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J C Bulinski

Publications and source records attributed to J C Bulinski.

At least 19 recordsLinked to original sources
PubMed2001

Accompanying protein alterations in malignant cells with a microtubule-polymerizing drug-resistance phenotype and a primary resistance mechanism.

Microtubules (MTs) are cytoskeletal components whose structural integrity is mandatory for the execution of many basic cell functions. Utilizing parental and drug-resistant ovarian carcinoma cell lines that have acquired point mutations in beta-tubulin and p53, we studied the level of expression and modification of proteins involved in apoptosis and MT integrity. Extending previous results, we demonstrated phosphorylation of pro-survival Bcl-x(L) in an epothilone-A resistant cell line, correlating it with drug sensitivity to tubulin-active compounds. Furthermore, Mcl-1 protein turned over more rapidly following exposure to tubulin-modifying agents, the stability of Mcl-1 protein paralleling the drug sensitivity profile of the paclitaxel or epothilone-A resistant cell lines. The observed decreases in Mcl-1 were not a consequence of G(2)M arrest, as determined by flow cytometry analysis, which showed prominent levels of Mcl-1 in the absence of any drug treatment in populations enriched in mitotic cells. We also observed that a paclitaxel-resistant cell line expressed Bax at a much lower level than the sensitive parental line [A2780(1A9)], consistent with its mutant p53 status. MT-associated protein-4 (MAP4), whose phosphorylation during specific phases of the cell cycle reduces its MT-polymerizing and -stabilizing capabilities, was phosphorylated in response to drug challenge without a change in expression. Phosphorylation of MAP4 correlated with sensitivity to tubulin-binding drugs and with a dissociation from MTs. We propose that the tubulin mutations, which result in a compromised paclitaxel:tubulin or epothilone:tubulin interaction and paclitaxel or epothilone resistance, indirectly inhibit downstream events that lead to cell death, and this, in turn, may contribute to the drug-resistance phenotype

Antineoplastic Agents, Phytogenic↗
PubMed2001

Abundant expression of the microtubule-associated protein, ensconsin (E-MAP-115), alters the cellular response to Taxol.

Correlation between expression level of a microtubule-associated protein called ensconsin (E-MAP-115) and degree of Taxol sensitivity in several cultured cell lines prompted us to investigate potential cause-and-effect relationships between ensconsin level and Taxol action. We used human MCF-7 or HeLa cells, which are sensitive to low Taxol concentrations (LD(50) of 30-35 and 3.5 nM, respectively) to prepare stably transfected populations of cells expressing heterogeneous levels of ensconsin chimeras, either green fluorescent protein (GFP) conjugated to full-length ensconsin (GFP-Ensc) or to ensconsin's microtubule-binding domain (GFP-EMTB). Both a subjective microscopic assay, i.e., scoring fluorescence of GFP-ensconsin chimeras following Taxol treatment, and a quantitative immunobiochemical assay, i.e., measuring level of GFP-ensconsin chimera in cells surviving treatment with Taxol, showed that cells expressing higher levels of GFP-ensconsin chimera were killed more readily by Taxol concentrations approaching the LD(50). In contrast, in TC-7 cells, which are relatively insensitive to Taxol (LD(50) > 600 nM), high-level expression of GFP-EMTB conferred no significant susceptibility to killing by Taxol. However, heightening the Taxol sensitivity of GFP-EMTB-TC-7 cells by pre-incubating cells with the p-glycoprotein inhibitor, verapamil, did result in selective killing of cells highly expressing GFP-EMTB. Taken together, results obtained in MCF-7, HeLa, and TC-7 cells suggest that elevated ensconsin level bestowed a selective disadvantage upon Taxol-sensitive cells. To probe potential mechanisms by which ensconsin could alter the Taxol response, we isolated microtubules from HeLa cells that were or were not pretreated with Taxol. In vivo Taxol treatment significantly tightened microtubule-binding of ensconsin, suggesting that Taxol alters ensconsin's microtubule-binding properties and may, in turn, alter the Taxol response of the microtubules. Our data support the hypothesis that Taxol works synergistically or in concert with microtubule-binding proteins in bringing about deleterious effects on the microtubule cytoskeleton.

