The pro-domain of the zebrafish Nodal-related protein Cyclops regulates its signaling activities

Abstract

Nodal proteins are secreted signaling factors of the transforming growth factor β (TGFβ) family with essential roles in embryonic development in vertebrates. Mutations affecting the Nodal factors have severe consequences in mammals and fish. Furthermore, increased Nodal levels have been associated with melanoma tumor progression. Like other TGFβ-related proteins, Nodal factors consist of a pro-domain and a mature domain. The pro-domain of mouse Nodal protein stabilizes its precursor. However, the mechanisms by which the pro-domains exert their activities are unknown. Here, we characterize the zebrafish Nodal-related factor Cyclops (Cyc) and find unexpected functions for the pro-domain in regulating Cyc activity. We identified a lysosome-targeting region in the Cyc pro-domain that destabilizes the precursor and restricts Cyc activity, revealing the molecular basis for the shortrange signaling activities of Cyc. We show that both the pro-and mature-domains of Cyc regulate its stability. We also characterize a mutation in the pro-domain of human NODAL (hNODAL) that underlies congenital heterotaxia. Heterologous expression of mutant hNODAL increases expression of Nodal-response genes. Our studies reveal unexpected roles for the pro-domain of the Nodal factors and provide a possible mechanism for familial heterotaxia.

Nodal enhances the activity of FoxO3a and its synergistic interaction with Smads to regulate cyclin G2 transcription in ovarian cancer cells

“…ALK4ca expression plasmid in HA-tagged pCMV5 was kindly provided by Dr Liliana Attisano (University of Toronto, ON, Canada). A plasmid expressing only the mature peptide of Nodal in pCS2 þ (Tian et al, 2008) was kindly provided by Dr Karuna Sampath (Nanyang Technological University, Singapore). Human Flag-tagged FoxO3a-WT and FoxO3a-AAA in a pcDNA3 vector (Ramaswamy et al, 2002) were obtained from Addgene (Cambridge, MA, USA).…”

Morphogen transport

“…Nodal and Lefty are members of the transforming growth factor β (TGFβ) superfamily and have signal sequences required for secretion (Zhou et al, 1993;Meno et al, 1996;Beck et al, 2002;Sakuma et al, 2002;Le Good et al, 2005;Jing et al, 2006;Blanchet et al, 2008;Tian et al, 2008;Marjoram and Wright, 2011;Müller et al, 2012). Fusion of the signal sequencecontaining N-terminal domains of Nodal and other morphogens to GFP causes secretion of GFP (Entchev et al, 2000;Yu et al, 2009;Müller et al, 2012).…”

Cytoplasmic mislocalization of p27 and CDK2 mediates the anti-migratory and anti-proliferative effects of Nodal in human trophoblast cells

“…Constitutively active ALK5 and ALK4 were kindly provided by Dr Liliana Attisano and Dr Jeff Wrana (University of Toronto, Canada) (Attisano et al, 1996;Wieser et al, 1995). pCS2-GFP was obtained from Dr Cunming Duan (University of Michigan, MI, USA) (Li et al, 2005) while N53 (encoding full-length Nodal in pCS2) and N56 (encoding mature Nodal in pCS2) were generous gifts from Dr Karuna Sampath (National University of Singapore, Singapore) (Tian et al, 2008). Wild-type p27 (p27-wt) was a gift from Dr Michele Pagano (New York University Cancer Institute, NY, USA) and two S10 mutants, p27-S10A and p27-S10D were provided by Dr Michelle Olive (National Institute of Health, DC, USA) (Boehm et al, 2002).…”