The leucine 10 residue in the pleckstrin homology domain of ceramide kinase is crucial for its catalytic activity.
Ceramide kinase (CERK) converts ceramide (Cer) to ceramide-1-phosphate (C1P), a newly recognized bioactive molecule capable of regulating diverse cellular functions. The N-terminus of the CERK protein encompasses a sequence motif known as a pleckstrin homology (PH) domain. However, little is known regarding the functional roles of this domain in CERK. In this study, we have demonstrated that the PH domain of CERK is essential for its enzyme activity. Using site-directed mutagenesis, we have further determined that Leu10 in the PH domain has an important role in CERK activity. Replacing this residue with a neutral alanine or isoleucine, caused a dramatic decrease in CERK activity to 1% and 29%, respectively, compared to CERK, but had no effect on substrate affinity. The study presented here suggests that the PH domain of CERK is not only indispensable for its activity but also act as a regulator of CERK activity.
Kim
Tack-Joong
TJ
Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan.
Mitsutake
Susumu
S
Kato
Mariko
M
Igarashi
Yasuyuki
Y
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D001798:Blood Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D006801:Humans; D007532:Isoleucine / Q000235:genetics; D007930:Leucine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D013379:Substrate Specificity
D001798:Blood Proteins; D010750:Phosphoproteins; C039077:platelet protein P47; D007532:Isoleucine; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase; D007930:Leucine; D000409:Alanine