Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain.
The N-terminus of ceramide kinase (CERK) is thought to be myristoylated and to contain a pleckstrin homology (PH) domain. We found that deletion of this region (DeltaPH-CERK) ablates activity. This is not due to prevention of N-terminal myristoylation since a G2A CERK mutant, which cannot be myristoylated, was active. CERK was able to bind liposomes, as well as the isolated unmyristoylated PH domain; DeltaPH-CERK was not. Upon analysis of EGFP-tagged proteins, CERK was found associated with the Golgi complex. Osmotic cell swelling induced translocation of CERK to the plasma membrane and formation of large vesicles displaying bound CERK. None of these features occurred with DeltaPH-CERK, which remained disseminated throughout the cytoplasm. These findings show that the PH domain of CERK is essential for localization, translocation, and activity of this lipid kinase.
Carré
Adeline
A
Novartis Institute for BioMedical Research Vienna, Brunnerstrasse 59, A-1235 Vienna, Austria.
Graf
Christine
C
Stora
Samantha
S
Mechtcheriakova
Diana
D
Csonga
Robert
R
Urtz
Nicole
N
Billich
Andreas
A
Baumruker
Thomas
T
Bornancin
Frédéric
F
D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D009997:Osmotic Pressure; D010750:Phosphoproteins / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport
D001798:Blood Proteins; D008081:Liposomes; D010750:Phosphoproteins; C039077:platelet protein P47; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase