Characterization of a ceramide kinase-like protein.
Ceramide is a key player governing cell fate, and its conversion to ceramide-1-phosphate by ceramide kinase (CERK) is emerging as an important mean to regulate apoptosis and inflammatory processes. We identified a new ceramide kinase homolog, designated CERK-like protein (CERKL) and we compared it to the known CERK. Real time-PCR analysis of human tissues revealed a restricted pattern of expression for CERKL mRNA. Surprisingly, various ceramides, known substrates for CERK, were not phosphorylated by CERKL in vitro. Upon 32P(i)-pulse labeling of COS-1 cells transiently expressing CERKL, or incubation with NBD-C6-ceramide, ceramide-1-phosphate was not detected. After recombinant expression in COS-1 cells, CERKL was partially recovered in the soluble fraction, as a phosphorylated protein. Live cell imaging indicated localization of GFP-tagged CERKL to many cell compartments, including specific association with nucleoli. Two splice variants of CERKL did not localize to nucleoli nor did a CERKL variant with a point mutation in the putative ATP binding site. We also studied a naturally occurring CERKL mutant (R257X), recently linked to the pathology of retinitis pigmentosa. It accumulated in the nucleus but was not associated with nucleoli. Treatment with the calcium ionophore A23187 led to clearing of CERKL from nucleoli, but had no effect on the R257X CERKL mutant. Collectively, although kinase activity of CERKL remains to be proven, these findings suggest a functional link between CERKL and its nucleolar localization. Furthermore, we propose that the cause for retinitis pigmentosa in patients bearing the CERKL R257X mutation might be the accumulation of a truncated CERKL protein in the nucleus.
Bornancin
Frédéric
F
Novartis Institutes for BioMedical Research, Brunnerstrasse 59, A-1235 Vienna, Austria. frederic.bornancin@pharma.novartis.com
Mechtcheriakova
Diana
D
Stora
Samantha
S
Graf
Christine
C
Wlachos
Alexander
A
Dévay
Piroska
P
Urtz
Nicole
N
Baumruker
Thomas
T
Billich
Andreas
A
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002458:Cell Fractionation; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012174:Retinitis Pigmentosa / Q000235:genetics; D016415:Sequence Alignment
D009687:Nuclear Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase