Genomic organization and alternative splicing of MAP1B

A keratan sulfate proteoglycan named claustrin and the mouse MAP1B protein share high homology, with claustrin representing a 5'-truncated fragment of MAP1B. In the present study, the relationship between claustrin and MAP1B is further studied. Described is the isolation of a cDNA encoding the 3'-region of MAP1B, which shares 3'-untranslated sequence, but not coding sequence, with claustrin. This partial cDNA has been called 3'-MAP1B-related clone (3'-MRC), since it is homologous to the 3'-region of the mouse MAP1B sequence. Distinct mRNAs are recognized by the claustrin and 3'-MRC cDNAs, and mRNAs encoding these distinct MAP1B-related molecules are present in embryonic chick brain and cardiac and smooth muscle. These data also suggest a higher level of expression of claustrin mRNA in astrocyte cultures, when compared to 3'-MRC. These data therefore provide new evidence that alternatively spliced variants of MAP1B are expressed in brain, and that at least one of these variants encodes the claustrin proteoglycan (Burg, 1997).

The genomic organization of the mouse and rat genes coding for the 2460-amino-acid microtubule-associated protein (MAP) 1B is reported. In addition to seven exons that encode full-length MAP1B, two alternative exons, exon 3A and the novel exon 3U, have been identified. Alternative MAP1B transcripts containing either exon 3A or exon 3U are expressed in a variety of mouse and rat tissues at about 1% to 10% of the level of regular transcripts. The alternative transcripts, if translated, would give rise to MAP1B isoforms truncated at the N-terminus. The exon/intron organization underlying the alternative transcripts and the N-terminal amino acid sequence of the putative truncated MAP1B isoforms resemble those of MAP1A, providing further evidence for an evolutionary relationship. The detection of alternative transcripts has implications for the interpretation of conflicting results recently obtained in MAP1B knockout mice (Kutschera, 1998).

Phosphorylation of MAP1B

A procedure for the purification of microtubule associated protein 1B (MAP1B) from calf brain has been described, and this study further characterizes the purified protein and its interaction with microtubules. Purified MAP1B (1) is thermostable; (2) is mainly phosphorylated at the casein kinase II (CKII) sites but only partially phosphorylated at the proline-directed protein kinase (PDPK) sites; (3) both the CKII and PDPK sites can be dephosphorylated by alkaline phosphatase; and (4) dephosphorylation results in an increased mobility on SDS-PAGE gels. The ability of MAP1B to interact with microtubules was also examined and shows that (1) phosphorylated (1B-P), alkaline phosphatase-treated (1B-AP), and heat-treated (1B-P), alkaline phosphatase-treated (1B-AP), and heat-treated (1B-HT) MAP1B bind to taxol-stabilized microtubules; (2) 1 mol of 1B-P, 1B-AP, or 1B-HT each binds about 13-14 tubulin dimers; (3) light chain interaction with MAP1B heavy chain is not affected by AP- or heat-treatment; (4) MAP1B can be displaced from taxol-stabilized microtubules by titration with salt; (5) higher salt concentrations are required to displace 1B-AP compared with 1B-P from taxol-stabilized microtubules; and (6) MAP2 is able to displace both 1B-P and 1B-AP from taxol-stabilized microtubules. The role of phosphorylation in regulating MAP1B interaction with microtubules and light chains is discussed (Pedrotti, 1996).

Neuronal morphogenesis depends on the organization of cytoskeletal elements among which microtubules play a very important role. The organization of microtubules is controlled by the presence of microtubule-associated proteins (MAPs); MAP activity is modulated by phosphorylation and dephosphorylation. One of these MAPs is MAP1B, which is very abundant within growing axons of developing neurons where it is found phosphorylated by several protein kinases including CK2. The expression of MAP1B is notably decreased after neuronal maturation in parallel with a change in the localization of the protein, which becomes largely concentrated in neuronal cell bodies and dendrites. Interestingly, MAP1B remains highly phosphorylated at sites targeted by protein kinase CK2 in mature neurons. The expression and localization of CK2 catalytic subunits during neuronal development has been examined. CK2alpha subunit appears early during development whereas CK2alpha' subunit appears within mature neurons at the time of dendrite maturation and synaptogenesis, in parallel with the change in the localization of MAP1B. CK2alpha subunit is found associated with microtubule preparations obtained from either grey matter or white matter from adult bovine brain, whereas CK2alpha' subunit is highly enriched in microtubules obtained from grey matter. These results lend support to the hypothesis that CK2alpha' subunit is concentrated in neuronal cell bodies and dendrites, where it associates with microtubules, thus contributing to the increased phosphorylation of MAP1B in this localization in mature neurons (Moreno, 1999).

Two major modes of MAP1B phosphorylation (I and II), respectively recognized by monoclonal antibodies 150 and 125, have been related to remodeling and formation of processes in the mature nervous system. To gain insight into the cytoskeletal modifications underlying peripheral nerve regeneration, the pattern of expression of both MAP1B phosphorylated modes was studied during this process. Sciatic nerves from adult Wistar rats were crushed and animals allowed to survive for 5, 7, 10 or 14 days. After those survival periods, damaged and undamaged sciatic nerves, dorsal root ganglia (DRG), and spinal cords, were subjected to immunohistochemistry and Western blot, using antibodies 150 and 125. At all survival periods analysed, MAP1B phosphorylated at mode I is concentrated at the distal region of regenerating nerves whereas mode II phosphorylation undergoes an overall decrease in regenerating axons that is less evident in more proximal nerve regions. Very high levels of MAP1B phosphorylated at mode II are detected in the bodies of DRG neurons and in bodies and dendrites of spinal motor neurons. This phosphorylation mode is also encountered in some Schwann cells and oligodendroglia associated with more proximal regions of regenerating axons. It is concluded that MAP1B is differentially phosphorylated depending on the cell type, subcellular compartment and stage of the regenerative process and the possible functional implications that differential expression of each MAP1B phosphorylation mode might have during nerve regeneration is discussed (Ramon-Cueto, 1999).

The function of the neuronal high molecular weight microtubule-associated proteins (MAPs) MAP1b and MAP2 is regulated by the degree of their phosphorylation, which in turn is controlled by the activities of protein kinases and protein phosphatases (PP). To investigate the role of PP in the regulation of the phosphorylation of MAP1b and MAP2, okadaic acid and cyclosporin A were used to selectively inhibit PP2A and PP2B activities, respectively, in metabolically competent rat brain slices. The alteration of the phosphorylation levels of MAP1b and MAP2 was examined by Western blots using several phosphorylation-dependent antibodies to these proteins. The inhibition of PP2A, and to a lesser extent of PP2B, was found to induce an increased phosphorylation of MAP1b and inhibit its microtubule binding activity. Immunocytochemically, a marked increase in neuronal staining in inhibitor-treated tissue was observed with antibodies to the phosphorylated MAP1b. The inhibition of PP2A but not of PP2B also induces phosphorylation of MAP2 at multiple sites and impairs its microtubule binding activity. These results suggest that PP2A might be the major PP that participates in regulation of the phosphorylation of MAP1b and MAP2 and their biological activities (Gong, 2000).

Phosphorylation at certain proline-directed sites on the microtubule-associated protein 1B (MAP1B) is a characteristic feature of mitotic neuronal precursor cells and developing neurons and is particularly abundant within growing axons. This mode of MAP1B phosphorylation disappears from mature neurons, except in those neurons that have a high regenerative potential, and is aberrantly up-regulated in degenerating neurons within the brains of Alzheimer's disease patients. This type of MAP1B phosphorylation is practically abolished in proliferating neuroblastoma cells that are treated with chemical inhibitors of cyclin-dependent kinases. In contrast, these drugs have no significant effect on MAP1B phosphorylation in either differentiated neuroblastoma cells or cerebellar granule neurons. Interestingly, lithium, which is a potent inhibitor of glycogen synthase kinase 3, suppresses this mode of MAP1B phosphorylation in differentiated neuroblastoma cells and cerebellar granule neurons. This is consistent with a major role of cyclin-dependent kinases in catalyzing this type of MAP1B phosphorylation in proliferating neural cells, whereas glycogen synthase kinase 3 would be largely responsible for this mode of MAP1B phosphorylation in postmitotic neurons that are extending axons. Both cyclin-dependent kinases and glycogen synthase kinase 3 might contribute to the aberrant MAP1B phosphorylation observed in Alzheimer's disease (Garcia-Perez, 1998).

Interaction of MAP1B and MAP light chain

Microtubule-associated proteins 1A (MAP1A) and MAP1B are abundant neuronal MAPs thought to be involved in neurite formation and stabilization. The relative levels of MAP1A and MAP1B change dramatically during development, with MAP1B expression highest in forming neurons, and MAP1A expression highest in mature neurons. The expression of light chain 3 (LC3), a subunit of both MAP1A and MAP1B was examined to see if its expression parallels that of either heavy chain. (For Drosophila proteins related to the light chain see CG12334 and CG1534), Anti-LC3 immunohistochemistry reveals that LC3 in rat brain is restricted to neurons that are expressing either the MAP1A or MAP1B heavy chain. Although LC3 is expressed exclusively in cells expressing heavy chains, developmental changes in the total amount of LC3 protein are not proportional to changes in the amount of either the MAP1A or MAP1B heavy chain. LC3 protein expression measured by quantitiative immunoblotting is twice as high in postnatal brain as in embryonic and adult brain. The localization of the LC3 gene to human chromosome 20cen-q13 demonstrates that LC3 is the only MAP1 subunit that is not linked to the heavy chain genes. Because LC3 is a component of both the MAP1A and MAP1B microtubule-binding domains, the heavy-chain independent regulation of LC3 expression might modify MAP1 microtubule-binding activity during development (Mann, 1996).

Previous studies on the role of microtubule-associated protein 1B (MAP1B) in adapting microtubules for nerve cell-specific functions have examined the activity of the entire MAP1B protein complex consisting of heavy and light chains and have revealed moderate effects on microtubule stability. The effects of the MAP1B light chain in the absence or presence of the heavy chain has been analyzed by immunofluorescence microscopy of transiently transfected cells. Distinct from all other MAPs, the MAP1B light chain induces formation of stable but apparently flexible microtubules resistant to the effects of nocodazole and taxol. Light chain activity is inhibited by the heavy chain. In addition, the light chain is found to harbor an actin filament binding domain in its COOH terminus. By coimmunoprecipitation experiments using epitope-tagged fragments of MAP1B it has been shown that light chains can dimerize or oligomerize. Furthermore, the domains for heavy chain-light chain interaction have been localized to regions containing sequences homologous to MAP1A. These findings assign several crucial activities to the MAP1B light chain and suggest a new model for the mechanism of action of MAP1B in which the heavy chain might act as the regulatory subunit of the MAP1B complex to control light chain activity (Togel, 1998).

Miscellaneous protein interactions of MAP1B

Dynein interacts with microtubules through an ATP-sensitive linkage mapped to a structurally complex region of the heavy chain following the fourth P-loop motif. Virtually nothing is known regarding how binding affinity is achieved and modulated during ATP hydrolysis. A detailed dissection of the microtubule contact site was performed, using fragment expression, alanine substitution, and peptide competition. Three clusters of amino acids have been identified as important for the physical contact with microtubules; two of these fall within a region sharing sequence homology with MAP1B, the third in a region just downstream. Amino acid substitutions within any one of these regions can eliminate or weaken microtubule binding (KK3379-80, E3385, K3387, K3397, KK3410-11, W3414, RKK3418-20, F3426, R3464, S3466, and K3467), suggesting that their activities are highly coordinated. A peptide that actively displaces MAP1B from microtubules perturbs dynein binding, supporting previous evidence for similar sites of interaction. Four amino acids have been identified whose substitutions affect release of the motor from the microtubule (E3413, R3444, E3460, and C3469). These suggest that nucleotide-sensitive affinity may be locally controlled at the site of contact. This work is the first detailed description of dynein-tubulin interactions and provides a framework for understanding how affinity is achieved and modulated (Koonce, 2000).

