Transforming growth factor-β (TGF-β) elicits its cellular effects through activation of type I and type II serine/threonine kinase receptors [1, 2]. The constitutively active type II receptor phosphorylates specific serine and threonine residues in the juxtamembrane region (so-called GS domain) of the type I receptor. Type I receptor acts downstream of type II receptor (Tβ R-II) and has been shown to determine signaling specificity within the heteromeric receptor complex. In most cell types, TGF-β signals via TGF-β type I receptor (Tβ R-I), also termed activin receptor-like kinase 5 (ALK5). In endothelial cells (ECs), however, TGF-β can signal via Tβ R-II and two different type I receptors, i.e. the broadly expressed ALK5 and the EC-restricted ALK1. Whereas ALK5 inhibits EC migration and proliferation, ALK1 stimulates both these processes . The activated type I receptor propagates the signal through phosphorylation of specific receptor-regulated Smads (R-Smads). Whereas ALK5 induces the phosphorylation of Smad2 and Smad3, ALK1 mediates the activation of Smad1 and Smad5 [4, 5]. Activated R-Smads can assemble into heteromeric complexes with common partner (Co-) Smad, i.e. Smad4 and translocate into the nucleus where they regulate the transcription of target genes [1, 2].
I-Smads (Smad6 and Smad7) are natural inhibitors of TGF-β signaling that prevent the activation of R- and Co-Smads [6–8]. They do so by interacting efficiently with the activated type I receptors preventing access and phosphorylation of R-Smads by the activated type I receptors. Smad6 has also been found to exert its inhibitory effect on signaling by competing with Smad4 for heteromeric complex formation with activated Smad1  and by recruiting the co-repressor CtBP and thereby repress transcription [10, 11]. I-Smads were found to interact with Smad ubiquitination-related factors, Smurfs, which are HECT-domain ubiquitin ligases that target the TGF-β receptors for degradation [12, 13]. The expression of I-Smads is quickly induced upon stimulation by members of the TGF-β family and upon shear stress of the endothelium [14, 15]. Thus, I-Smads may be part of negative feedback control mechanisms.
A key event in TGF-β signaling is serine phosphorylation of Tβ R-I by Tβ R-II, and of R-Smads by Tβ R-I. These phosphorylations are tightly controlled, e.g. the immunophilin FKBP-12 binds to Tβ R-I and thereby inhibits phosphorylation of Tβ R-I by Tβ R-II . C-terminal phosphorylation of Smad2 and Smad3 is strongly facilitated by Smad anchor for receptor activation (SARA).
Serine/threonine protein phosphatases (PPs) are likely involved in the dephosphorylation of these phosphorylated signaling components. PPs consist of a catalytic subunit that binds to one or two regulatory subunits that generate holoenzymes with unique localizations and specificities . One of the major PPs is PP1 consisting of a PP1 catalytic subunit (PP1c) that can form complexes with more than 50 regulatory subunits . Four mammalian isoforms of the PP1c gene have thus far been described, i.e. PP1α, PP1β and two splice variants of PP1γ. Studies in Drosophila melanogaster suggest that PP1 binds to the decapentaplegic (dpp) type I receptor with the aid of SARA and negatively regulates dpp signaling . Very recently, it was reported that the TGF-β- induced Smad7 can interact with the growth arrest and DNA damage protein 34 (GADD34) (21), which is a regulatory subunit of PP1. The Smad7- GADD34 complex was shown to recruit PP1c to Tβ R-I, and thereby dephosphorylate and inactivate it .
In the present report, we have investigated the molecular mechanisms that underlie the TGF-β-induced transient ALK1-mediated Smad1/5 phosphorylation versus sustained ALK5-mediated Smad2 phosphorylation in ECs. Analysis of the effect of various chemical inhibitors on the TGF-β/ALK1 response, suggested an important contribution of PP1 in the negative regulation of ALK1 signaling, but not ALK5 signaling in ECs. Our data suggest that Smad7, induced by ALK1 activation, recruits PP1α to ALK1 and thereby inhibits TGF-β/ALK1-induced Smad1/5 phosphorylation in ECs.