Amyloid-beta peptide binds to microtubule-associated protein 1B (MAP1B)

Extracellular and intraneuronal formation of amyloid-beta aggregates have been demonstrated to be involved in the pathogenesis of Alzheimer's disease. However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of targets have deleterious effects on cellular functions. In the present study we have shown for the first time that amyloid-beta 1-42 bound to a peptide comprising the microtubule binding domain of the heavy chain of microtubule-associated protein 1B by the screening of a human brain cDNA library expressed on M13 phage. This interaction may explain, in part, the loss of neuronal cytoskeletal integrity, impairment of microtubule-dependent transport and synaptic dysfunction observed previously in Alzheimer's disease.     

Chicken microtubule-associated protein 4 (MAP4): a novel member of the MAP4 family

 Chicken gizzard smooth muscle has often been used as a source of proteins of the contractile and cytoskeletal apparatus. In the present study, we isolated a hitherto unknown doublet of proteins, with apparent molecular weights of 200 kDa, from embryonic chicken gizzard and showed its association with the microtubular cytoskeleton by cosedimentation with microtubules (MTs) and by immunofluorescence staining of cultured cells. Immunoblot analysis also revealed the ubiquitous expression of this protein in all embryonic chicken tissues examined. Molecular cloning techniques allowed its identification as the chicken homologue of the microtubule-associated protein 4 (MAP4), known from mammalian species, and revealed approximately 90% of its amino acid sequence. MAP4 is the major MAP of non-neuronal tissues and cross-species comparisons clearly demonstrated its highly conserved overall structure, consisting of a basic C-terminal MT-binding region and an acidic N-terminal projection domain of unknown function. Despite these conserved features, overall sequence homologies to its mammalian counterparts are rather low and focused to distinct regions of the molecule. Among these are a conserved 18-amino acid motif, which is known to mediate binding to MTs and a part of the MT-binding domain known as the proline-rich region, which is thought to be the regulatory domain of MAP4. The N-terminal 59 amino acids are a conserved and unique feature of the MAP4 sequence and might be an indication that MAP4 performs other functions besides the enhancement of MT assembly. Accepted: 13 March 1996     

A protein kinase bound to the projection portion of MAP 2 (microtubule-associated protein 2)

In previous work we have demonstrated that the microtubule-associated protein 2 (MAP 2) molecule consists of two structural parts. One part of the molecule, referred to as the assembly-promoting domain, binds to the microtubule surface and is responsible for promoting microtubule assembly; the other represents a filamentous projection observed on the microtubule surface that may be involved in the interaction of microtubules with other cellular structures. MAP 2 is known to be specifically phosphorylated as the result of a protein kinase activity that is present in microtubule preparations. We have now found that the activity copurifies with the projection portion of MAP 2 itself. Kinase activity coeluted with MAP 2 when microtubule protein was subjected to either gel- filtration chromatography on bio-gel A-15m or ion-exchange chromatography on DEAE- Sephadex. The activity was released from microtubules by mild digestion with chymotrypsin in parallel with the removal by the protease of the MAP 2 projections from the microtubule surface. The association of the activity with the projection was demonstrated directly by gel filtration chromatography of the projections on bio-gel A-15m. Three protein species (M(r) = 39,000, 55,000, and 70,000) cofractionated with MAP 2, and two of these (M(r) = 39,000 and 55,000) may represent the subunits of an associated cyclic AMP- dependent protein kinase. The projection-associated activity was stimulated 10-fold by cyclic AMP and was inhibited more than 95 percent by the cyclic AMP-dependent protein kinase inhibitor from rabbit skeletal muscle. It appeared to represent the only significant activity associated with microtubules, almost no activity being found with tubulin, other MAPs, or the assembly-promoting domain of MAP 2, and was estimated to account for 7-22 percent of the total brain cytosolic protein kinase activity. The location of the kinase on the projection is consistent with a role in regulating the function of the projection, though other roles for the enzyme are also possible.     

MAP3: characterization of a novel microtubule-associated protein

Using monoclonal antibodies we have characterized a brain protein that copurifies with microtubules. We identify it as a microtubule-associated protein (MAP) by the following criteria: it copolymerizes with tubulin through repeated cycles of microtubule assembly in vitro; it is not associated with any brain subcellular fraction other than microtubules; in double-label immunofluorescence experiments antibodies against this protein stain the same fibrous elements in cultured cells as are stained by antitubulin; and this fibrous staining pattern is dispersed when cytoplasmic microtubules are disrupted by colchicine. Because it is distinct from previously described MAPs we designate this novel species MAP3. The MAP3 protein consists of a closely spaced pair of polypeptides on SDS gels, Mr 180,000, which are present in both glial (glioma C6) and neuronal (neuroblastoma B104) cell lines. In brain the MAP3 antigen is present in both neurons and glia. In nerve cells its distribution is strikingly restricted: anti-MAP3 staining is detectable only in neurofilament-rich axons. It is not, however, a component of isolated brain intermediate filaments.     

Binding of a C-terminal fragment (residues 369 to 435) of vitamin D-binding protein to actin

The vitamin D-binding protein (DBP) binds to monomeric actin with high affinity. The variation in DBP isoforms is due to genetic polymorphism and varying glycosylation. To obtain a homogeneous preparation, the cDNA for human DBP and truncations thereof were cloned and various systems were applied for heterologous bacterial and yeast expression. The full-length protein and the N- and C-terminal halves of DBP remained insoluble probably because the protein did not fold to its native three-dimensional structure due to formation of accidental intra- and inter-molecular disulfide bonds during expression in bacteria or yeast. This problem was overcome by cloning of a C-terminal fragment comprising residues 369 to 435 that did not contain disulfide bonds and was completely soluble. Binding of the C-terminal fragment to monomeric actin was demonstrated by comigration with actin during native polyacrylamide gel electrophoresis and surface plasmon resonance, however, at considerably lower affinity than full-length DBP. This suggests that in addition to the C-terminal amino acid sequence other parts (amino acid residues or sugar moieties) of DBP participate in actin binding. The C-terminal fragment was found to inhibit denaturation of actin and to decrease the rate of actin polymerisation both at the barbed and at the pointed end in a concentration-dependent manner. According to a quantitative analysis of the polymerisation kinetics, association of actin monomers to nucleate filaments was not prevented by binding of the C-terminal fragment to actin. These data suggest that the sites on the surface of actin that are involved in actin nucleation and elongation are different.     

Developmental regulation of two microtubule-associated proteins (MAP2 and MAP5) in the embryonic avian retina

Previous studies with the mammalian brain have shown that the expression of a number of neuronal microtubule-associated proteins (MAPs) is developmentally regulated. For example, the low-molecular-weight form of MAP2 (MAP2c) is abundant in neonatal rat brains and is less abundant in adults. Similarly, MAP5 levels decrease during postnatal development. Using monoclonal antibodies, we have followed the time of first appearance, cellular distribution, and molecular form of MAP2 and MAP5 during the morphogenesis of the quail retina. MAP2 first appears in ganglion cell bodies and in the axons of the optic fibre layer (OFL) at embryonic day 4 (E4). Anti-MAP2 staining remains restricted to these sites until E10, when staining appears in the inner plexiform layer (IPL). At E14, one day before hatching, anti-MAP2 staining is found in three broad laminae in the IPL, as well as in photosensitive cells. MAP5 is present in ganglion cell axons from the onset of neurite elongation at E3 and is limited to the OFL until E10. The intensity of anti-MAP5 staining in the OFL and optic nerve decreases after E7, which corresponds with a decrease in the number of actively growing ganglion cell axons. By E14, anti-MAP5 stains five layers in the IPL that correspond with layers of amacrine cell process arborizations. Western blots of E10 brain microtubule proteins show that MAP2 is represented by both a 260 x 10(3) Mr protein and a 60-65 x 10(3) Mr protein; the latter is much more abundant. Anti-MAP5 recognizes a 320 x 10(3) Mr brain microtubule protein in both the quail and the rat. We conclude that the cellular distribution, developmental regulation and molecular forms of MAP2 and MAP5 are similar in the rat and quail, suggesting that these molecules have conserved and hence fundamental roles in the growth and differentiation of neuronal processes.     

The phosphorylation of a putative sperm microtubule-associated protein 2 (MAP2) is uniquely sensitive to regulation.

