Lefty proteins exhibit unique processing and activate the MAPK pathway

Lefty polypeptides, novel members of the transforming growth factor-beta (T GF-beta) superfamily, are involved in the formation of embryonic lateral pa tterning. Members of the TGF-beta superfamily require processing fear their activation, suggesting cleavage to be an essential step for lefty activati on. Transfection of different cell lines with lefty resulted in expression of a 42-kDa protein, which was proteolytically processed to release two pol ypeptides of 34 and 28 kDa, Since members of the proprotein convertase (PC) family cleave different TGF-beta factors and are involved in the establish ment of embryonic laterality, we studied their role in lefty processing. Co transfection analysis showed that PC5A processed the lefty precursor to the 34-kDa form in vivo, whereas furin, PACE4, PC5B, and PC7 had a limited act ivity. None of these PCs showed activity in the processing of the lefty pol ypeptide to the 28-kDa lefty form. The mutation of the consensus sequences for PC cleavage in the lefty protein allowed the lefty cleavage sites to be identified. Mutations of the sequence RGKR to (G) under bar GK (G) under b ar (amino acids 74-77) and of RHGR to GHGR (amino acids 132-135) prevented the proteolytic processing of the lefty precursor to the 34- and 28-kDa for ms, respectively. To identify the biologically active form of lefty, we stu died the effect of lefty treatment on pluripotent P19 cells. Lefty did not induce Smad2 or Smad5 phosphorylation, Smad2/Smad4 heterodimerization, or n uclear translocation of Smad2 or Smad4, but activated the MAPK pathway in a time- and dose-dependent fashion, Further analysis showed the 28-kDa (but not the 34-kDa) polypeptide to induce MAPK activity, Surprisingly, the 42-k Da lefty protein was also capable of inducing MAPK activity, indicating tha t the lefty precursor is biologically active. The data support a molecular model of processing as a mechanism for regulation of lefty signaling.