Physical and functional interactions between type I transforming growth factor beta receptors and Balpha, a WD-40 repeat subunit of phosphatase 2A.

We have previously shown that a WD-40 repeat protein, TRIP-1, associates with the type II transforming growth factor beta (TGF-beta) receptor. In this report, we show that another WD-40 repeat protein, the Balpha subunit of protein phosphatase 2A, associates with the cytoplasmic domain of type I TGF-beta receptors. This association depends on the kinase activity of the type I receptor, is increased by coexpression of the type II receptor, which is known to phosphorylate and activate the type I receptor, and allows the type I receptor to phosphorylate Balpha. Furthermore, Balpha enhances the growth inhibition activity of TGF-beta in a receptor-dependent manner. Because Balpha has been characterized as a regulator of phosphatase 2A activity, our observations suggest possible functional interactions between the TGF-beta receptor complex and the regulation of protein phosphatase 2A.

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 kinases, 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 Escherichia coli and baculovirus-infected insect cells. Anti-phosphotyrosine Western blotting demonstrated that the type II receptor kinase can autophosphorylate on tyrosine. Following an in vitro kinase reaction, the autophosphorylation of the cytoplasmic domain and phosphorylation of exogenous substrate was shown by phosphoamino acid analysis to occur not only on serine and threonine but also on tyrosine. The dual kinase specificity of the receptor was also demonstrated using immunoprecipitated receptors expressed in mammalian cells and in vivo 32P labeling showed phosphorylation of the receptor on serine and tyrosine. In addition, the kinase activity of the cytoplasmic domain was inhibited by the tyrosine kinase inhibitor tyrphostin. Tryptic mapping and amino acid sequencing of in vitro autophosphorylated type II receptor cytoplasmic domain allowed the localization of the sites of tyrosine phosphorylation to positions 259, 336, and 424. Replacement of all three tyrosines with phenylalanines strongly inhibited the kinase activity of the receptor, suggesting that tyrosine autophosphorylation may play an autoregulatory role for the kinase activity of this receptor. These results demonstrate that the type II TGF-beta receptor can function as a dual specificity kinase and suggest a role for tyrosine autophosphorylation in TGF-beta receptor signaling.

A WD-domain protein that is associated with and phosphorylated by the type II TGF-beta receptor.

Transforming growth factor-beta (TGF-beta) is the prototype for a family of extracellular polypeptides that affect cell proliferation and differentiation, and tissue morphogenesis. TGF-beta signalling is mediated by two types of serine/threonine kinase receptors, the type I and II receptors, which are able to form a heteromeric complex. No cytoplasmic proteins that associate with these receptors in vivo, or are their kinase targets, have yet been described. We have now identified a WD-domain-containing protein, TRIP-1, which specifically associates with the type II TGF-beta receptor in a kinase-dependent way. TRIP-1 does not interact with the type II activin or type I receptors, but associates with the heteromeric TGF-beta receptor complex. TRIP-1 is phosphorylated on serine and threonine by the receptor kinase, strongly suggesting that it has a role in TGF-beta signalling. This is supported by coexpression of TRIP-1 and type II receptor during development. The existence of TRIP-1 homologues in plant and yeast suggests a conserved function in all eukaryotes.

Phosphorylation-dependent interaction of the cytoplasmic domains of the type I and type II transforming growth factor-beta receptors.

Transforming growth factor-beta (TGF-beta) transduces signals through its type I and type II receptors. Both receptor types have previously been shown to interact in a heteromeric complex in the presence of TGF-beta. We have now characterized these interactions between both receptor types using a combination of yeast two-hybrid interaction assays and coimmunoprecipitation analyses. Our results indicate a direct association between the cytoplasmic domains of the two receptor types. Mutation analysis of these cytoplasmic domains reveals that this direct interaction requires kinase activity and, thus, depends on phosphorylation, probably via a transphosphorylation mechanism. Furthermore, the two receptor types already have an inherent affinity for each other in the absence of TGF-beta, and the heteromeric complex can be detected in coimmunoprecipitations under these conditions. Taken together, our results reveal a novel mechanism of receptor complex formation, whereby two different cytoplasmic domains directly associate with each other. This interaction may play a major role in activation of serine/threonine kinase receptors.

A new proline-rich early nodulin from Medicago truncatula is highly expressed in nodule meristematic cells.

We cloned and characterized MtPRP4, a new member of the repetitive proline-rich protein gene family in Medicago truncatula. The sequence of MtPRP4 predicts a 62-kD protein consisting of a 22-amino acid N-terminal signal peptide and a 527-amino acid repetitive proline-rich domain composed of three repetitive pentapeptide motifs arranged into two decapeptide repeats: PPVEKPPVHK and PPVEKPPVYK. MtPRP4 is the largest PRP described to date and contains repeated motifs that have not previously been found together in a single polypeptide. RNA gel blot experiments detected MtPRP4 transcripts in symbiotic root nodules, but not in roots, hypocotyls, or leaves. Accumulation of MtPRP4 transcript was an early response to Rhizobium inoculation and did not depend on nodule infection. In situ hybridization experiments demonstrated that MtPRP4 was expressed early in the development of the nodule meristem and that expression was highest in the meristematic cells of mature indeterminate nodules. These data support the proposition that an important early response of legume host roots to Rhizobium involves remodeling the host extracellular matrix and that proline-rich wall proteins play an important role in this architectural modification.