THE TYPE-II TRANSFORMING GROWTH-FACTOR-BETA RECEPTOR AUTOPHOSPHORYLATES NOT ONLY ON SERINE AND THREONINE BUT ALSO ON TYROSINE RESIDUES

The type I and type II receptors for transforming growth factor-beta ( TGF-beta) are structurally related transmembrane serine/threonine kina ses, which are able to physically interact with each other at the cell surface. To help define the initial events in TGF-beta signaling, we characterized the kinase activity of the type II TGF-beta receptor. A recombinant cytoplasmic domain of the receptor was purified from Esche richia coli and baculovirus-infected insect cells. Anti-phosphotyrosin e Western blotting demonstrated that the type II receptor kinase can a utophosphorylate on tyrosine. Following an in vitro kinase reaction, t he autophosphorylation of the cytoplasmic domain and phosphorylation o f exogenous substrate was shown by phosphoamino acid analysis to occur not only on serine and threonine but also on tyrosine. The dual kinas e specificity of the receptor was also demonstrated using immunoprecip itated receptors expressed in mammalian cells and in vivo P-32 labelin g showed phosphorylation of the receptor on serine and tyrosine. In ad dition, the kinase activity of the cytoplasmic domain was inhibited by the tyrosine kinase inhibitor tyrphostin. Tryptic mapping and amino a cid sequencing of in vitro autophosphorylated type II receptor cytopla smic domain allowed the localization of the sites of tyrosine phosphor ylation to positions 259, 336, and 424. Replacement of all three tyros ines with phenylalanines strongly inhibited the kinase activity of the receptor, suggesting that tyrosine autophosphorylation may play an au toregulatory role for the kinase activity of this receptor. These resu lts demonstrate that the type II TGF-beta receptor can function as a d ual specificity kinase and suggest a role for tyrosine autophosphoryla tion in TGF-beta receptor signaling.