Open AccessA critical β6–β7 loop in the pleckstrin homology domain of ceramide kinase
Author(s) -
Philipp Rovina,
Markus Jaritz,
Siegfried Höfinger,
Christine Gräf,
Piroska Dévay,
Andreas Billich,
Thomas Baumruker,
Frédéric Bornancin
Publication year - 2006
Publication title -
biochemical journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.706
H-Index - 265
eISSN - 1470-8728
pISSN - 0264-6021
DOI - 10.1042/bj20060316
Subject(s) - pleckstrin homology domain , ceramide , biochemistry , homology modeling , amino acid , biology , mutant , cysteine , chemistry , kinase , enzyme , apoptosis , gene
CerK (ceramide kinase) produces ceramide 1-phosphate, a sphingophospholipid with recognized signalling properties. It localizes to the Golgi complex and fractionates essentially between detergent-soluble and -insoluble fractions; however, the determinants are unknown. Here, we made a detailed mutagenesis study of the N-terminal PH domain (pleckstrin homology domain) of CerK, based on modelling, and identified key positively charged amino acid residues within an unusual motif in the loop interconnecting beta-strands 6 and 7. These residues are critical for CerK membrane association and polyphosphoinositide binding and activity. Their mutagenesis results in increased thermolability, sensitivity to proteolysis, reduced apparent molecular mass as well as propensity of the recombinant mutant protein to aggregate, indicating that this loop impacts the overall conformation of the CerK protein. This is in contrast with most PH domains whose function strongly relies on charges located in the beta1-beta2 loop.