Article Figures & Data
Figures
- Fig. 1.
JIP2 is a member of the JIP group of scaffold proteins. (A) The structure of the JIP group of scaffold proteins is illustrated schematically. The JBD, the SH3 domain, and the PTB domain are indicated. Comparison of the NH2-terminal region of JIP2 (residues 1 to 603) with the corresponding region of JIP1 indicates 41% identity and 46% similarity (Line-up Program, Wisconsin package, version 9.1; Genetics Computer Group). The SH3 domains of JIP2 (residues 604 to 665) and JIP1 are 66% identical and 80% similar. The PTB domains of JIP2 (residues 683 to 812) and JIP1b are 64% identical and 74% similar. (B) The human JIP2 gene consists of 12 exons and is located on chromosome 22q13 (GenBank accession no. U62317 ). The structure of the gene is illustrated schematically. Solid boxes, coding regions; open boxes, noncoding regions of exons 1 and 12. (C) The primary sequence of human JIP2 deduced from the sequence of cDNA clones is presented in single-letter code and compared to the sequences of JIP1 and JIP1b. Periods, residues that are identical to JIP2 residues; dashes, deletions; pound signs (#), termination codons. The JBD, the SH3 domain, and the PTB domain are indicated. The sequences of JIP1, JIP1b, and JIP2 have been deposited in GenBank under accession no.AF003115 , AF054611 , and AF136382 , respectively. (D) The expression of JIP2 mRNA was examined by Northern blot analysis of different human tissues (Clontech Inc.) using JIP2 cDNA as a probe. (E) Comparison of the tissue distribution of the expression of the human JIP1and JIP2 genes. Immobilized human mRNA was hybridized with human JIP1 and JIP2 cDNA probes labeled with 32P by random priming. The data were quantitated by PhosphorImager analysis and are presented graphically. The data are normalized to the expression in the whole brain (arbitrarily set at 1.0). JIP1 and JIP2 are represented by open and filled bars, respectively.
- Fig. 2.
Selective binding of JIP1 and JIP2 to the mixed-lineage group of MAPKKKs. (A) Epitope-tagged JIP1 or JIP2 was expressed in cells together with epitope-tagged MAPKKKs. Control experiments were performed by transfection of the empty expression vector instead of the JIP expression vector. The expression of JIP and MAPKKK proteins was examined by immunoblot analysis of the cell lysates. The MAPKKK proteins were immunoprecipitated with the M2 monoclonal antibody to the Flag epitope. The presence of JIP proteins in the immunoprecipitates (IP) was examined by immunoblot analysis using a monoclonal antibody that binds the T7-Tag epitope. Coimmunoprecipitation was not observed in experiments using MEKK1, MEKK3, or MEKK4. In contrast, JIP1 and JIP2 coimmunoprecipitated with the MLKs DLK, MLK2, and MLK3. (B) The COOH-terminal regions of JIP1 (residues 283 to 660) and JIP2 (residues 499 to 824) were expressed in cells as GST fusion proteins together with epitope-tagged MLK. Control experiments were performed by transfection with the GST expression vector pEBG. The amounts of the GST proteins and MLK in the cell lysates were examined by protein immunoblot analysis. The GST fusion proteins were precipitated from cell lysates with glutathione-agarose, and MLK present in the pellet was detected by protein immunoblot analysis.
- Fig. 3.
Selective binding of JIP1 and JIP2 to the MAPKK MKK7. (A) Epitope-tagged JIP1 and JIP2 were expressed in cells together with epitope-tagged MEK1, MKK3, MKK4, MKK6, or MKK7. Control experiments were performed by transfection of the empty expression vector instead of the JIP expression vector. The expression of JIP and MAPKK proteins was examined by immunoblot analysis of the cell lysates. The MAPKK proteins were immunoprecipitated, and the presence of JIP1 and JIP2 in the immunoprecipitates (IP) was examined by immunoblot analysis using a monoclonal antibody that binds the T7-Tag epitope. (B) The COOH-terminal regions of JIP1 (residues 283 to 660) and JIP2 (residues 499 to 824) were expressed in cells as GST fusion proteins together with epitope-tagged MKK7. Control experiments were performed by transfection with the GST expression vector pEBG. The amounts of the GST proteins and MKK7 in the cell lysates were examined by protein immunoblot analysis. The GST fusion proteins were precipitated from cell lysates with glutathione-agarose, and MKK7 present in the pellet was detected by protein immunoblot analysis using an antibody to the Flag epitope.
- Fig. 4.
