Experimental testing of predicted myristoylation targets involved in asymmetric cell division and calcium-dependent signalling
Authors: Wolfgang Benetka
Manfred Koranda
Ralph Neumüller
Michaela Sammer
Frank Eisenhaber
Norbert Mehlmer
Sebastian Maurer-Stroh
Jörg Betschinger
Jürgen A Knoblich
Markus Teige
Journal: Cell Cycle (2008)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Evolutionary conservation; N-terminal N-myristoylation; protein families; lipid posttranslational modification; MYRbase study
Affiliations: Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.

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. 

Retracted: No

PubPeer comments:
 This article reports on five bistable benzimidazole derivatives, two of which display ferroelectricity in the bulk state up to *ca*. 400 K. This is obviously of great interest, since, for example, 2-methylbenzimidazole (MBI, ferroelectric) is an amphoteric, cheap, stable, and commercially available compound, with a large dipole moment. The authors assume that the polarization reversal mechanism is based on proton tautomerism in the imidazole moiety. By changing some substituents on the imidazole and benzene rings, hysteresis loops in polarization-electric field curves are modified. This work is thus a nice handling of the u201cstructure-properties relationshipu201d paradigm. Unfortunately, in order to support the proton tautomerization hypothesis, X-ray structures were systematically refined in subgroups of the actual space groups. As a consequence, imidazolic protons are ordered, allowing polarity in planes of hydrogen-bonded molecules. In the case of MBI, the authors state that: u201cThe reported MBI crystal structure ([18] ) belongs to the tetragonal system with the symmetric space group *P*4(2) *n* (no. 86). The protons are disordered over two sites, and their hydrogen bonds form straight bands running along either [110]tetra or the crystallographically equivalent [1-10]tetra direction. In our analysis, the tetragonal lattice is barely lowered to the monoclinic system with the space group *Pn* (no. 7), which is one of the maximal subgroups of *P*4(2) *n* by interchanging the crystallographic *b* and *c* axes. Because the as-grown MBI crystals are not in a single-domain state but easily mix the tetragonal *a* and *b* axes, the axes and plane indices are presented here according to the tetragonal symmetry. The proton ordering is responsible for the polarity within the (001)tetra plane in the bulk (Fig. 1d).u201d Even if the presence of low-symmetry microdomains in MBI samples could explain the observed ferroelectric hysteresis, I believe that refining the *bulk* crystal structures using an artificially lowered crystal symmetry seems to be a bit far-fetched. In the case of MBI, the asymmetric unit in space group *Pn* includes four u201cindependentu201d molecules, which are obviously related by a 4(2) screw axis. The monoclinic cell is reported with *a* = *c* and *beta* = 90u00b0. Theoretically, this is not impossible, but hard to believe in the case of the MBI crystal reported here. This crystal is described as a monoclinic system having a u201cpseudo-tetragonal crystal symmetryu201d, however, the opposite is the correct description: the unit cell used for the refinement has a pseudo-monoclinic crystal symmetry, while the crystal is actually tetragonal. This strategy was used for the five studied derivatives. The following table shows the reported (wrong) space groups and the actual space groups determined using [ADDSYM](http: www.platonsoft.nl platon pl000401.html). This algorithm provides the correct space groups with a confidence of 100%, as reported by the [checkcif](http: checkcif.iucr.org ) tool. ![file] Forcing a refinement in an unrealistic space group is not really a strong evidence within a structure-properties relationship study. After all, an extreme strategy could be to refine all structures in *P*1 (this is *always* possible) to rationalize (almost) any physical property. On the other hand, crystal structures published in wrong space groups pollute the CSD with wrong polymorphs. Other databases are also tainted. For MBI, check for example the crystal structure reported in the PubChem database, [here] . ,