title: A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase.
abstract: 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.
authors.affiliation: Novartis Institutes for BioMedical Research, Brunnerstrasse 59, A-1235 Vienna, Austria.
authors.forename: Philipp
authors.initials: P
authors.lastname: Rovina
authors.forename: Markus
authors.initials: M
authors.lastname: Jaritz
authors.forename: Siegfried
authors.initials: S
authors.lastname: Höfinger
authors.forename: Christine
authors.initials: C
authors.lastname: Graf
authors.forename: Piroska
authors.initials: P
authors.lastname: Dévay
authors.forename: Andreas
authors.initials: A
authors.lastname: Billich
authors.forename: Thomas
authors.initials: T
authors.lastname: Baumruker
authors.forename: Frédéric
authors.initials: F
authors.lastname: Bornancin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D001798:Blood Proteins; D010750:Phosphoproteins; C039077:platelet protein P47; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase