A new role for sphingosine: Up-regulation of Fam20C, the genuine casein kinase that phosphorylates secreted proteins

• We demonstrate that sphingosine upregulates Fam20C activity.
• Sphingosine protects Fam20C against thermal inactivation.
• Blockage of sphingosine biosynthesis inactivates endogenous Fam20C.
• Fingolimod, an immunosuppressive drug, is an activator of Fam20C.
• Sphingosine and fingolimod significantly up-regulate Fam20C T268M defective mutant.

Fam20C is an atypical kinase implicated in bio-mineralization and phosphate homeostasis disorders, and has recently been shown to account for the activity of an orphan enzyme (“genuine casein kinase”, G-CK) previously characterized for its ability to phosphorylate casein and a plethora of secreted proteins at serine residues specified by the S-x-E/pS motif. Fam20C/G-CK activity is only appreciable in the presence of high Mn2 + concentration (> 1 mM), and is negligible if Mn2 + is replaced by physiological Mg2 + concentrations. Here we show that sphingosine (but not its biological precursor ceramide) not only stimulates several-fold Fam20C activity in the presence of Mn2 +, but also confers a comparable activity to Fam20C assayed with Mg2 +. Activation by sphingosine is evident using a variety of substrates, and is accounted for by both higher Vmax and decreased Km(ATP), as judged from kinetics run with the β(28–40) substrate peptide and a physiological substrate, BMP-15. Sphingosine also protects Fam20C from thermal inactivation. Consistent with the in vitro results, by treating Fam20C expressing HEK293T cells with myriocin, a potent inhibitor of the sphingosine biosynthetic pathway, the activity of Fam20C released into the conditioned medium is substantially decreased corroborating the concept that sphingosine (or related metabolite(s)) is a co-factor required by Fam20C to optimally display its biological functions. None of the small molecule kinase inhibitors tested so far were able to inhibit Fam20C. Interestingly however fingolimod, an immunosuppressive drug structurally related to sphingosine, used for the treatment of multiple sclerosis, is a powerful activator of Fam20C, both wild type and its pathogenic, loss of function, T268M mutant. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.