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. Author manuscript; available in PMC: 2009 Feb 27.
Published in final edited form as: Nat Cell Biol. 2008 May 11;10(6):654–664. doi: 10.1038/ncb1728

Figure 2.

Figure 2

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