JSAP1, a novel jun N-terminal protein kinase (JNK)-binding protein that functions as a Scaffold factor in the JNK signaling pathway

Abstract

The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38alpha), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.

FIG. 1

(A) Deduced amino acid sequence of JSAP1. The region (residues 115 to 504) isolated in the yeast two-hybrid system is shown by L-shaped arrows. The leucine residues of the leucine zipper are boxed. (B) Expression of JNK3 and JSAP1 mRNAs in mouse tissues. Poly(A)⁺ RNA samples isolated from various mouse tissues were examined by Northern blotting analysis. The positions of 28S and 18S rRNAs are indicated on the left. β-Actin mRNA was included as a loading control.

FIG. 2

(A) Binding of JSAP1 to mammalian MAPKs. COS-7 cells were transiently cotransfected with 0.4 μg of pFlag-CMV2-JNK1, -JNK2, -JNK3, -p38α or -ERK2 along with 1.1 μg of either pcDNA3-His-S empty vector or pcDNA3-His-S-JSAP1. Cell lysates were precipitated with S-protein-linked agarose (S-PA) and analyzed by immunoblotting with anti-Flag antibody. Expression of Flag-MAPKs and His-S-JSAP1 was examined by immunoblotting 1/10 of the cell lysates used in the binding reactions. (B) Mapping of the JNK3-binding region on JSAP1. In vitro-translated ³⁵S-labeled JNK3 was incubated with 0.5 μg of either immobilized Trx-His-S protein or Trx-His-S-tagged segments of JSAP1. The protein complexes were extensively washed, and the bound JNK3 was detected by SDS-PAGE and autoradiography. One-tenth of the ³⁵S-labeled protein used in the binding reactions was loaded as a positive control.

FIG. 3

Phosphorylation of JSAP1 by JNK3 (A) and its effect on the interaction with JNK3 (B). (A) COS-7 cells were transiently transfected with 1.5 μg of pFlag-CMV2-JNK3 with or without 0.05 μg of pEF-3XHA-ΔMEKK. Cell lysates were immunoprecipitated with anti-Flag antibody, and the in vitro kinase assay was carried out as described in Materials and Methods. GST–c-Jun (residues 1 to 79) and Trx-His-S-JSAP1 (residues 115 to 274) were used as substrates. The mutants of JSAP1 (T-0, T-1, T-2, and T-3) were as indicated. WT, wild type. The concentration of each substrate was 1 pmol/μl in 30 μl of total volume. (B) COS-7 cells were transiently transfected with 1.5 μg of pFlag-CMV2-JNK3 with or without 0.05 μg of pEF-3XHA-ΔMEKK. The unstimulated (lanes 1 to 3) and stimulated (lanes 4 to 6) Flag-JNK3 were precipitated from cell lysates with 0.5 μg of immobilized Trx-His-S protein (lanes 1 and 4) or the unphosphorylated (lanes 2 and 5) or phosphorylated (lanes 3 and 6) Trx-His-S-JSAP1 and analyzed by immunoblotting with anti-Flag or anti-His antibody. Expression of Flag-JNK3 was examined by immunoblotting 1/10 of the cell lysates used in the binding reactions. The phosphorylated form of Flag-JNK3 was detected with a phospho-specific JNK (P-JNK) antibody.

FIG. 4

Binding of JSAP1 to SEK1 MAPKK and mapping of the SEK1-binding region on JSAP1. COS-7 cells were transiently cotransfected with 0.4 μg of pcDNA3-Flag-SEK1 and 1.1 μg of pcDNA3-His-S-JSAP1, -Δ1, -Δ2, or -Δ3 with or without 0.04 μg of pEF-3XHA-ΔMEKK, as indicated. Cell lysates were precipitated with S-protein-linked agarose (S-PA) and analyzed by immunoblotting with anti-Flag antibody. Expression of Flag-SEK1 and His-S-JSAP1, -Δ1, -Δ2, and -Δ3 was examined by immunoblotting 1/10 of the cell lysates used in the binding reactions. The phosphorylated form of Flag-SEK1 was detected using a phospho-specific SEK1 (P-SEK1) antibody.

FIG. 5

(A) Binding of JSAP1 to mammalian MAPKKs. COS-7 cells were transiently cotransfected with 0.4 μg of pcDNA3-Flag-SEK1, -MKK7, -MEK1, or -MKK6 with 1.1 μg of either pcDNA3-His-S empty vector or pcDNA3-His-S-JSAP1 in the absence or presence of 0.04 μg of pEF-3XHA-ΔMEKK, as indicated. Cell lysates were precipitated with S-protein-linked agarose (S-PA) and analyzed as for Fig. 2A. Expression of Flag-MAPKKs and His-S-JSAP1 was examined by immunoblotting 1/10 of the cell lysates used in the binding reactions. (B) Competition analysis of SEK1 and MEK1 in the interaction with JSAP1. COS-7 cells were transiently cotransfected with 0.2 μg of pcDNA3-Flag-SEK1, 0.02 μg of pEF-3XHA-ΔMEKK, and 0.2 μg of either pcDNA3-His-S empty vector or pcDNA3-His-S-JSAP1 with different amounts of pcDNA3-Myc-MEK1 (0, 0, 0.05, 0.2, and 0.5 μg in lanes 1 to 5, respectively). COS-7 cells were transiently cotransfected with 0.1 μg of pcDNA3-Myc-MEK1, 0.02 μg of pEF-3XHA-ΔMEKK, and 0.2 μg of either pcDNA3-His-S empty vector or pcDNA3-His-S-JSAP1 with different amounts of pcDNA3-Flag-SEK1 (0, 0, 0.1, 0.3, and 0.6 μg in lanes 6 to 10, respectively). Total DNA was kept at 1.5 μg per transfection with pcDNA3-His-S empty vector. Cell lysates were precipitated with S-protein-linked agarose (S-PA) and analyzed as for Fig. 2A. Expression of Flag-SEK1, Myc-MEK1, and His-S-JSAP1 was examined by immunoblotting 1/10 of the cell lysates used in the binding reactions.