Animals↗
PubMed2001

Phosphorylation of MAP4 affects microtubule properties and cell cycle progression.

In human cells, MAP4, a microtubule-associated protein ubiquitously expressed in proliferating cells, has been shown to undergo in vivo phosphorylation. Two phosphorylation sites, serines 696 and 787, lie within the proline-rich region of its microtubule-binding domain. To test the hypothesis that phosphorylation at these sites influences microtubule properties or cell cycle progression, we prepared stable cell lines that inducibly express versions of MAP4 in which phosphorylation of these two serines was prevented by their replacement with alanine, lysine, or glutamate residues (AA-, KK-, or EE-MAP4). All nonphosphorylatable mutant forms of MAP4 expressed in mouse Ltk- cells were localized to MT arrays that were unremarkable in appearance. Expression of nonphosphorylatable mutants of MAP4 did not affect cell doubling time; however, expression of some mutants altered progression into or through cell division. Interactions of mutant MAP4 with MTs were examined in vitro. KK mutant MAP4 bound MTs more avidly than its wild-type counterpart, WT-MAP4. In vivo MT polymer also differed among the mutants: MTs in cells expressing the KK- and AA-MAP4 forms were more resistant to nocodazole depolymerization than those in cells expressing EE- or WT-MAP4 forms. Our results demonstrate that phosphorylation alters MAP4 properties and suggest a raison d'être for phosphorylation of the MAP4 microtubule-binding domain during cell cycle progression.

Amino Acid Sequence↗
PubMed2001

Rapid dynamics of the microtubule binding of ensconsin in vivo.

Microtubule-associated proteins (MAPs) are proteins that reversibly bind to and regulate microtubule dynamics and functions in vivo. We examined the dynamics of binding of a MAP called ensconsin (E-MAP-115) to microtubules in vivo. We used 5xGFP-EMTB, a construct in which the microtubule-binding domain of ensconsin (EMTB) is fused to five copies of green fluorescent protein (GFP), as a reporter molecule amenable to the use of fluorescent speckle microscopy. Fluorescent speckle microscopy (FSM) sequences and kymograph analyses showed rapid dynamics of speckles comprised of 5xGFP-EMTB in untreated cells. By contrast, in detergent-lysed cytoskeletons, speckles were not dynamic. Since detergent-lysed cytoskeletons differ from living cells in that they lack both ATP and dynamic microtubules, we used azide treatment to substantially reduce the level of ATP in living cells and we used Taxol to halt microtubule dynamics. Both treatments slowed the dynamics of 5xGFP-EMTB speckles observed by FSM. We also used fluorescence recovery after photobleaching (FRAP) to quantify the half-time of binding and dissociation of the 5xGFP-EMTB chimera and to compare this half-time to that of the full-length MAP molecule. In untreated cells, the t(g) of either 5xGFP-EMTB or full-length GFP-ensconsin was similarly rapid (approximately 4 seconds), while in ATP-reduced and Taxol-treated cells, t(g) was increased to 210 seconds and 40 seconds, respectively. In detergent-extracted cells no recovery was seen. Consistent with the rapid dynamics of 5xGFP-EMTB measured with fluorescent speckle microscopy and FRAP, we estimated that the affinity of the MAP for microtubules is approximately 40 microM in untreated living cells, compared with approximately 1 microM in vitro. However, K(D,app) was not significantly changed in the presence of azide and was increased to 110 microM in the presence of Taxol. To test whether changes in the phosphorylation state of cellular proteins might be responsible for altering the dynamics of ensconsin binding, we used FSM to monitor staurosporine-treated cells. Staurosporine treatment substantially halted dynamics of 5xGFP-EMTB speckles along MTs. Our results show that ensconsin is highly dynamic in its association with microtubules, and its microtubule association can be altered by in vivo phosphorylation events.

Animals↗
PubMed2000

Ser787 in the proline-rich region of human MAP4 is a critical phosphorylation site that reduces its activity to promote tubulin polymerization.

p34cdc2 kinase-phosphorylation sites in the microtubule (MT)-binding region of MAP4 were determined by peptide sequence of phosphorylated MTB3, a fragment containing the carboxy-terminal half of human MAP4. In addition to two phosphopeptides containing Ser696 and Ser787 which were previously indicated to be in vivo phosphorylation sites, two novel phosphopeptides, containing Thr892 or Thr901 and Thr917 as possible phosphorylation sites, were isolated, though only in in vitro phosphorylation. The role of phosphorylation at Ser696 and Ser787, which were differently phosphorylated during the cell cycle (Ookata et al., (1997). Biochemistry, 36: 15873-15883), was investigated in MT-polymerization, using MAP4 Ser to Glu mutants, which mimic phosphorylation at each site. Mutation of Ser787 to Glu strikingly reduced the MAP4's MT-polymerization activity, while Glu-mutation at Ser696 did not. These results suggest that Ser787 could be the critical phosphorylation site causing MTs to be dynamic at mitosis.

Amino Acid Sequence↗
PubMed1999

Microtubule-associated protein 4 (MAP4) regulates assembly, protomer-polymer partitioning and synthesis of tubulin in cultured cells.

We depleted MAP4, a ubiquitously expressed microtubule (MT)-associated protein previously shown to be capable of stabilizing MTs, from HeLa cells by stably expressing antisense RNA. These HeLa-AS cells, in which the MAP4 level was decreased to 33% of the wild-type level, displayed decreased content of total tubulin (65% of the wild-type level). The partitioning of cellular tubulin into protomer and polymer was altered in HeLa-AS cells: polymeric tubulin was decreased to 46% of the level in control cells, while protomeric tubulin was increased to 226% of the level in control cells. Tubulin protein synthesis was decreased, consistent with the tubulin autoregulation model, which proposes that tubulin protomer inhibits its own synthesis. Following release from drug-induced depolymerization, MTs in HeLa-AS cells reformed more slowly, and showed an increased focus on the centrosome, as compared to control cells. HeLa-AS cells also appeared to be less bipolar in shape and flatter than control cells. Our data suggest that MAP4 regulates assembly level of MTs and, perhaps through this mechanism, is involved in controlling spreading and shape of cells.

Biopolymers↗
PubMed1999

E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo and is not a physiological modulator of microtubule dynamics.

Microtubule-associated proteins (MAPs) have been hypothesized to regulate microtubule dynamics and/or functions. To test hypotheses concerning E-MAP-115 (ensconsin) function, we prepared stable cell lines expressing conjugates in which the full-length MAP (Ensc) or its microtubule-binding domain (EMTB) was conjugated to one or more green fluorescent protein (GFP) molecules. Because both distribution and microtubule-binding properties of GFP-Ensc, GFP-EMTB, and 2x, 3x, or 4xGFP-EMTB chimeras all appeared to be identical to those of endogenous E-MAP-115 (ensconsin), we used the 2xGFP-EMTB molecule as a reporter for the behavior and microtubule-binding function of endogenous MAP. Dual wavelength time-lapse fluorescence imaging of 2xGFP-EMTB in cells microinjected with labeled tubulin revealed that this GFP-MAP chimera associated with the lattice of all microtubules immediately upon polymerization and dissociated concomitant with depolymerization, suggesting that dynamics of MAP:microtubule interactions were at least as rapid as tubulin:microtubule dynamics in the polymerization reaction. Presence of both GFP-EMTB chimeras and endogenous E-MAP-115 (ensconsin) along apparently all cellular microtubules at all cell cycle stages suggested that the MAP might function in modulating stability or dynamics of microtubules, a capability shown previously in transiently transfected cells. Although cells with extremely high expression levels of GFP-EMTB chimera exhibited stabilized microtubules, cells expressing four to ten times the physiological level of endogenous MAP exhibited microtubule dynamics indistinguishable from those of untransfected cells. This result shows that E-MAP-115 (ensconsin) is unlikely to function as a microtubule stabilizer in vivo. Instead, this MAP most likely serves to modulate microtubule functions or interactions with other cytoskeletal elements.

Female↗
PubMed1998

Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells.

