The JIP1 signalosome: oligomerization of MAP kinases and VRK2 proteins in the presence of JIP1.

<p>(A) Composition of endogenous signalosome formed by JIP1, VRK2 proteins and JNK in non transfected Cos1 cells. (B). Detection of JIP1 oligomeric complexes in Cos1 cells transfected with only 3 µg of pGST-JIP1. (C) Reconstitution of JIP1 signalosome in the absence of VRK2 proteins in Cos1 cells transfected with 3 µg of pGST-JIP1, 50 ng of pHA-TAK1, 50 ng of pFlag-TAB1, 0,2 µg of pFlag-MKK7, and 4 µg of pFlag-JNK. (D) Effects of VRK2A on the JIP1 signalosome. Cos1 cells were transfected with the plasmids indicated in C plus 4 µg of pCEFL-HA-VRK2A. (E) Effects of VRK2B on the JIP1 signalosome. Cos1 cells were transfected with the plasmids indicated in C plus 4 µg of pCEFL-HA-VRK2B. Cell extracts were fractionated in a Superose 12 10/300 GL column that fractionates proteins in the range from 50 to 1500 kDa. The fractions were analyzed in western blots with antibodies for the specific epitope in each protein. The corresponding molecular weight of the fractions is indicated above. The calculated molecular weights of each monomeric protein are; JIP1 77 kDa, VRK2A 55 kDa, VRK2B 43 kDa, TAK1 64 kDa, TAB1 55 kDa, MKK7 48 kDa and JNK1 46 kDa.