Lefty proteins exhibit unique processing and activate the MAPK pathway.

Area of Science:

• Developmental Biology
• Molecular Biology
• Cell Signaling

Background:

• Lefty polypeptides are novel transforming growth factor-beta (TGF-beta) superfamily members essential for embryonic lateral patterning.
• Activation of TGF-beta superfamily members typically requires proteolytic processing, indicating cleavage is vital for lefty function.

Purpose of the Study:

• To investigate the role of proprotein convertases (PCs) in lefty polypeptide processing.
• To identify the specific PC enzymes involved in lefty cleavage.
• To determine the biologically active form(s) of lefty and their signaling pathways.

Main Methods:

• Transfection of cell lines with lefty and various PC enzymes.
• Cotransfection analysis to assess PC activity on lefty precursor.
• Site-directed mutagenesis to identify lefty cleavage sites.
• Treatment of pluripotent P19 cells with different lefty forms to analyze cellular responses (Smad phosphorylation, MAPK activation).

Main Results:

• PC5A was identified as the primary enzyme processing the lefty precursor to a 34-kDa form; other PCs showed limited activity.
• Specific mutations in lefty (RGKR to GGKG and RHGR to GHGR) blocked processing to 34-kDa and 28-kDa forms, respectively.
• The 28-kDa lefty form and the 42-kDa lefty precursor, but not the 34-kDa form, activated the MAPK pathway.
• Lefty did not affect Smad2/Smad5 signaling pathways.

Conclusions:

• Lefty processing by PCs, particularly PC5A, is a key regulatory mechanism.
• The 28-kDa form and the precursor itself are biologically active, signaling through the MAPK pathway.
• Lefty processing regulates its biological activity and signaling, supporting a model of processing-dependent regulation.