Fluorescence microscopic visualization of fluorophore-conjugated proteins that have been microinjected or expressed in living cells and have incorporated into cellular structures has yielded much information about protein localization and dynamics [1]. This approach has, however, been limited by high background fluorescence and the difficulty of detecting movement of fluorescent structures because of uniform labeling. These problems have been partially alleviated by the use of more cumbersome methods such as three-dimensional confocal microscopy, laser photobleaching and photoactivation of fluorescence [2]. We report here a method called fluorescent speckle microscopy (FSM) that uses a very low concentration of fluorescent subunits, conventional wide-field fluorescence light microscopy and digital imaging with a low-noise, cooled charged coupled device (CCD) camera. A unique feature of this method is that it reveals the assembly dynamics, movement and turnover of protein assemblies throughout the image field of view at diffraction-limited resolution. We found that FSM also significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells. Our initial applications include the measurement of microtubule movements in mitotic spindles and actin retrograde flow in migrating cells.

Animals↗
PubMed1998

Identification of kinesin-like molecules in myogenic cells.

Numerous organelles are repositioned during myogenic differentiation and are maintained in an asymmetric distribution throughout the life span of a myotube. It is likely that members of the kinesin superfamily may be responsible for some or all of these microtubule-dependent movements. Consequently, we have attempted to identify kinesin-like molecules expressed throughout myogenesis. Using a standard PCR-based strategy, we cloned two kinesin-like molecules from a rat myogenic cell line, L6. Sequence analysis of the first of these, KIF3C, defines it as a novel member of the KIF3 subfamily of kinesin-like proteins. KIF3C is expressed throughout myogenesis as well as in numerous rat tissues. Like other members of the KIF3 subfamily, KIF3C has an N-terminal motor domain. The second molecule identified is a rat homolog of murine KIF1B, a putative mitochondrial transporter. KIF1B is also expressed ubiquitously both in myogenic cells at all stages and in a variety of rat tissues.

Amino Acid Sequence↗
PubMed1998

Mechanisms of trafficking in axons and dendrites: implications for development and neurodegeneration.

In the area of routing and sorting of dendritic traffic, the current phenomenological data beg questions about the cellular mechanisms utilized not only to transport material but also to modulate activity in a process, even apoptosis. To aid in formulating testable hypotheses, many plausible models are developed here and linked with some of the preliminary data that supports them. We first assume that in long dendrites the sorting of membranous proteins into transport vesicles also involves the linkage of motor proteins to the vesicles. Second, we assume that the cytoskeleton in dendrites is altered from the cytoskeleton in axons and the cell body. Viral glycoproteins, MAP2 and specific mRNA sorting into dendrites provide the simplest models for analyzing vesicular, cytoskeletal and RNA sorting. In the case of viral glycoproteins, initial sorting appears to occur at the Golgi but additional routing steps involve further complexities that could best be served by an additional sorting step at the junction of the cell body and the process. Transport of the specialized cytoskeletal proteins and specific mRNAs as well as vesicular material could be controlled by a similar gatekeeper at the mouth of a process. Studies of the microtubule-organelle motor complex, regulation of microtubule-based motility by microtubule-associated proteins, and slow axonal transport all provide insights into important aspects of the routing and sorting. These processes are in turn controlled by extracellular signals such as those generated by matrix molecules or their hydrolysis products in the case of amyloid precursor protein (APP). Routing and sorting mechanisms may be central to the development of Alzheimer's disease in view of evidence that APP processing is affected, transport is disturbed, and intracellular vesicles (early endosomes) hypertrophied. Further it is possible that routing mechanisms play a role in cell-cell interactions as, for example, the possibility that pathogenic/cellular stress signals may be passed along circuits transsynaptically.

Animals↗
PubMed1998

Changes in dendritic structure and function following hippocampal lesions: correlations with developmental events?

Recovery after nervous system lesions may lead to partial re-institution of developmental schemes and processes. Here we review several of these proposed schemes, with the conclusion that though some processes may involve re-expression of embryonic phenotypes, there are many processes invoked during recovery from lesions that do not mirror developmental phenomena. The inability to fully revert to embryonic schemes because of adult phenotype may partially account for the decreased recovery observed in adults compared to that noted after lesions during development.

Animals↗
PubMed1997

MAP4 is the in vivo substrate for CDC2 kinase in HeLa cells: identification of an M-phase specific and a cell cycle-independent phosphorylation site in MAP4.

