Figure 2 - The kinase activities of TRI and TRII are required for TRI sumoylation.

(a) Activated TRI is more sumoylated than wild-type TRI. In vitro sumoylation of immunopurified Flag-tagged wild-type and activated (ca) TRI in the presence or absence of recombinant SUMO-1, Aos1/Uba2 (E1), and Ubc9. The reaction mixture was analysed by western blotting for TRI. (b) Effects of the TRI kinase inhibitor and TRI dephosphorylation on TRI sumoylation. In vitro sumoylation was performed as in a with wild-type or activated (ca) TRI, as indicated, in the presence or absence of the TRI kinase inhibitor SB431542. The phosphates were removed from TRI with lambda phosphatase before in vitro sumoylation. (c) The kinase activities of TRII and TRI are required for efficient TRI sumoylation. 293T cells co-expressed a cytoplasmic receptor chimaera TRII-RI, in which the TRI cytoplasmic domain follows the TRII cytoplasmic domain, or chimaeras in which the TRII and/or TRI kinase activities were inactivated by point mutation (KR), with Myc-tagged (M-) SUMO-1 and Ubc9. Sumoylation of the chimaera was analysed by western blotting. (d) In vitro sumoylation of immunopurified cytoplasmic receptor chimaeras or each of the kinase-defective receptor chimaeras, used in c. (e) Diagram showing TGF--induced sumoylation of TRI in the receptor complex.