Selective Inhibitory Effects of Smad6 on Bone Morphogenetic Protein Type I Receptors

Abstract

The inhibitory Smads, Smad6 and Smad7, play pivotal roles in negative regulation of transforming growth factor-␤ (TGF-␤) family signaling as feedback molecules as well as mediators of cross-talk with other signaling pathways. Whereas Smad7 acts as a ubiquitous inhibitor of Smad signaling, Smad6 has been shown to effectively inhibit bone morphogenetic protein (BMP) signaling but only weakly TGF-␤/activin signaling. In the present study, we have found that Smad6 inhibits signaling from the activin receptor-like kinase (ALK)-3/6 subgroup in preference to that from the ALK-1/2 subgroup of BMP type I receptors. The difference is attributable to the interaction of Smad6 with these BMP type I receptors. The amino acid residues responsible for Smad6 sensitivity of ALK-3 were identified as Arg-238, Phe-264, Thr-265, and Ala-269, which map to the N-terminal lobe of the ALK-3 kinase domain. Although Smad6 regulates BMP signaling through multiple mechanisms, our findings suggest that interaction with type I receptors is a critical step in the function of Smad6.

The role of bone morphogenetic proteins in myeloma cell survival

“…[27] SMAD7 inhibits both TGF-β and BMP signaling, whereas SMAD6 mainly inhibits BMP signaling, and more efficiently signaling by ALK3/6 than ALK1/2. [29] BMP signaling is also regulated by phosphatases that dephosphorylate C-terminal phosphorylated R-SMADs. [27] Other intracellular regulatory mechanisms also exist, but will not be discussed here.…”

Smad7 Inhibits Transforming Growth Factor-β Family Type I Receptors through Two Distinct Modes of Interaction

“…Preparation of cell lysates, immunoprecipitation, and immunoblotting were performed as described previously (24). The antibodies used were as follows: anti-FLAG antibody (M2; Sigma), anti-HA antibody (3F10; Roche Diagnostics), anti-Myc antibody (9E10; Pharmingen), anti-phospho-Smad1/5 antibody (Cell Signaling), and anti-␣-tubulin antibody (DM1A; Sigma).…”

“…Luciferase Reporter Assay-Luciferase reporter assay was performed as described previously (24). A BMP-responsive reporter construct, BRE-Luc (28), an activin-responsive reporter construct, AR3-Lux, and a TGF-␤-responsive reporter construct, (CAGA) 12 -MLP-Luc (14,29), were used.…”

“…In contrast, Smad6 efficiently inhibits BMP signaling from ALK-3 but only weakly inhibits that from ALK-2 (24). By utilizing these differential effects of Smad6 and Smad7 on ALK-2 signaling, we investigated the molecular basis of the Smad7-ALK-2 interaction.…”

Activation of c-Jun NH2-terminal kinase 1 increases cellular responsiveness to BMP-2 and decreases binding of inhibitory Smad6 to the type 1 BMP receptor

“…(8)(9)(10) Inhibitory Smads6 and -7 potently antagonize the BMP/Smad pathway by interacting with activated BMPR-I and preventing activation of R-Smads or by competing with activated R-Smads to form complexes with Smad4. (11)(12)(13)(14) BMP responsiveness may be modulated in part by complex cross-talk with other signaling pathways, including mitogenactivated protein kinases (MAPKs). (15,16) MAPKs are a group of well-described serine/threonine kinases that transduce extracellular signals to intracellular targets.…”