b-Arrestin 2: A Receptor-Regulated MAPK Scaffold for the Activation of JNK3 Patricia H. McDonald, Chi-Wing Chow, William E. Miller, Stéphane A. Laporte, Michael E. Field, Fang-Tsyr Lin, Roger J. Davis, and Robert J. Lefkowitz

Supplementary Material

Supplemental Figure 1a. b-arrestins specifically bind ct-JNKs. (A) The amino acid sequence of the yeast two-hybrid cDNA clones isolated as binding partners for b-arrestin 2 and their location within the JNK molecule are indicated. The solid line represents the start of the JNK2 clones and the broken line represents the start of the JNK3 clone. *TPY* indicates the position of the threonine and tyrosine residues phosphorylated by the MAPKK. (B) ct-JNK2 or ct-JNK3 plasmids were cotransformed with pAS2-1 or pAS2-1 vectors containing b-arrestin 1, b-arrestin 2, or Lamin [as indicated by the diagram (top left)]. All yeast transformants grew on tryptophan- and leucine-deficient plates (bottom left). However, only b-arrestin 1 or b-arrestin 2 cotransformed with clones for ct-JNK2 or ct-JNK3 grew on either adenine- (top right) or histidine-deficient (bottom right) plates, indicative of a positive interaction in the yeast two-hybrid system. Note the much stronger growth with b-arrestin 2 compared with that of b-arrestin 1.

Medium version | Full size version

---

Supplemental Figure 1b.

Medium version | Full size version

---

Supplemental Figure 2a. b-arrestin 2 enhances the activation of JNK3 by ASK1. (A) Demonstration that overexpression of b-arrestin 2 leads to an enhancement of JNK3 phosphorylation. Whole cell lysates were prepared from cells expressing HA-JNK3 with either b-arrestin 1 (bArr1) or b-arrestin 2 (bArr2) or empty vector (control) in the presence of 0.1 to 1 mg of ASK1. Active JNK was detected in the cell lysates on immunoblots probed with an anti-active JNK antibody (top panel). The relative amounts of ASK1 and the b-arrestins in the lysates were examined by immunoblot analysis (lower panels). Data shown are representative of four independent experiments. (B) Graphical representation of the data shown in (A). The data represent the average of four independent experiments and error bars represent SEM.

Medium version | Full size version

---

Supplemental Figure 2b.

Medium version | Full size version

---

Supplemental Figure 3. b-arrestin 2 that lacks the ability to bind either ASK1 or JNK3 is defective in enhancing ASK1-mediated JNK3 phosphorylation. (A) COS-7 cells were transiently transfected with HA-ASK1 only (control), or HA-ASK1 with wild-type (WT) Flag-b-arrestin 2, b-arr2_1-185, or b-arr2_186-410. The b-arrestins were immunoprecipitated using M2 affinity agarose beads and the presence of ASK1 in the immunoprecipitates (IP) was detected by protein blot (IB) analysis (upper panel). COS-7 cells were transiently transfected with HA-JNK3 only (control), or withHA-JNK3 and wild-type Flag-b-arrestin 2, b-arr2_1-185, or b-arr2_186-410. The presence of JNK3 in the immunoprecipitates was detected by immunoblot analysis (lower panel). (B) COS-7 cells were transiently transfected with HA-JNK3, wild-type Flag-b-arrestin 2, b-arr2_1-185, or b-arr2_186-410 with increasing amounts of ASK1. The amount of active JNK in the cell lysates was assessed by immunoblot analysis using antibody to phosphorylated JNK. Data shown are representative of three independent experiments.

Medium version | Full size version