The ionotropic type-A and type-C receptors for the neurotransmitter gamma-aminobutyric acid [GABA(A) and GABA(C) receptors] are the principal sites of fast synaptic inhibition in the central nervous system, but it is not known how these receptors are localized at GABA-dependent synapses. GABA(C) receptors, which are composed of rho-subunits, are expressed almost exclusively in the retina of adult vertebrates, where they are enriched on bipolar cell axon terminals. The microtubule-associated protein 1B (MAP-1B) specifically interacts with the GABA(C) rho1 subunit but not with GABA(A) receptor subunits. Furthermore, GABA(C) receptors and MAP-1B co-localize at postsynaptic sites on bipolar cell axon terminals. Co-expression of MAP-1B and the rho1 subunit in COS cells results in a dramatic redistribution of the rho1 subunit. These observations suggest a novel mechanism for localizing ionotropic GABA receptors to synaptic sites. This mechanism, which is specific for GABA(C) but not GABA(A) receptors, may allow these receptor subtypes, which have distinct physiological and pharmacological properties, to be differentially localized at inhibitory synapses (Hanley, 1999).

Subcellular location of MAP1B

Microtubule-associated protein (MAP) 1B is a high-molecular-weight cytoskeletal protein that is abundant in developing neuronal processes and appears to be necessary for axonal growth. Various biochemical and immunocytochemical results are reported, indicating that a significant fraction of MAP1B is expressed as an integral membrane glycoprotein in vesicles and the plasma membrane of neurons. MAP1B is present in microsomal fractions isolated from developing rat brain and fractionates across a sucrose gradient in a manner similar to synaptophysin, a well-known vesicular and plasma membrane protein. MAP1B is also in axolemma-enriched fractions (AEFs) isolated from myelinated axons of rat brain. MAP1B in AEFs and membrane fractions from cultured dorsal root ganglion neurons (DRGNs) remains membrane-associated following high-salt washes and contains sialic acid. Furthermore, MAP1B in intact DRGNs is readily degraded by extracellular trypsin and is labeled by the cell surface probe sulfosuccinimidobiotin. Immunocytochemical examination of DRGNs shows that MAP1B is concentrated in vesicle-rich varicosities along the length of axons. Myelinated peripheral nerves immunostained for MAP1B show an enrichment at the axonal plasma membrane. These observations demonstrate that some of the MAP1B in developing neurons is an integral plasma membrane glycoprotein (Tanner, 2000).

Transport of MAP1B

Cytoskeletal proteins are axonally transported with slow components a and b (SCa and SCb). In peripheral nerves, the transport velocity of SCa, which includes neurofilaments and tubulin, is 1-2 mm/d, whereas SCb, which includes actin, tubulin, and numerous soluble proteins, moves as a heterogeneous wave at 2-4 mm/d. Two isoforms of microtubule-associated protein 1B (MAP1B), which can be separated on SDS polyacrylamide gels on the basis of differences in their phosphorylation states (band I and band II), are transported at two different rates. All of band I MAP1B moves as a coherent wave at a velocity of 7-9 mm/d, distinct from slow axonal transport components SCa and SCb. Several other proteins were detected within the component that moves at the velocity of 7-9 mm/d, including the leading wave of tubulin and actin. The properties of this component define a distinct fraction of the slow axonal transport that is termed the slow component c (SCc). The relatively fast transport of the phosphorylated MAP1B isoform at 7-9 mm/d may account for the high concentration of phosphorylated MAP1B in the distal end of growing axons. In contrast to band I MAP1B, the transport profile of band II is complex and contains components moving with SCa and SCb and a leading edge at SCc. Thus, MAP1B isoforms in different phosphorylation states move with distinct components of slow axonal transport, possibly because of differences in their abilities to associate with other proteins (Ma, 2000).

Effects of MAP1B on microtubule dynamics, growth cone dynamics and neuronal migration

For the development of the nervous system it is crucial that growth cones detect environmental information and react by altering their growth direction. The latter process is thought to depend on local stabilization of growth cone microtubules. Evidence has been obtained for a role for the microtubule-associated protein MAP1B, in particular a mode 1 phosphoisoform of the molecule, P1-MAP1B, in this process. P1-MAP1B is tightly associated with the cytoskeleton and is present at highest concentrations in the distal axon and the growth cone of chick retinal ganglion cells. In growth cones turning at nonpermissive substrate borders, P1-MAP1B is restricted to regions which are stabilized. Unilateral neutralization of P1-MAP1B in one-half the growth cone by microscale chromophore-assisted laser inactivation, changes growth cone motility, morphology, and growth direction. The results indicate a functional role for P1-MAP1B in local growth cone stabilization and thus growth cone steering (Mack, 2000).

