Novel dominant negative Smad antagonists to TGFbeta signaling.
We previously identified a critical serine/threonine residue within the linker domain of Smad2/3, phosphorylated by the kinase GRK2 which plays a critical role in regulating Smad signaling. To define the mechanism by which GRK2-mediated phosphorylation modifies Smad2/3 behavior at the molecular level, we generated mutant Smads where the GRK2 phosphorylation site was replaced with an aspartic acid (D) to mimic the properties of a phospho-residue or an alanine (A) as a control. Interestingly, overexpression of either the D or A mutant inhibits TGFbeta signaling, but through two distinct mechanisms. The D mutant is properly localized and released from the plasma membrane upon ligand stimulation, but fails to interact with the type I receptor kinase. The A mutant properly interacts with and is phosphorylated by the type I receptor, translocates to the nucleus and homodimerizes with wild-type R-Smads, but it fails to form a heterocomplex with Smad4. As a result, both mutants act as antagonists of endogenous TGFbeta signaling through divergent mechanisms. The D mutant acts by blocking endogenous R-Smads phosphorylation and the A mutant acts by preventing endogenous R-Smad/Smad4 heterocomplexes. Thus, mutation of the GRK2 phosphorylation site within the Smad generates dominant negative Smads that efficiently inhibit TGFbeta responses.
Ho
Joanne
J
Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, McGill University, 687 Pine Avenue West, Montreal, Canada.
Chen
Hui
H
Lebrun
Jean-Jacques
JJ
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D015870:Gene Expression; D005799:Genes, Dominant; D018716:Molecular Mimicry; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D015398:Signal Transduction; D051785:Smad Proteins / Q000235:genetics* / Q000378:metabolism*; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D051901:Smad4 Protein / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors*; D051552:beta-Adrenergic Receptor Kinases / Q000378:metabolism
D050505:Mutant Proteins; D051785:Smad Proteins; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D016212:Transforming Growth Factor beta; D051552:beta-Adrenergic Receptor Kinases; D020559:Monomeric GTP-Binding Proteins