Fam20C is under the control of sphingolipid signaling in human cell lines

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Fam20C (dentin matrix protein 4, Golgi casein kinase) is an atypical protein kinase involved in phosphorylation of secreted proteins.
• Fam20C is implicated in biomineralization, phosphate homeostasis, and neoplasia.
• The regulatory mechanisms of Fam20C activity remain largely unknown, with in vitro studies suggesting stimulation by sphingosine.

Purpose of the Study:

• To investigate the in vivo physiological relevance of sphingosine in regulating Fam20C activity.
• To determine if sphingolipid signaling controls Fam20C function within cellular systems.

Main Methods:

• Utilized CRISPR/Cas9 technology to generate Fam20C knockout cell lines.
• Examined Fam20C activity in wild-type and knockout cells under basal conditions and after sphingosine depletion using myriocin.
• Assessed the impact of exogenous sphingosine and ceramide supplementation on Fam20C activity.

Main Results:

• Fam20C activity was detected in wild-type cells and responsive to sphingosine and analogs, but undetectable in Fam20C knockout cells.
• Myriocin treatment, which depletes endogenous sphingosine, led to negligible Fam20C activity in both cell lysates and conditioned media.
• Supplementation with exogenous sphingosine or ceramide partially restored Fam20C activity following myriocin treatment, without altering Fam20C protein levels.

Conclusions:

• Fam20C activity is physiologically regulated by sphingolipid signaling pathways in vivo.
• Sphingosine and its precursor ceramide play a crucial role in modulating Fam20C kinase function.
• These findings elucidate a novel regulatory mechanism for Fam20C, impacting understanding of its role in health and disease.