Glycogen synthase kinase 3beta (GSK3beta) phosphorylates the microtubule-associated protein (MAP) 1B in an in vitro kinase assay and in cultured cerebellar granule cells. Mapping studies have identified a region of MAP1B high in serine-proline motifs that is phosphorylated by GSK3beta. COS cells, transiently transfected with both MAP1B and GSK3beta, express high levels of the phosphorylated isoform of MAP1B (MAP1B-P) generated by GSK3beta. To investigate effects of MAP1B-P on microtubule dynamics, double transfected cells were labelled with antibodies to tyrosinated and detyrosinated tubulin markers for stable and unstable microtubules. High levels of MAP1B-P expression are associated with the loss of a population of detyrosinated microtubules in these cells. Transfection with MAP1B protects microtubules in COS cells against nocodazole depolymerization. However, this protective effect is greatly reduced in cells containing high levels of MAP1B-P, following transfection with both MAP1B and GSK3beta. Since MAP1B binds to tyrosinated, but not to detyrosinated, microtubules in transfected cells, it is proposed that MAP1B-P prevents tubulin detyrosination and subsequent conversion of unstable to stable microtubules and that this involves binding of MAP1B-P to unstable microtubules. The highest levels of MAP1B-P are found in neuronal growth cones: therefore, these findings suggest that a primary role of MAP1B-P in growing axons may be to maintain growth cone microtubules in a dynamically unstable state, a known requirement of growth cone microtubules during pathfinding. To test this prediction, the levels of MAP1B-P in neuronal growth cones of dorsal root ganglion cells in culture were reduced by inhibiting GSK3beta with lithium. In confirmation of the proposed role of MAP1B-P in maintaining microtubule dynamics it was found that lithium treatment dramatically increases the numbers of stable (detyrosinated) microtubules in the growth cones of these neurons (Goold, 1999).

The signaling cascades governing neuronal migration and axonal guidance link extracellular signals to cytoskeletal components. MAP1B is a neuron-specific microtubule-associated protein implicated in the crosstalk between microtubules and actin filaments. Netrin 1 regulates, both in vivo and in vitro, mode I MAP1B phosphorylation, which controls MAP1B activity, in a signaling pathway that depends essentially on the kinases GSK3 and CDK5. map1B-deficient neurons from the lower rhombic lip and other brain regions have reduced chemoattractive responses to Netrin 1 in vitro. Furthermore, map1B mutant mice have severe abnormalities, similar to those described in netrin 1-deficient mice, in axonal tracts and in the pontine nuclei. These data indicate that MAP1B phosphorylation is controlled by Netrin 1 and that the lack of MAP1B impairs Netrin 1-mediated chemoattraction in vitro and in vivo. Thus, MAP1B may be a downstream effector in the Netrin 1-signaling pathway (Del Río, 2004).

Mutation of MAP1B

Microtubules play an important role in establishing cellular architecture. Neuronal microtubules are considered to have a role in dendrite and axon formation. Different portions of the developing and adult brain microtubules are associated with different microtubule-associated proteins (MAPs). The roles of each of the different MAPs are not well understood. One of these proteins, MAP1B, is expressed in different portions of the brain and has been postulated to have a role in neuronal plasticity and brain development. To ascertain the role of MAP1B, mutant mice were generated by gene-targeting methods. Mice that are homozygous for the modification die during embryogenesis. The heterozygotes exhibit a spectrum of phenotypes, including slower growth rates, lack of visual acuity in one or both eyes, and motor system abnormalities. Histochemical analysis of the severely affected mice reveals that their Purkinje cell dendritic processes are abnormal; they do not react with MAP1B antibodies, and show reduced staining with MAP1A antibodies. Similar histological and immunochemical changes occur in the olfactory bulb, hippocampus, and retina, providing a basis for the observed phenotypes (Edelmann, 1996).

