DEFINITION
Ropp120 (restrictedly overexpressed proliferation-associated protein 120 kDa) is a human WD repeat protein of 120 kDa that, because of its significant sequence homology with other members of the EMAP proteins, was named
EML4 (Echinoderm microtubule- associated protein like protein 4)
Analysis of the translated amino acid sequence (981 amino acids) shows that EML4 contains:
- four WD repeats located at the C-terminus;
- an active site that is well conserved in serine proteases from the trypsin family (amino acids 469–480);
- a histidine acid phosphate site (amino acids 688–705);
- an EF-hand calciumbinding domain (amino acids 384–396);
- 6 N-glycosylation sites;
- more than 20 different phosphorylation sites, including 2 tyrosine kinase phosphorylation sites.
Instead, the amino-terminal portion of EML4 (amino acids 1–249) is essential for the association with microtubules promoting their formation and stabilization.
CELLULAR LOCALIZATION
From the overall distribution of EML4 along MTs during interphase, it may be assumed that EML4 has a MT stabilizing function like that known for MAP2 and tau.
In addition EML4 may have an essential function for the formation of the mitotic spindle. In fact it was monstred that GFP-Eml4 colocalizes with the mitotic spindle of dividing cells. Such colocalization to the mitotic spindle has also been reported for other members of the EMAP protein family. This renders Eml4 likely to play a role during cell division. In fact siRNA-mediated knockdown of EML4 in HeLa cells led to a significant decrease in the number of cells. Most importantly, EML4 negative cells showed a completely modified microtubule network and it results on an obvious increase in apoptotic cells, indicating that EML4 is necessary for correct microtubule formation, normal cell proliferation and survival.
REGULATION
EML4 Phosphorylation
Similar to EMAP, the function of EML4 is regulated by cell cycle-dependent phosphorylation pathways. It was monstred that mitotic EML4 is significantly phosphorylated on Ser/Thr residues. Microtubule dynamics and organization change markedly during the interphase-M phase transition of the cell cycle. Serine/threonine phosphorylation may cause a change in the structure and function of EML4 during mitosis and might be essential for the binding to the mitotic spindle.
EML4 expression is developmentally regulated
EML4 is highly expressed in mouse embryos and expression declines throughout development:
- in mouse E11 embryos high expression levels are observed in developing nervous system.
- In E15 embryos, Eml4 is also highly expressed in the developing CNS (e.g. neopallial cortex, ventricular zone, roof of midbrain, cerebellar primordium and spinal cord) and peripheral nervous system (e.g. ganglions). Other regions in which Eml4 is expressed include, liver, thymus, intestines, eye, kidney, salivary gland and paws.
- In E18 embryos, Eml4 expression is mainly restricted to regions of the developing nervous system e.g. in neopallial cortex (the future cerebral cortex), hippocampus, diencephalon, roof of midbrain, cerebellum and spinal cord. Expression of Eml4 is also observed within the developing intestine, cerebellum and eye.
- In adult mouse brain Eml4 is expressed albeit at low level in olfactory bulb, hippocampus and cerebellum.
Thus the EML4 expression is highly developmentally regulated and that EML4 expression becomes predominantly restricted to regions of the developing CNS. In addition, EML4 expression persists in adult mouse brain in neurons of olfactory bulb, hippocampus and cerebellum.
Thus, what is the role of EML4 in the neuronal morphogenesis ?
The structure and integrity of the cytoplasm of eukaryotic cells depend on the assembly and organization of microtubules, actin filament and intermediate filaments.
The protein-to-protein interactions that play a key role in this process include:
i) assembly of tubulin leading to microtubule formation
ii) interactions between cytoskeletal polymers and microtubule-associated proteins (MAPs).
iii) involvement of the extracellular matrix (ECM) in the determination of modulatory signals that affect the neuronal cytoskeleton in processes like neuritogenesis, and the possible role of MAPs in mediating these cellular changes.
Neuronal microtubules constitute major structures directly involved in neuronal morphogenesis, with different subsets of tubulin specifically expressed in neuronal and glial cells. In general, the biological function of microtubules is based on the ability of tubulin to polymerize and depolymerize.
image
Microtubule ends can interconvert between slow elongation and rapid shortening, a process called dynamic instability, because of the presumed gain and loss of a small region of tubulin-liganded GTP at the microtubule end. Instead, along the microtubule axis, tubulin heterodimers are joined end-to-end to form protofilaments, with alternating alfa and beta subunits which have a nucleotide binding site:
1- alfa-Tubulin has a bound molecule of GTP, that does not hydrolyze;
2- beta-Tubulin may have bound GTP or GDP. Under certain conditions beta-tubulin can hydrolyze its bound GTP to GDP.
The minus end of an alfa-subunit may serve as GAP (GTPase activating protein) for beta-tubulin of the adjacent dimer in a protofilament. The inability of GTP to dissociate from the alfa-subunit is consistent with occlusion by a loop from the beta subunit. The loss of the cap results in a transition from growth to shortening (called a catastrophe), whereas the reacquisition of the GTP cap results in a transition from shortening to growing (called a rescue).
The microtubule-associated proteins (including EML4) are known to regulate microtubule dynamics by stabilizing microtubules in part, by suppressing the rate and extent of microtubule shortening and by suppressing the catastrophe frequency and increasing the rescue frequency. Post-translational modifications of microtubules are directly involved in the determination of morphogenesis in neurons. The fact that these microtubules are enriched in acetylated tubulin suggests that this post-translational modification of tubulin renders microtubules more resistant to depolymerization.
It was shown that specific phosphorylations of MAP-1 occur during neuronal outgrowth in neuroblastoma cells, and this modification is related to an enhancement of microtubule assembly. The splicing of a common RNA transcript, as well as post-translational modification are the main regulatory mechanisms for EML activity on neurons. Instead it is monstred that EML4 is subjected to alternative splicing of different exons (2, 5, 6 and 8) and that the abundance of individual splice variants differs between specific brain areas. In summary, EML4 protein expression is developmentally regulated and expression persists in distinct regions of the adult mouse brain.
It was found the role of tau in mediating microtubule-actin filament interactions, an activity that could be involved in regulating the actin cortex and association with plasma membrane in the establishment of architectural patterns during neuronal growth. EML4 could exerces a similar role.
Conclusions
1- The expression of EML4 is essential for the formation of an intact MT network. The binding of EML4 to MTs leads to:
-reduction in catastrophe rate
-reduction in shrinkage rate
-increase in rescue frequency
-increase in growth rate
2- EML4 is most likely regulated via differential phosphorylation during cell cycle.
3- EML4 is essential for normal cell proliferation and cell survival.
4- Its expression is developmentally regulated
5- EML4 fusions with the anaplastic lymphoma kinase (ALK) occur in a subset of non-small cell lung cancers and adenocarcinomas of the lungs [ EML4-ALK in NSCLC ]
So far, however, the physiological meccanisme of function of this protein remains to be determined.
Identifiers
symbols: EML4
Location: Chr2:42.25-42.41Mb
Entrez: 27436
OMIM: 607442
EnsemblGeneView: ENSG00000143924
uniprot: Q9HC35
Refseq: NM_019063 (mRNA) NP_061936 (protein)
Reference:
Human EML4, a novel member of the EMAP family, is essential for microtubule formation
Echinoderm microtubule-associated protein like protein 4, a member of the echinoderm microtubule-associated protein family, stabilizes microtubules
G protein alpha subunits activate tubulin GTPase and modulate microtubule polymerization dynamics
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/microtub.htm
Contraction due to microtubule disruption is associated with increased phosphorylation of myosin regulatory light chain
Cloning and localization of C2orf2, a previously unknown WD repeat protein