We have identified a bovine sperm phosphoprotein, pp255 (Mr = 255,000), which reacts strongly and specifically with an antibody to rat brain microtubule-associated protein 2 (MAP2). The phosphorylation state of this putative sperm MAP2 in intact bovine epididymal sperm is uniquely sensitive to regulation by intracellular pH (pHi), calcium, isobutyl-3-methylxanthine (MIX), H-8, and fluoride. Increasing pHi by approximately 0.4 units or exposure to calcium (0.1 microM with the ionophore A23187) or to the protein kinase inhibitor, H-8, decreases sperm MAP2 phosphorylation. Decreasing sperm pHi or exposure to MIX or fluoride increases MAP2 phosphorylation. Numerous other detectable sperm phosphoproteins are either unresponsive to most of these modulators or are considerably less sensitive to them. This phosphoprotein co-sediments with the particulate sperm heads during subcellular fractionation, and is not detectable in other sperm fractions. Two-dimensional electrophoresis separates sperm MAP2 into multiple species, indicative of varying degrees of phosphorylation. Sperm MAP2 is phosphorylated on serine residues, changes electrophoretic mobility slightly on one-dimensional gels with changes in phosphorylation levels, and exhibits the highest specific radioactivity of any sperm phosphoprotein observed. The phosphorylation state of sperm MAP2 can be uncoupled from sperm motility levels under several conditions. The co-localization of sperm MAP2 with the head fraction and the unique sensitivity of its phosphorylation level to modulators, which are known to regulate capacitation and the acrosome reaction, suggest that sperm MAP2 phosphorylation may be an intermediate step in the regulation of one or both of these sperm processes.     

Phosphorylation determines the binding of microtubule-associated protein 2 (MAP2) to microtubules in living cells

The influence of phosphorylation on the binding of microtubule-associated protein 2 (MAP2) to cellular microtubules was studied by microinjecting MAP2 in various phosphorylation states into rat-1 fibroblasts, which lack endogenous MAP2. Conventionally prepared brain MAP2, containing 10 mol of endogenous phosphate per mol (MAP2-P10), was completely bound to cellular microtubules within 2-3 min after injection. MAP2 prepared in the presence of phosphatase inhibitors, containing 25 mol/mol of phosphate (MAP2-P25), also bound completely. However, MAP2 whose phosphate content had been reduced to 2 mol phosphate per mol by treatment with alkaline phosphatase in vitro (MAP2-P2) did not initially bind to microtubules, suggesting that phosphorylation of certain sites in MAP2 is essential for binding to microtubules. MAP2-P10 was further phosphorylated in vitro via an endogenously bound protein kinase activity, adding 12 more phosphates, giving a total of 22 mol/mol. This preparation (MAP2-P10+12) also did not bind to microtubules. Assay of the binding of these preparations to taxol-stabilized tubulin polymers in vitro confirmed that their binding to tubulin depended on the state of phosphorylation, but the results obtained in microinjection experiments differed in some cases from in vitro binding. The results suggest that the site of phosphate incorporation rather than the amount is the critical factor in determining microtubule binding activity of MAP2. Furthermore, the interaction of MAP2 with cellular microtubules may be influenced by additional factors that are not evident in vitro.     

Regulated association of microtubule-associated protein 2 (MAP2) with Src and Grb2: evidence for MAP2 as a scaffolding protein

Microtubule-associated protein 2 (MAP2) and tau, which is involved in Alzheimer's disease, are major cytoskeletal proteins in neurons. These proteins are involved in microtubule assembly and stability. To further characterize MAP2, we took a strategy of identifying potential MAP2 binding partners. The low molecular weight MAP2c protein has 11 PXXP motifs that are conserved across species, and these PXXP motifs could be potential ligands for Src homology 3 (SH3) domains. We tested for MAP2 interaction with SH3 domain-containing proteins. All neuronal MAP2 isoforms bound specifically to the SH3 domains of c-Src and Grb2 in an in vitro glutathione S-transferase-SH3 pull-down assay. Interactions between endogenous proteins were confirmed by co-immunoprecipitation using brain lysate. All three proteins were also found co-expressed in neuronal cell bodies and dendrites. Surprisingly, the SH3 domain-binding site was mapped to the microtubule-binding domain that contains no PXXP motif. Src bound primarily the soluble, non-microtubule-associated MAP2c in vitro. This specific MAP2/SH3 domain interaction was inhibited by phosphorylation of MAP2c by the mitogen-activated protein kinase extracellular signal-regulated kinase 2 but not by protein kinase A. This phosphorylation-regulated association of MAP2 with proteins of intracellular signal transduction pathways suggests a possible link between cellular signaling and neuronal cytoskeleton, with MAP2 perhaps acting as a molecular scaffold upon which cytoskeleton-modifying proteins assemble and dissociate in response to neuronal activity.     

Purification of brain microtubule-associated protein MAP1A

Brain microtubule-associated protein MAP1A has been purified until homogeneity by using a novel procedure involving copolymerization with microtubules, treatment with poly-l-aspartic acid and FPLC. The purified protein retains its capacity to facilitate microtubule assembly.     