We reported previously that cdc2 kinase decreased the microtubule-stabilizing ability of a major HeLa cell microtubule-associated protein, MAP4, by phosphorylation in vitro [Ookata, K., et al. (1995) J. Cell Biol. 128, 849-862]. An important question raised by this study is whether MAP4 is indeed phosphorylated by cdc2 kinase at mitosis in vivo. We present here evidence that cdc2 kinase is the major M-phase MAP4 kinase, and, further, we identify two phosphorylation sites within the proline-rich domain of MAP4. Metabolic 32P labeling showed the increased phosphorylation of MAP4 at mitosis. A specific inhibitor of cdc2 kinase, butyrolactone I, inhibited phosphorylation of MAP4 both in mitotic HeLa cells and in the mitotic HeLa cell extract. The phosphopeptide map analysis revealed the high similarity of in vivo labeled mitotic MAP4 to that phosphorylated by cdc2 kinase in vitro. Ser-696 and Ser-787, both of which lie within SPXK consensus sequences for cdc2 kinase, were identified as phosphorylation sites in the proline-rich region of MAP4 in vivo and in vitro. Immunoblotting with antibodies that recognize the phosphorylation state of Ser-696 or Ser-787 showed that Ser-787 in the SPSK sequence was specifically phosphorylated at mitosis while Ser-696 in the SPEK sequence was phosphorylated both at mitosis and in interphase. These results suggest that cdc2 kinase directly regulates microtubule dynamics at mitosis through phosphorylation of MAP4 at a number of sites, including Ser-787.

4-Butyrolactone↗
PubMed1997

Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 regulates cytoskeletal organization and chemotaxis via catalytic and microtubule-specific interactions.

The extracellular signal-regulated kinases (ERKs) 1 and 2 are mitogen-activated protein kinases that act as key components in a signaling cascade linking growth factor receptors to the cytoskeleton and the nucleus. ERK2 mutants have been used to alter cytoskeletal regulation in Chinese hamster ovary cells without affecting cell growth or feedback signaling. Mutation of the unique loop L6 (residues 91-95), which is in a portion of the molecule that is cryptic upon the binding of ERK2 to the microtubules (MTs), generated significant morphological alterations. Most notable phenotypes were observed after expression of a combined mutant incorporating changes to both L6 and the TEY phosphorylation lip, including a 70% increase in cell spreading. Actin stress fibers in these cells, which normally formed a single broad parallel array, were arranged in three or more orientations or in fan-like arrays. MTs, which ordinarily extend longitudinally from the centrosome, spread radially, covering a larger surface area. Single, but not the double, mutations of the Thr and Tyr residues of the TEY phosphorylation lip caused a ca. 25% increase in cell spreading, accompanied by a threefold increase in chemotactic cell migration. Mutation of Lys-52 triggered a 48% increase in cell spreading but no alteration to chemotaxis. These findings suggest that wild-type ERK2 inhibits the organization of the cytoskeleton, the spreading of the cell, and chemotactic migration. This involves control of the orientation of actin and MTs and the positioning of focal adhesions via regulatory interactions that may occur on the MTs.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Overexpression of full- or partial-length MAP4 stabilizes microtubules and alters cell growth.

To investigate the in vivo functions of MAP4, a microtubule-associated protein expressed almost ubiquitously in vertebrate cells, we prepared stably transfected clonal mouse Ltk- cell lines expressing full-length MAP4 (L-MAP4 cells) or its MT-binding domain (L-MTB cells). Although transfectants showed no dramatic defect in morphology, organellar distribution, or level of MT polymer, as compared to naive Ltk- cells or L-MOCK cells (transfected with vector alone), MTs in L-MAP4 and L-MTB cells showed greater stability than those in control cells, as monitored by the level of post-translationally detyrosinated alpha-tubulin and by a quantitative nocodazole-resistance assay. In vivo, the MT-binding domain of MAP4 stabilized MTs less potently than full-length MAP4, in contrast to the equivalent efficacy demonstrated in studies of in vitro MT polymerization (Aizawa et al. (1991), J. Biol. Chem. 266, 9841-9846), L-MAP4 and L-MTB cells grew significantly more slowly than control cells; this growth inhibition was not due to mitotic arrest or cell death. L-MAP4 and L-MTB cells also exhibited greater tolerance to the MT-depolymerizing agent, nocodazole, but not to the MT-polymerizing agent, Taxol. Our results demonstrate that MAP4 and its MT-binding domain are capable of MT stabilization in vivo, and that increasing the intracellular level of MAP4 affects cell growth parameters.