Selective binding of JIP1 and JIP2 to the JNK group of MAPKs. (A) Epitope-tagged (T7-Tag) JIP1 and JIP2 were expressed in cells with the HA-tagged MAPKs ERK2, p38α, and JNK2α2. Control experiments were performed by transfection of the empty expression vector instead of the JIP expression vectors. The MAPKs were immunoprecipitated with an antibody to HA. The presence of JIP in the immunoprecipitates (IP) was detected on immunoblots probed with an antibody to T7-Tag. The amounts of JIP and MAPKs in the cell lysates were examined by protein immunoblot analysis. (B) The NH2-terminal regions of JIP1 (residues 1 to 282) and JIP2 (residues 1 to 229) were expressed in cells as GST fusion proteins together with epitope-tagged JNK1α1 or JNK2α2. Control experiments were performed by transfection with the GST expression vector pEBG instead of the JIP expression vector. The amounts of the GST fusion proteins and JNK in the cell lysates were examined by protein immunoblot analysis. The GST fusion proteins were precipitated from cell lysates with glutathione-agarose, and JNK present in the pellet was detected by protein immunoblot analysis using an antibody to the HA epitope. (C) Comparison of the binding of 10 human JNK isoforms to JIP1 and JIP2. The JNK protein kinases were prepared by in vitro translation in the presence of [35S]methionine. The JNK proteins were incubated with GST-JIP1, GST-JIP2, or GST immobilized on glutathione-agarose beads. The beads were washed with lysis buffer. Bound JNK was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The amount of JNK bound was quantitated by PhosphorImager analysis. The data are presented graphically as relative binding by defining the amount of JNK1α1 binding to JIP1 as 1.0.
- Fig. 5.
JIP2 binds JNK, MKK7, and MLK3 in vitro. Purified bacterially expressed GST and GST-JIP2 fusion proteins were immobilized on glutathione-agarose and incubated with purified bacterially expressed JNK2, MKK7, or MLK3 (residues 1 to 204). Bound JNK2, MKK7, and MLK3 were detected by immunoblot analysis.
- Fig. 6.
JIP1 and JIP2 enhance JNK activation by MLK3. Epitope-tagged (HA) JNK1α1 (A), JNK2α2 (B), and JNK3α2 (C) were each expressed alone or coexpressed with JIP1 and/or JIP2. JNK activity was examined by using c-Jun as a substrate. The effect of coexpression with MLK3 was examined. The amounts of JIP, MLK, and JNK expressed were examined by immunoblot analysis.
- Fig. 7.
JIP proteins form homo- and hetero-oligomeric scaffold complexes. Epitope-tagged JIP1 and JIP2 proteins (Flag and T7-Tag) were expressed in COS-7 cells. The expression of epitope-tagged JIP proteins was examined by immunoblot analysis of cell lysates with antibodies to Flag and T7-Tag. Coimmunoprecipitation analysis was performed by immunoblot analysis (T7-Tag antibody) of immunoprecipitates (IP) prepared by using the M2 monoclonal antibody to the Flag epitope.
- Fig. 8.
The interaction of JIP1 and JIP2 is mediated by the COOH-terminal region. Epitope-tagged JIP1 was expressed in COS-7 cells together with GST or GST-JIP2 fusion proteins. The expression of JIP1 was detected by immunoblot analysis using an antibody to the T7 epitope tag. GST and GST-JIP2 fusion proteins were detected by immunoblot analysis using an antibody that binds GST. The interaction of JIP1 with JIP2 was examined by isolation of GST and GST-JIP2 complexes using glutathione-agarose and immunoblot analysis of the bound T7-tagged JIP1. Deletion analysis demonstrated that the COOH-terminal regions, but not the NH2-terminal regions, of JIP1 and JIP2 were sufficient for the formation of hetero-oligomeric complexes.
- Fig. 9.
Identification of endogenous oligomeric JIP complexes. (A) Characterization of monoclonal antibodies to JIP1 and JIP2. COS-7 cell lysates containing T7-Tag-labeled JIP proteins were examined by protein immunoblot analysis using antibodies to the T7-Tag and monoclonal antibodies to JIP1 and JIP2. (B) JIP1 and JIP2 interact in vivo. Extracts were prepared from Rin5F insulinoma cells and examined by protein immunoblot analysis by probing with murine antibodies to JIP1 and JIP2. The endogenous JIP1 and JIP2 proteins were detected in the cell lysates and in immunoprecipitates (IP) prepared by using antibodies to JIP1 and JIP2.
- Fig. 10.
Cytoplasmic location of endogenous JIP proteins. (A) The endogenous JIP1 and JIP2 proteins expressed by Rin5F insulinoma cells were detected by confocal immunofluorescence analysis using monoclonal antibodies to JIP1 and JIP2 (red). The nucleus was detected by staining DNA with SYTOX green. (B) Double-label immunofluorescence analysis of JIP1 (green) and JIP2 (red) was performed by conventional microscopy. The nucleus was detected by staining DNA with 4,6-diamidino-2-phenylindole (blue).