MAP 4: a microtubule-associated protein specific for a subset of tissue microtubules

The cytological distribution of microtubule-associated protein 4 (MAP 4) (L. M. Parysek, C. F. Asnes, J. B. Olmsted, 1984, J. Cell Biol., 99:1309-1315) in mouse tissues has been examined. Adjacent 0.5-0.9- micron sections of polyethylene glycol-embedded tissues were incubated with affinity-purified MAP 4 or tubulin antibodies, and the immunofluorescent images were compared. Tubulin antibody labeling showed distinct microtubules in all tissues examined. MAP 4 antibody also labeled microtubule-like patterns, but the extent of MAP 4 reactivity was cell type-specific within each tissue. MAP 4 antibody labeled microtubules in vascular elements of all tissues and in other cells considered to have supportive functions, including Sertoli cells in the testis and glial elements in the nervous system. Microtubule patterns were also observed in cardiac, smooth, and skeletal (eye) muscle, podocytes in kidney, Kuppfer cells in liver, and spermatid manchettes. The only MAP 4-positive cells in which the pattern was not microtubule-like were the principal cells of the collecting ducts in kidney cortex, in which diffuse fluorescence was seen. MAP 4 antibody did not react with microtubule-rich neuronal elements of the central and peripheral nervous system, skeletal muscle from anterior thigh, liver parenchymal cells, columnar epithelial cells of the small intestine, and absorptive cells of the tubular component of the nephron. These observations indicate that MAP 4 may be associated with only certain kinds of cell functions as demonstrated by the preferential distribution with microtubules of defined cell types.     

Regulation of a major microtubule-associated protein by MPF and MAP kinase.

The interphase-M phase transition of microtubule dynamics is thought to be induced by phosphorylation reactions mediated by MPF and by MAP kinase functioning downstream of MPF. We have now identified and purified from Xenopus eggs a major microtubule-associated protein, p220, that may be a target protein for these two M phase-activated kinases. p220, when purified from interphase cells, potently bound to microtubules and stimulated tubulin polymerization, whereas p220 purified from M phase cells showed little or no such activities. Cell staining with a monoclonal anti-p220 antibody revealed that p220 is localized on cytoplasmic microtubule networks during interphase, while it is distributed rather diffusely throughout the cell during M phase. We have further found that p220 is phosphorylated specifically in M phase. Moreover, p220 purified from interphase cells served as a good substrate for MAP kinase and MPF in vitro, and two-dimensional phosphopeptide mapping pattern of the p220 phosphorylated in vitro was very similar to that of p220 phosphorylated at M phase in vivo. These results suggest that the drastic change in p220 activity during the transition from interphase to M phase may be induced by its phosphorylation in M phase probably catalyzed by MAP kinase and MPF.     

Phosphorylation-dependent localization of microtubule-associated protein MAP2c to the actin cytoskeleton

Microtubule-associated protein 2 (MAP2) is a neuronal phosphoprotein that promotes net microtubule growth and actin cross-linking and bundling in vitro. Little is known about MAP2 regulation or its interaction with the cytoskeleton in vivo. Here we investigate the in vivo function of three specific sites of phosphorylation on MAP2. cAMP-dependent protein kinase activity disrupts the MAP2-microtubule interaction in living HeLa cells and promotes MAP2c localization to peripheral membrane ruffles enriched in actin. cAMP-dependent protein kinase phosphorylates serines within three KXGS motifs, one within each tubulin-binding repeat. These highly conserved motifs are also found in homologous proteins tau and MAP4. Phosphorylation at two of these sites was detected in brain tissue. Constitutive phosphorylation at these sites was mimicked by single, double, and triple mutations to glutamic acid. Biochemical and microscopy-based assays indicated that mutation of a single residue was adequate to disrupt the MAP2-microtubule interaction in HeLa cells. Double or triple point mutation promoted MAP2c localization to the actin cytoskeleton. Specific association between MAP2c and the actin cytoskeleton was demonstrated by retention of MAP2c-actin colocalization after detergent extraction. Specific phosphorylation states may enhance the interaction of MAP2 with the actin cytoskeleton, thereby providing a regulated mechanism for MAP2 function within distinct cytoskeletal domains.     

Domain structure and antiparallel dimers of microtubule-associated protein 2 (MAP2).