FIG. 6

Binding of JSAP1 to MEKK1 MAPKKK, mapping of the MEKK1-binding region on JSAP1, and binding of MEKK1 MAPKKK to SEK1 MAPKK. (A) COS-7 cells were transiently cotransfected with 0.4 μg of pcDNA3-Flag-MEKK-N with 1.1 μg of pcDNA3-His-S empty vector or pcDNA3-His-S-JSAP1, -Δ1, or -Δ4. Cell lysates were precipitated with S-protein-linked agarose (S-PA) and analyzed as for Fig. 2A. Expression of Flag-MEKK-N and His-S-JSAP1, -Δ1, and -Δ4 was examined by immunoblotting 1/10 of the cell lysates used in the binding reactions. (B) In vitro-translated ³⁵S-labeled full-length MEKK was incubated with 0.5 μg of either immobilized Trx-His-S protein or Trx-His-S-tagged segments of JSAP1 and analyzed as for Fig. 2B. One-tenth of the ³⁵S-labeled protein used in the binding reactions was loaded as a positive control. (C) COS-7 cells were transiently transfected with 1.5 μg of pcDNA3-Flag-ΔMEKK. Cell lysate was precipitated with 0.5 μg of either immobilized Trx-His-S protein (lane 2) or Trx-His-S-SEK1 (lane 3) and analyzed by immunoblotting with anti-Flag antibody. One-tenth of the cell lysate used in the binding reactions was loaded as a control (lane 1).

FIG. 7

(A) Binding of JSAP1 to mammalian MAPKKKs. COS-7 cells were transiently cotransfected with 0.4 μg of pcDNA3-Flag-Raf-N, -Raf-C, or -MEKK-N with 1.1 μg of either pcDNA3-His-S empty vector or pcDNA3-His-S-JSAP1. (B) Competition analysis of MEKK1 and Raf-1 in the interaction with JSAP1. COS-7 cells were transiently cotransfected with 0.7 μg of pcDNA3-Flag-Raf-C and 0.2 μg of either pcDNA3-His-S empty vector or pcDNA3-His-S-JSAP1 with different amounts of pcDNA3-Flag-MEKK-N (0, 0, 0.05, 0.2 and 0.6 μg in lanes 1 to 5, respectively). Total DNA was kept at 1.5 μg per transfection with pcDNA3-His-S empty vector. Cell lysates were precipitated with S-protein-linked agarose (S-PA) and analyzed as for Fig. 2A. Expression of Flag-MAPKKKs and His-S-JSAP1 was examined by immunoblotting 1/10 of the cell lysates used in the binding reactions.

FIG. 8

JSAP1 enhances the activation of JNK3 by MEKK1 and SEK1. (A) Effect of overexpressing JSAP1 on JNK activation by MEKK1. COS-7 cells were transiently transfected with 0.2 μg of pFlag-CMV2-JNK1 (lane 1), -JNK2 (lane 4), or -JNK3 (lane 7) alone or with 0.02 μg of pcDNA3-His-S-MEKK1 in the absence or presence of 1 μg of pcDNA3-His-S-JSAP1, as indicated. (B) Effect of overexpressing SEK1 and MEK1 on JNK3 activation by JSAP1 and MEKK1. COS-7 cells were transiently cotransfected with 0.2 μg of pFlag-CMV2-JNK3, 0.06 μg of pcDNA3-His-S-MEKK1, and 1 μg of pcDNA3-His-S-JSAP1 in the absence or presence of 0.2 μg of either pcDNA3-His-S-SEK1 or pcDNA3-His-S-MEK1, as indicated. (C) Effect of overexpressing JSAP1 mutants on JNK3 activation by MEKK1 and SEK1. COS-7 cells were transiently cotransfected with 0.2 μg of pFlag-CMV2-JNK3, 0.06 μg of pcDNA3-His-S-MEKK1, and 0.2 μg of pcDNA3-His-S-SEK1 with 1 μg of pcDNA3-His-S empty vector and pcDNA3-His-S-JSAP1, -ΔJ, -ΔM, or -ΔS, as indicated. Total DNA was kept at 1.5 μg per transfection with pcDNA3-His-S empty vector. Cell lysates were immunoprecipitated with anti-Flag antibody, and kinase activity was measured by using GST–c-Jun (residues 1 to 79) as the substrate. Expression of Flag-JNKs, His-S-JSAP1, His-S-SEK1, and His-S-MEK1 was examined by immunoblotting 1/10 of the cell lysates used in the kinase assays.