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Disruption of transforming growth factor beta signaling by a mutation that prevents transphosphorylation within the
J Cárcamo1, A Zentella, J Massagué
1Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Abstract:
T beta R-II (transforming growth factor beta [TGF-beta] type II receptor) is a transmembrane serine/threonine kinase that acts as the primary TGF-beta receptor. Ligand binding to T beta R-II leads to the recruitment and phosphorylation of T beta R-I, a distantly related transmembrane kinase that acts as a downstream signaling component. T beta R-I phosphorylation by T beta R-II is shown here to be essential for signaling. A mutant T beta R-II that binds ligand but lacks signaling activity was identified. This mutant was identified by screening with a TGF-beta-inducible vector a series of mink lung epithelial cell clones that have normal TGF-beta binding activity but have lost antiproliferative and transcriptional responses to TGF-beta. When transiently cotransfected with T beta R-II, one of these cell lines, S-21, recovered TGF-beta responsiveness. cDNA cloning and sequencing of T beta R-II from S-21 cells revealed a point mutation that changes proline 525 to leucine in kinase subdomain XI. A recombinant receptor containing this mutation, T beta R-II(P525L), is similar to wild-type T beta R-II in its abilities to bind ligand, support ligand binding to T beta R-I, and form a complex with T beta R-I in vivo. T beta R-II(P525L) has autophosphorylating activity in vitro and in vivo; however, unlike the wild-type receptor, it fails to phosphorylate an associated T beta R-I. These results suggest that T beta R-II(P525L) is a catalytically active receptor that cannot recognize T beta R-I as a substrate. The close link between T beta R-I transphosphorylation and signaling activity argues that transphosphorylation is essential for signal propagation via T beta R-I.
Insights
Transforming growth factor beta type II receptor (TβR-II) signaling requires TβR-II to phosphorylate TβR-I. A specific TβR-II mutation (P525L) impairs this transphosphorylation, blocking TGF-β responses despite normal ligand binding.
Area of Science:
- Cellular biology
- Molecular signaling
- Receptor tyrosine kinases
Background:
- Transforming growth factor beta (TGF-β) signaling is crucial for cellular processes.
- TGF-β signaling relies on a receptor complex involving TβR-II and TβR-I.
- The precise mechanism of TβR-I activation by TβR-II is under investigation.
Purpose of the Study:
- To identify mutations in TβR-II that disrupt TGF-β signaling.
- To elucidate the role of TβR-I phosphorylation in TGF-β signal transduction.
- To characterize a novel TβR-II mutant with impaired signaling.
Main Methods:
- Screening of mink lung epithelial cells for TGF-β unresponsiveness.
- Identification and sequencing of mutant TβR-II from unresponsive cell lines.
- Expression and characterization of recombinant wild-type and mutant TβR-II.
- In vitro and in vivo kinase assays to assess TβR-I phosphorylation.
Main Results:
- A TβR-II mutant (P525L) was identified that binds TGF-β but fails to induce cellular responses.
- This mutant receptor retains ligand binding, TβR-I complex formation, and autophosphorylation.
- Crucially, TβR-II(P525L) cannot phosphorylate TβR-I, indicating a substrate recognition defect.
- TβR-I transphosphorylation by TβR-II is demonstrated to be essential for signal propagation.
Conclusions:
- The P525L mutation in TβR-II disrupts its kinase activity towards TβR-I.
- TβR-I transphosphorylation is a critical step for initiating TGF-β signal transduction.
- This study provides direct evidence linking TβR-I phosphorylation to downstream signaling events.