title: Experimental testing of predicted myristoylation targets involved in asymmetric cell division and calcium-dependent signalling.
abstract: Evolutionary conservation of N-terminal N-myristoylation within protein families indicates significant functional impact of this lipid posttranslational modification for function. In the MYRbase study (Maurer-Stroh et al., (2004) Genome Biology 5, R21), protein families with relevance to asymmetric cell division in animals and the group of plant calcium-dependent protein kinases (CPKs) have surfaced with many predicted myristoylated members. Here, we describe experimental in vitro verification of predicted myristoylation and explore its impact on subcellular localization for these targets in vivo. Our results confirm that, indeed, Numb isoform A, Neuralized isoforms C and D from Drosophila melanogaster and two Neuralized-like homologues from Mus musculus have the capability for N-terminal myristoylation in vitro and in vivo (in fly tissue and in mouse 3T3 cells respectively) whereas other isoforms such as Neuralized A and B have not. The latter two cases are an examples of different potential of various isoforms for posttranslational modifications. Additionally, the Arabidopsis thaliana CDPKs CPK6, CPK9 and CPK13 are shown to be substrates for myristoylation in vitro, which also affects their subcellular localization (in Arabidopsis protoplasts and tobacco leaves). At the same time, CPK6 and CPK13 do not appear to be substrates of a NMT1-like enzyme; the reasons for differing substrate specificities of NMT homologues in plants are derived from the evolutionary divergence of their N-myristoyl transferase sequences. As a methodical advance, we describe a fast and very sensitive technique (compared to traditional autoradiography) for in vitro testing of myristoylation based on thin layer chromatography read-out of the incorporated radioactive myristoyl anchor with subsequent Western blotting detection for protein yield determination using the same membrane.
authors.affiliation: Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Benetka
authors.forename: Norbert
authors.initials: N
authors.lastname: Mehlmer
authors.forename: Sebastian
authors.initials: S
authors.lastname: Maurer-Stroh
authors.forename: Michaela
authors.initials: M
authors.lastname: Sammer
authors.forename: Manfred
authors.initials: M
authors.lastname: Koranda
authors.forename: Ralph
authors.initials: R
authors.lastname: Neumüller
authors.forename: Jörg
authors.initials: J
authors.lastname: Betschinger
authors.forename: Jürgen A
authors.initials: JA
authors.lastname: Knoblich
authors.forename: Markus
authors.initials: M
authors.lastname: Teige
authors.forename: Frank
authors.initials: F
authors.lastname: Eisenhaber
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry; D020013:Calcium Signaling; D002455:Cell Division; D002855:Chromatography, Thin Layer; D029721:Drosophila Proteins / Q000378:metabolism; D004331:Drosophila melanogaster / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D007605:Juvenile Hormones / Q000378:metabolism; D007814:Larva / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D029721:Drosophila Proteins; D007605:Juvenile Hormones; D020033:Protein Isoforms; C060606:numb protein, Drosophila; D019814:Myristic Acid; D044767:Ubiquitin-Protein Ligases; C071006:neur protein, Drosophila