We have studied the microtubule-associated protein MAP2 from porcine brain and its subfragments by limited proteolysis, antibody labeling, and electron microscopy. Two major chymotryptic fragments start at lys 1528 and arg 1664, generating microtubule-binding fragments of Mr 36 kDa (303 residues, analogous to the "assembly domain" of Vallee, 1980) and 18 kDa (167 residues). These fragments can be labeled with the antibody 2-4 which recognizes the last internal repeat of MAP2 (Dingus et al., 1991). The epitope of another monoclonal antibody, AP18 (Binder et al., 1986), was mapped to the first 151 residues of MAP2. The interaction with AP18 is phosphorylation dependent; dephosphorylated MAP2 is not recognized. Intact MAP2 forms rod-like particles of 97 nm mean length, similar to Gottlieb and Murphy's (1985) observations. Both antibodies bind near an end of the rod, suggesting that the sequence and the structure are approximately colinear. There is a pronounced tendency for MAP2 to form dimers whose components are nearly in register but of opposite polarity. MAP2 can also fold in a hairpin-like fashion, generating 50-nm rods, and it can self-associate into oligomers and fibers. The 36-kDa microtubule-binding fragment also has a rod-like shape; its mean length is 49 nm, half of the intact molecule, even though the fragment contains only one-sixth of the mass. The antibody 2-4 decorates one end of the rod, similar to the intact protein. The fragment also forms antiparallel dimers, but its tendency for higher self-assembly forms is much lower than with intact MAP2.     

Truncation of the projection domain of MAP4 (microtubule-associated protein 4) leads to attenuation of microtubule dynamic instability

MAP4, a ubiquitous heat-stable MAP, is composed of an asymmetric structure common to the heat-stable MAPs, consisting of an N-terminal projection (PJ) domain and a C-terminal microtubule (MT)-binding (MTB) domain. Although the MTB domain has been intensively studied, the role of the PJ domain, which protrudes from MT-wall and does not bind to MTs, remains unclear. We investigated the roles of the PJ domain on the dynamic instability of MTs by dark-field microscopy using various PJ domain deletion constructs of human MAP4 (PJ1, PJ2, Na-MTB and KDM-MTB). There was no obvious difference in the dynamic instability between the wtMAP4 and any fragments at 0.1 microM, the minimum concentration required to stabilize MTs. The individual MTs stochastically altered between polymerization and depolymerization phases with similar profiles of length change as had been observed in the presence of MAP2 or tau. We also examined the effects at the increased concentrations of 0.7 microM, and found that in some cases the dynamic instability was almost entirely attenuated. The length of both the polymerization and depolymerization phases decreased and "pause-phases" were occasionally observed, especially in the case of PJ1, PJ2 or Na-MTB. No obvious change was observed in the increased concentration of wtMAP4 and KDM-MTB. Additionally, the profiles of MT length change were quite different in 0.7 microM PJ2. Relatively rapid and long depolymerization phases were sometimes observed among quite slow length changes. Perhaps, this unusual profile could be due to the uneven distribution of PJ2 along the MT lattice. These results indicate that the PJ domain of MAP4 participates in the regulation of the dynamic instability.     

Bovine platelets contain a 280 kDa microtubule-associated protein antigenically related to brain MAP 2.

Resting bovine platelets contain a microtubule coil which reorganizes into linear arrays upon thrombin activation. Microtubule arrays in both resting and activated platelets are extensively cross-linked. In an effort to determine the proteins responsible for this cross-linking, we have developed a method to isolate taxol-stabilized microtubule coils directly from platelet-rich plasma. Negatively stained coils are still cross-linked, and fine filamentous projections are seen between adjacent microtubules. Critical-point-dried rotary shadowed replicas of these coils most clearly demonstrate the projections radiating from individual microtubules as well as along the microtubule coil. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of isolated coils shows many microtubule-associated proteins (MAPs) present in addition to tubulin. One of these proteins, a 280 kDa MAP, cross-reacts with an antibody to bovine brain MAP 2 by immunoblot analysis. Immunofluorescence localization of this protein with both monoclonal and polyclonal antibodies demonstrates that it is associated with the microtubule coil in resting platelets and with the linear microtubule array present after thrombin activation. Immunoelectron microscopic localization demonstrates that projections from individual microtubules are labeled by the antibodies. We suggest that this MAP, along with several other potential MAPs, is responsible for the cross-linking and stability of bovine platelet microtubules.     

Phosphorylation of the microtubule-associated protein MAP2 by GTP.

The chick brain microtubule-associated protein MAP2 can be phosphorylated in vitro to the extent of 12 mol/mol with GTP at the same sites as can be labelled by the cyclic AMP-independent protein kinase utilizing [gamma-32P]ATP as the phosphoryl donor. Consequently, the microtubule protein is chemically modified by the conditions usually employed for studies of microtubule assembly, so that the derived kinetic parameters may not relate to steady-state conditions.