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• W2000999799 abstract "We had previously described the leucine-rich acidic nuclear protein (LANP) as a candidate mediator of toxicity in the polyglutamine disease, spinocerebellar ataxia type 1 (SCA1). This was based on the observation that LANP binds ataxin-1, the protein involved in this disease, in a glutamine repeat-dependent manner. Furthermore, LANP is expressed abundantly in purkinje cells, the primary site of ataxin-1 pathology. Here we focused our efforts on understanding the neuronal properties of LANP. In undifferentiated neuronal cells LANP is predominantly a nuclear protein, requiring a bona fide nuclear localization signal to be imported into the nucleus. LANP translocates from the nucleus to the cytoplasm during the process of neuritogenesis, interacts with the light chain of the microtubule-associated protein 1B (MAP1B), and modulates the effects of MAP1B on neurite extension. LANP thus could play a key role in neuronal development and/or neurodegeneration by its interactions with microtubule associated proteins. We had previously described the leucine-rich acidic nuclear protein (LANP) as a candidate mediator of toxicity in the polyglutamine disease, spinocerebellar ataxia type 1 (SCA1). This was based on the observation that LANP binds ataxin-1, the protein involved in this disease, in a glutamine repeat-dependent manner. Furthermore, LANP is expressed abundantly in purkinje cells, the primary site of ataxin-1 pathology. Here we focused our efforts on understanding the neuronal properties of LANP. In undifferentiated neuronal cells LANP is predominantly a nuclear protein, requiring a bona fide nuclear localization signal to be imported into the nucleus. LANP translocates from the nucleus to the cytoplasm during the process of neuritogenesis, interacts with the light chain of the microtubule-associated protein 1B (MAP1B), and modulates the effects of MAP1B on neurite extension. LANP thus could play a key role in neuronal development and/or neurodegeneration by its interactions with microtubule associated proteins. Spinocerebellar ataxia type 1 (SCA1) 1The abbreviations used are: SCA1, spinocerebellar ataxia type 1; LANP, leucine-rich acidic nuclear protein; LRR, leucine-rich repeat; MAP, microtubule-associated protein; HA, hemagglutinin; IP, immunoprecipitation; NLS, nuclear localization signal; DAPI, 4′,6-diamidino-2-phenylindole. belongs to a group of disorders in which a polyglutamine expansion in the disease protein launches a cascade of events that causes relentless neurodegeneration. We had previously proposed that the leucine-rich acidic nuclear protein (LANP) stands out as a particularly appealing candidate mediator of toxicity in SCA1 based on its ability to interact with ataxin-1 in a glutamine repeat-dependent manner (1Matilla A. Koshy B. Cummings C.J. Isobe T. Orr H.T. Zoghbi H.Y. Nature. 1997; 389: 974-978Crossref PubMed Scopus (231) Google Scholar). Moreover, LANP is expressed at particularly high levels in purkinje cells, the seat of SCA1 pathology. Thus, one could envisage a scenario where the functions of LANP could be altered upon binding to ataxin-1, triggering downstream toxic events. This could also account for the regional toxicity of ataxin-1, despite its own ubiquitous expression. Since its first description in 1994, LANP has been implicated in myriad cellular functions from the cell surface to the nucleus. First described as a putative human leukocyte antigen class II-associated protein (and hence called PHAPI), it was suspected to be involved in signal transduction in lymphocytes (2Vaesen M. Barnikol-Watanabe S. Gotz H. Awni L.A. Cole T. Zimmermann B. Kratzin H.D. Hilschmann N. Biol. Chem. Hoppe-Seyler. 1994; 375: 113-126Crossref PubMed Scopus (95) Google Scholar). Matsuoka et al. (1994) independently described this protein in the developing cerebellum, and noting that it contained a leucine-rich repeat, called it by the acronym LANP. With a modular architecture reminiscent of a tadpole, LANP consists of a globular head formed by the N-terminal leucine-rich domain containing five leucine-rich repeats (LRR) and a C-terminal tail formed by the remaining length of acidic residues (3Matsuoka K. Taoka M. Satozawa N. Nakayama H. Ichimura T. Takahashi N. Yamakuni T. Song S.-Y. Isobe T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9670-9674Crossref PubMed Scopus (75) Google Scholar). As such, it belongs to a large and very interesting family of proteins that contain LRRs crucial for protein interactions, by forming a very characteristic secondary structure designed for protein-protein interactions (4Kobe B. Deisenhofer J. Trends Biochem. Sci. 1994; 19: 415-421Abstract Full Text PDF PubMed Scopus (1047) Google Scholar, 5Kobe B. Deisenhofer J. Nature. 