Subcellular localization of ceramide kinase and ceramide kinase-like protein requires interplay of their Pleckstrin Homology domain-containing N-terminal regions together with C-terminal domains.
Ceramide kinase (CERK) and the ceramide kinase-like protein (CERKL), two related members of the diacylglycerol kinase family, are ill-defined at the molecular level. In particular, what determines their distinctive subcellular localization is not well understood. Here we show that the Pleckstrin Homology (PH) domain of CERK, which is required for Golgi complex localization, can substitute for the N-terminal region of CERKL and allow for wild-type CERKL localization, which is typified by nucleolar accumulation. This demonstrates that determinants for localization of these two enzymes do not lie solely in their PH domain-containing N-terminal regions. Moreover, we present evidence for a previously unrecognized participation of CERK distal sequences in structural stability, localization and activity of the full-length protein. Progressive deletion of CERK and CERKL from the C-terminus revealed similar sequential organization in both proteins, with nuclear import signals in their N-terminal part, and nuclear export signals in their C-terminal part. Furthermore, mutagenesis of individual cysteine residues of a CERK-specific CXXXCXXC motif severely compromised both exportation of CERK from the nucleus and its association with the Golgi complex. Altogether, this work identifies conserved domains in CERK and CERKL as well as new determinants for their subcellular localization. It further suggests a nucleocytoplasmic shuttling mechanism for both proteins that may be defective in CERKL mutant proteins responsible for retinal degenerative diseases.
Rovina
Philipp
P
Novartis Institutes for BioMedical Research, Brunnerstrasse 59, A-1235 Vienna, Austria.
Schanzer
Andrea
A
Graf
Christine
C
Mechtcheriakova
Diana
D
Jaritz
Markus
M
Bornancin
Frédéric
F
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002466:Cell Nucleolus / Q000201:enzymology; D017124:Conserved Sequence; D006801:Humans; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012162:Retinal Degeneration / Q000201:enzymology / Q000473:pathology; D016415:Sequence Alignment; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000201:enzymology
D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase