A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase.
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.
Rovina
Philipp
P
Novartis Institutes for BioMedical Research, Brunnerstrasse 59, A-1235 Vienna, Austria.
Jaritz
Markus
M
Höfinger
Siegfried
S
Graf
Christine
C
Dévay
Piroska
P
Billich
Andreas
A
Baumruker
Thomas
T
Bornancin
Frédéric
F
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
D001798:Blood Proteins; D010750:Phosphoproteins; C039077:platelet protein P47; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase