Microtubule formation and function is regulated by a variety of proteins that mediate the structural and regulatory interactions between these microtubules and their cargo, and catalyze their assembly and disassembly. This dynamics process is necessary for a variety of essential processes including cell polarity, migration, division and mechano-transduction. This dynamism is regulated by the interaction with microtubule-associated proteins (MAPs). The MAPs can generally be classified into two categories:
1- microtubule- stabilizing proteins (e.g. MAP2, cytoskeleton-associated protein 2, tau)
2- microtubule-destabilizing proteins (e.g. ketanin, stathmin).
In dividing sea urchin eggs the most abundant MAP is echinoderm microtubule-associated protein (EMAP). It is a 75-kDa protein associated with the mitotic spindle apparatus. Homologous of EMAP, EMLs (EMAP-like proteins) have been identified in a variety of eukaryotic organisms, such as sea urchins, fruit flies, rodents, and humans.
In humans, five EMAP-like proteins (ELPs) have been identified: EML1, EML2, EML3, EML4 and EML5.
The protein sequences of all five EMLs are highly conserved. They all contain:
- a hydrophobic EMAP like domain (HELP) at the N-terminal region that is unique to this family and that is necessary for the binding to microtubule (MT);
- multiple WD40 domains located within the central and C-terminal region. WD repeat motifs are approximately 40 amino acids long, beginning in glycine-histidine (GH) and ending in tryptophan-aspartate (WD). WD repeat proteins belong to a large family of proteins with diverse cellular functions including signal transduction, vesicular trafficking, and cytoskeletal assembly. Each WD repeat folds into a structure of b-strands and b-turns that takes on the appearance of an airplane propeller. The variable regions of the WD repeats are predicted to be exposed on the surface of the propellers where they can present a changeable surface for protein-protein interaction.
EMAP with its 10 divergent WD repeats may fold into a compact structure with up to 10 individual propellers and the capacity to interact with up to 10 different partner proteins.
Although evidence suggests that EMAP and EML proteins associate with microtubules and several are implicated in regulating microtubule stability during mitosis. Their expression is not limited to dividing cells, however. The function of EMAP-like proteins in post-mitotic cells such as neurons is poorly understood.
The in vivo function of EMAP and EMAP-like proteins is unknown. However there are indications that loss or alteration of EMAP function may lead to human disease. In fact,
- EML1 has been discussed as a potential candidate for the most severe form (type 1) of the degenerative hereditary disease Usher syndrome, which manifests as a combination of hearing impairment and retinitis pigmentosa. Furthermore, in certain patients with T-cell acute lymphoblastic leukemia (TALL), the gene encoding the nonreceptor protein kinase (c-ABL1) is fused to the EML1 gene on chromosome 14, which causes expression of an EML1-ABL1 fusion protein, that functions as a dysregulated tyrosine kinase. The EML1-ABL1 fusion protein constitutively activates the ERK, Stat5, and Src signalling pathways
- EML2 was described as a microtubule destabilizer that interferes with nucleation of microtubules and simultaneously promotes their depolymerization in a concentration-dependent manner, presumably via a lateral destabilization mechanism. It was found that human EML2 RNA is abundant in cancer cell lines including chronic myelogenous leukemia (K-562), lymphoblastic leukemia (MOLT-4), colorectal adenocarcinoma (SW480), and lung carcinoma (A549).
- EML3 is a nuclear microtubule-binding protein required for the correct alignment of chromosomes in metaphase.
- The recently published EML5 is likely involved in regulating cytoskeletal rearrangements both in the adult brain and during neuronal development in rats.
- Kinase oncogenes are not restricted to fusions with EML1. 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 ]
While EML4 is essential for microtubule formation and stabilization, other members of the EMAP protein family, EML2 and EMAP, have been shown to destabilize microtubule: EML2 reduces growth rate and increases the frequency of catastrophe (the transition from growing to shortening microtubules), whereas EMAP destabilizes microtubules by reducing the frequency of rescue (the transition from shortening to growing microtubule).
In mammalian cells, MAP phosphorylation during mitosis correlates to a dramatic decrease in the microtubule polymer level . Since MAPs are known to stabilize microtubules against disassembly, it is suggested that the introduction of negative charge by phosphorylation reduces the affinity of MAPs for microtubules and thereby increases microtubule instability.
EMAP contains several divergent WD-40 repeats that may be involved in protein-protein interactions, as well as a number of phosphorylation consensus sites for protein kinases, including the p34cdc2 protein kinase.
In the unfertilized egg, EMAP is constitutively phosphorylated on 5 serine residues. Although the unfertilized egg is considered to be metabolically inactive, protein kinases are active at this time. Considering that there are few detectable microtubules in the unfertilized egg, these phosphorylation events may favor microtubule disassembly. In addition, a constitutive level of phosphorylation may be necessary for the proper folding of the EMAP polypeptide. The change in EMAP phosphorylation corresponds to the time of nuclear envelope breakdown, the formation of the mitotic apparatus, and an increase in microtubule dynamics. The phosphorylation of EMAP during mitosis is consistent with EMAP being a target for the p34cdc2 kinase or a kinase activated downstream of the p34cdc2 kinase. Sequence analysis of a cDNA clone for EMAP reveals a number of phosphorylation consensus sites corresponding to several different serine/threonine kinases including two consensus sites for the p34cdc2 kinase and one for the mitogen-activated protein (MAP) kinase, all within the first 50 amino acids of EMAP.
Cell cycle-dependent phosphorylation of the 77 kDa echinoderm microtubuleassociated
protein (EMAP) in vivo and association with the p34cdc2 kinase
The conservation of the EMAP-like protein family amongst metazoans and the direct correlation between EML translocations and cancer indicates that this novel protein family may perform an important function in cells and tissues. So far, however, the physiological function of this protein family remains unknown.
- Purification of a WD repeat protein, EMAP, that promotes microtubule dynamics through an inhibition of rescue
- Cell Cycle Tyrosine Phosphorylation of p34cdc2 and a Microtubule-Associated Protein Kinase Homolog in Xenopus Oocytes and Eggs
- EMAP, an echinoderm microtubule-associated protein found in microtubule-ribosome complexes