Animals↗
PubMed1997

Overexpression of MAP4 inhibits organelle motility and trafficking in vivo.

We previously prepared cell lines that inducibly overexpress MAP4, a microtubule (MT)-associated protein widely expressed in non-neuronal cells. Overexpression of either the full-length MAP4 molecule or its MT-binding domain, MTB, stabilized MTs and retarded cell growth, suggesting that overexpressed MAP4 impacts on MT-dependent functions in vivo. To test this hypothesis, we examined MT-based vesicle movements in living cells, using high resolution DIC microscopy. Overexpression of either MAP4 or MTB yielded a dose-dependent reduction in the frequency of MT-dependent organelle movements, relative to control cells. At steady state, both MAP4- and MTB-overexpressing cells showed unusual distributions of transferrin, LDL, dextran, and Golgi elements, as compared to control cells. MAP4 preferentially inhibited receptor-dependent uptake and degradation of LDL, and repositioning of Golgi elements after disruption by the drug, brefeldin A. L-MOCK cells treated with Taxol to stabilize the MTs to an extent equivalent to MAP4 overexpression did not show similar inhibition of vesicle motility or organellar trafficking, suggesting that deficits in organelle movements in vivo represent a direct effect of the presence of MAP4 or MTB, rather than an indirect effect of the stabilization of MTs by overexpressed MAP constructs. Our results show that MAP4 has the capacity to affect transport along MTs in vivo; these findings suggest a potential mechanism by which MAP4 could contribute to polarization or morphogenesis of cells.

Biological Transport↗
PubMed1996

Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level.

Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT-dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system.

Animals↗
PubMed1995

Differential expression of alternatively spliced forms of MAP4: a repertoire of structurally different microtubule-binding domains.

We previously reported that the microtubule (MT)-binding domain of microtubule-associated protein 4 (MAP4) contains three 18-amino acid imperfect repeats that are homologous to the repeats found in the MT-binding domains of the neuronal MAPs, MAP2 and tau [Chapin, S. J., & Bulinski, J. C. (1991) J. Cell Sci. 98, 27-36]. Here we report the isolation of clones encoding additional isoforms of MAP4, which differ in the number of repeated elements contained within their MT-binding domains. In addition to clones encoding three repeats, we isolated clones encoding a four-repeat isoform, whose MT-binding domain bears a striking similarity to the four-repeat isoform of tau. Other MAP4 clones that we isolated encode five repeats. The additional repeat in the five-repeat isoform of MAP4 is quite unusual in its amino acid sequence; this unusual repeat was also found by Aizawa et al. [Aizawa, H., et al. (1990) J. Biol. Chem. 265, 13849-13855] among the repeats encoded by bovine MAP4 clones possessing four repeats. In humans, MAP4 was recently shown to be encoded by a single-copy gene [West, R. R., et al. (1991) J. Biol. Chem. 266, 21886-21896]; we demonstrated that the human MAP4 gene is located on human chromosome 3p21. Expression of multiple MAP4 isoforms from this gene, which appears to result from alternative RNA splicing, was investigated by RNase protection analysis of mammalian cell lines and rat tissues. The five-repeat isoform was the only form detectable in most cell lines, and it was the most abundant isoform expressed in rat lung, liver, kidney, spleen, and testis. However, in rat brain, heart, and skeletal muscle, although the five-repeat isoform was expressed at all developmental stages examined, the tau-like four-repeat isoform was also expressed, and its expression increased during development. The three-repeat isoform was expressed in heart and, to a lesser extent, in brain, skeletal muscle, and lung. Our results demonstrate that several different MAP4 isoforms are expressed in the rat in different tissues and at various developmental stages. Furthermore, our data suggest that differential expression of MAP4 isoforms possessing distinct MT-binding domains may be involved in the changes in MT dynamics or function that are known to accompany differentiation.

Alternative Splicing↗