Microtubule-associated protein 1B (MAP1B), one of the microtubule-associated proteins (MAPs), is a major component of the neuronal cytoskeleton. It is expressed at high levels in immature neurons during growth of their axons, which indicates that it plays a crucial role in neuronal morphogenesis and neurite extension. To better define the role of MAP1B in vivo, gene targeting was used to disrupt the murine MAP1B gene. Heterozygotes of this MAP1B disruption exhibit no overt abnormalities in their development and behavior, while homozygotes show a slightly decreased brain weight and delayed nervous system development. These data indicate that while MAP1B is not essential for survival, it is essential for normal time course development of the murine nervous system. These conclusions are very different from those of a previous MAP1B gene-targeting study (Edelmann, 1996). In this previous effort, homozygotes died before reaching 8-d embryos, while heterozygotes showed severely abnormal phenotypes in their nervous systems. Because the gene targeting event in these mice produced a gene encoding a 571-amino acid truncated product of MAP1B, it seems likely that the phenotypes seen arise from the truncated MAP1B product acting in a dominant-negative fashion, rather than a loss of MAP1B function (Takei, 1997).

Microtubule-associated proteins such as MAP1B have long been suspected to play an important role in neuronal differentiation, but proof has been lacking. Generation of a complete MAP1B null allele has been accomplished. Mice heterozygous for this MAP1B deletion are not affected. Homozygous mutants are viable but display a striking developmental defect in the brain, the selective absence of the corpus callosum, and the concomitant formation of myelinated fiber bundles consisting of misguided cortical axons. In addition, peripheral nerves of MAP1B-deficient mice have a reduced number of large myelinated axons. The myelin sheaths of the remaining axons are of reduced thickness, resulting in a decrease of nerve conduction velocity in the adult sciatic nerve. The anticipated involvement of MAP1B in retinal development and gamma-aminobutyric acid C receptor clustering is not substantiated. These results demonstrate an essential role of MAP1B in development and function of the nervous system (Meixner, 2000).

MAP1B expression dynamics

MAP1B is a major cytoskeletal protein in growing axons and is strongly regulated during brain development. The expression of MAP1B mRNA, the protein, and its phosphorylated isoform in spinal cord and dorsal root ganglia (DRGs) was compared with expression in the brain. In spinal cord and brain, MAP1B mRNA levels are highest in early stages of development, decreasing several fold during postnatal development, and remaining low in adults. In contrast, there are no significant changes of MAP1B mRNA levels during development of DRG and they remain high in adults. The levels of MAP1B protein decreases in brain and spinal cord in parallel with the changes of their mRNA. The protein levels in DRG remain relatively high but decline in the sciatic nerve. Phosphorylated MAP1B is expressed at high levels during the early stages of development in brain, spinal cord, and sciatic nerve and decreases rapidly to undetectable levels postnatally except for sciatic nerve where it remains detectable. Immunohistochemical analysis shows that phosphorylated MAP1B is absent from DRG cell bodies at all stages but is present in axons of DRG and motor neurons in both spinal cord and sciatic nerve. Immunostaining also confirms Western blot analysis indicating that MAP1B is initially abundant within the spinal cord but is at later stages present only in motor neurons and the central processes of DRG neurons. These results reflect differential distribution of MAP1B isoforms at different stages of development and in different regions of the nervous system (Ma, 1997).

Microtubule-associated protein IB (MAP1B) is the first MAP to be detected in the developing nervous system, and it becomes markedly down-regulated postnatally. Its expression, particularly that of its phosphorylated isoform, is associated with axonal growth. To determine whether adult central nervous system (CNS) areas that retain immunoreactivity for MAP1B are associated with morphological plasticity, the distribution of a phosphorylated MAP1B isoform (MAP1B-P) was compared to the distribution of total MAP1B protein and MAP1B-mRNA. Although these are present only at very low levels, both protein and message are found ubiquitously in almost all adult CNS neurons. The intensity of staining, however, varies markedly among different regions, with only a few nuclei retaining relatively high levels. MAP1B-P is restricted to axons, whereas total MAP1B is present in cell bodies and processes. Relative to total MAP1B protein and its mRNA, MAP1B-P levels decrease more dramatically with maturation, and they are detectable in only a few specific areas that undergo structural modifications. These included primary afferents and motor neurons, olfactory tubercles, habenular and raphe projections to interpeduncular nuclei, septum, and the hypothalamus. The distribution pattern of MAP1B-P was compared to that of the embryonic N-CAM rich in polysialic acid (PSA-NCAM). PSA-NCAM immunostaining is largely overlapped with that of MAP1B-P in the adult CNS. These results suggest that, like PSA-NCAM, MAP1B may be one of the molecules expressed during brain development that also plays a role in structural remodeling in the adult (Nothias, 1996).

