLC) were immobilized on glutathione-agarose and incubated with equal amounts of in vitro-translated and [³⁵S]methionine-labeled JIP3 or JIP4. Control experiments were performed using [³⁵S]methionine-labeled MLK3 and luciferase. The bound proteins were detected by SDS-PAGE and autoradiography.

Comparison of protein interactions of JIP3 and JIP4. T7-tagged JIP3 or JIP4 was expressed in COS cells together with HA-tagged JNK1, p38α MAP kinase, and ERK2 (A), Flag-tagged MKK7 (B), HA-tagged MLK3 (C), or HA-tagged ASK1 (D). The JIP proteins were isolated from the cells by immunoprecipitation (IP) with the T7 antibody. JNK, MLK3, and MKK7 binding were examined by immunoblot (IB) analysis.

JIP4 forms homo-oligomers but does not interact with other JIP proteins. The interactions between JIP proteins were examined. Flag-tagged JIP3 and JIP4 proteins were coexpressed with T7-tagged JIP3 or JIP4 (A) and JIP1 or JIP2 (B). The JIP3 and JIP4 proteins were immunoprecipitated (IP) with the Flag (M2) or T7 antibody. The coimmunoprecipitation of other JIP proteins was examined by immunoblot (IB) analysis. JIP3/4, JIP3 and JIP4; JIP1/2, JIP1 and JIP2.

JIP4 can potentiate p38α MAP kinase activation. (A) JIP4 can interact with p38 MAP kinase. JNK and p38α MAP kinases were in vitro translated in the presence of [³⁵S]methionine and quantitated. Equal amounts of these proteins were incubated with bacterially expressed GST, GST-JIP3 (residues 190 through 380), and GST-JIP4 (residues 1 through 150) immobilized on glutathione-agarose beads. The beads were washed, and bound proteins were detected by SDS-PAGE and autoradiography. (B) The effect of JIP3 and JIP4 on ASK1-stimulated p38 activity was examined in cotransfection assays in COS7 cells by using HA epitope-tagged p38α MAPK. The effect of the expression of ASK1, JIP3, or JIP4 was examined. The levels of expression of ASK1, JIP3, JIP4, and p38α MAP kinase were examined by immunoblot analysis. The p38α MAP kinase was immunoprecipitated, and its activity was measured in an immune complex kinase assay with [γ-³²P]ATP and ATF2 as substrates. The phosphorylated ATF2 was detected by SDS-PAGE and autoradiography and was quantitated by a PhosphorImager analysis. (C) The effects of JIP3 and JIP4 on ASK1-stimulated JNK activity were examined in a similar assay as shown in panel B, using HA-tagged JNK1 and c-Jun as the substrates.