Transcription regulation of MAP1B

The MAP1B (Mtap1b) promoter presents two evolutionary conserved overlapping homeoproteins and Hepatocyte nuclear factor 3ß (HNF3ß/Foxa2) cognate binding sites (defining putative homeoprotein/Fox sites, HF1 and HF2). Accordingly, the promoter domain containing HF1 and HF2 is recognized by cerebellum nuclear extracts containing Engrailed and Foxa2 and has regulatory functions in primary cultures of embryonic mesmetencephalic nerve cells. Transfection experiments further demonstrate that Engrailed and Foxa2 interact physiologically in a dose-dependent manner: Foxa2 antagonizes the Engrailed-driven regulation of the MAP1B promoter, and vice versa. This led to an investigation to see if Engrailed and Foxa2 interact directly. Direct interaction was confirmed by pull-down experiments, and the regions participating in this interaction were identified. In Foxa2 the interacting domain is the Forkhead box DNA-binding domain. In Engrailed, two independent interacting domains exist: the homeodomain and a region that includes the Pbx-binding domain. Finally, Foxa2 not only binds Engrailed but also Lim1, Gsc and Hoxa5 homeoproteins and in the four cases Foxa2 binds at least the homeodomain. Based on the involvement of conserved domains in both classes of proteins, it is proposed that the interaction between Forkhead box transcription factors and homeoproteins is a general phenomenon (Foucher, 2003).

Mapping of the interacting domains identified the Forkhead box binding domain in Foxa2 as the only domain interacting with Engrailed, Hoxa5, Lim1, and Gsc and Otx2. Similarly, for all homeoproteins tested, the homeodomain alone binds Foxa2. However, and in contrast with Foxa2, four out of these five homeoproteins contained additional Foxa2-interacting regions: Engrailed, Hoxa5, Gsc (in all three cases in the N-terminal sequence) and Otx2 [in its C-terminal sequence]. A detailed analysis of the interacting domains has been done for Engrailed only and the mapping of the other homeoproteins has been limited to the homeodomain, and its flanking N- and C-terminal regions, at large. In the case of Engrailed, in addition to the homeodomain, a short sequence (amino acids 146-199) overlapping the Pbx-interacting domain also binds Foxa2. This latter domain and the homeodomain bind independently to Foxa2 and the possibility that they interact with different sub-regions of the Forkhead box domain was not investigated. Such an additional non-homeodomain Foxa2 interacting domain was also present in the N-terminal sequences of Hoxa5 and Gsc, but not in Lim1. With the exception of the hexapeptide sequence present in Engrailed and Hoxa5, no further similarities were found between the Foxa2-binding domains identified outside the homeodomain in Engrailed, Hoxa5, Gsc and Otx2. It is thus possible that, in addition to the homeodomain, different homeoproteins have evolved separate Foxa2-binding regions with regulatory functions (Foucher, 2003).

In this context it is interesting that the fragment 146-199 of Engrailed includes the EH2 (homologous to hexapeptide in Hox proteins) and EH3 domains of Engrailed, both of which are implicated in functional interactions with Exd/Pbx homeoproteins. The same observation also holds for Hoxa5, for which the N-terminal sequence containing the hexapeptide sequence binds Foxa2. Both Pbx and Foxa2 might bind Engrailed (or Hoxa5) to form a tripartite complex or, alternatively, that Foxa2 and Pbx binding are mutually exclusive. Also intriguing is the fact that Engrailed and Gsc, as well as different Forkhead box proteins -- including BF1 and Foxa2 -- interact with co-factors of the Groucho/TLE family. Since the Groucho/TLE-interacting domains of Engrailed and Foxa2 have been mapped to the EH1 and CRII domains, respectively (two domains not involved in the Foxa2-Engrailed interaction) it is possible that larger complexes involving Groucho/TLE proteins, Forkhead transcription factors and homeoproteins form in vivo (Foucher, 2003).

futsch : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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