1995; 374: 183-186Crossref PubMed Scopus (576) Google Scholar, 6Kobe B. Deisenhofer J. Curr. Opin. Struct. Biol. 1995; 5: 409-416Crossref PubMed Scopus (322) Google Scholar). It was therefore proposed to be a modulator of signaling pathways in cerebellar morphogenesis. LANP has since been implicated in a number of other functions: as a phosphorylated protein, LANP (known in this context as phosphoprotein 32 or pp32) was suggested to act as tumor suppressor (7Chen T.H. Brody J.R. Romantsev F.E. Yu J.G. Kayler A.E. Voneiff E. Kuhajda F.P. Pasternack G.R. Mol. Biol. Cell. 1996; 7: 2045-2056Crossref PubMed Scopus (78) Google Scholar, 8Bai J. Brody J.R. Kadkol S.S. Pasternack G.R. Oncogene. 2001; 20: 2153-2160Crossref PubMed Scopus (64) Google Scholar, 9Kadkol S.S. Brody J.R. Pevsner J. Bai J. Pasternack G.R. Nat. Med. 1999; 5: 275-279Crossref PubMed Scopus (5) Google Scholar, 10Kadkol S.S. El Naga G.A. Brody J.R. Bai J. Gusev Y. Dooley W.C. Pasternack G.R. Breast Cancer Res. Treat. 2001; 68: 65-73Crossref PubMed Scopus (26) Google Scholar); LANP has been shown to bind and shuttle the RNA-binding protein HuR, which is involved in RNA stability and transport. More recently it has been described as an inhibitor of histone acetylation and thus a transcriptional regulator (11Seo S.B. McNamara P. Heo S. Turner A. Lane W.S. Chakravarti D. Cell. 2001; 104: 119-130Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar) and in a very different role as a modulator of apoptosis (12Fan Z. Beresford P.J. Oh D.Y. Zhang D. Lieberman J. Cell. 2003; 112: 659-672Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar, 13Fan Z. Beresford P.J. Zhang D. Lieberman J. Mol. Cell. Biol. 2002; 22: 2810-2820Crossref PubMed Scopus (116) Google Scholar, 14Jiang X. Kim H.E. Shu H. Zhao Y. Zhang H. Kofron J. Donnelly J. Burns D. Ng S.C. Rosenberg S. Wang X. Science. 2003; 299: 223-226Crossref PubMed Scopus (353) Google Scholar). Ulitzur et al. (15Ulitzur N. Humbert M. Pfeffer S.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5084-5089Crossref PubMed Scopus (51) Google Scholar, 16Ulitzur N. Rancaño C. Pfeffer S.R. J. Biol. Chem. 1997; 272: 30577-30582Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) were the first to suggest that LANP may also have cytoplasmic functions: in biochemical assays it binds to the microtubule-associated proteins (MAPs) MAP2, MAP4, and tau, stimulating the microtubule- and dynein-dependent localization of the Golgi apparatus in semi-intact cells. Since it is unclear which of the functions of LANP, if any, might be perturbed in SCA1 pathogenesis, we sought to understand the neuronal properties of LANP. Here we report that LANP is typically nuclear in undifferentiated neuro2a cells. This compartmentalization is dependent on a nuclear localization signal in its C-terminal domain. LANP tends to be drawn to the cytoplasm during the process of neuronal differentiation. Intriquingly, in a search for LANP interacting proteins, we have identified the microtubule-associated protein, MAP1B, as a cytoplasmic protein that interacts with LANP. This interaction occurs via its light chain. Moreover the effects of MAP1B on neurite extension are altered by its interaction with LANP. This interaction has the potential to not only modulate neuritogenesis during neuronal development, but could also contribute to the loss of neurites and cytoarchitectural disarray seen in SCA1. Yeast Two-hybrid Plasmids and Yeast Strains—Full-length mouse LANP cDNA was subcloned into the NcoI-SmaI sites of the yeast two-hybrid bait plasmid pGBKT7, and the plasmid DNA was transformed into yeast strain AH109 (Clontech). Transformed AH109 yeast strains containing the LANP bait plasmid were mated to the Y187 yeast strain pretransformed with a mouse brain pACT2 library. We used high stringency auxotrophic selection (using media deficient in adenine and histidine) to select for interacting clones. Positive clones were also tested for beta galactosidase activity in an overlay assay for the interaction of the bait and prey (library clone) interactions independently. To delimit the region of interaction of LANP we generated N- and C-terminally deleted LANP constructs. The C-terminally deleted LANP construct was engineered by digesting pGBK-T7 LANP with BsmI-SmaI to remove the C-terminal acidic domain, preserving the complete LRR region and then religating the backbone. To generate the N-terminally deleted construct we used a PCR-based strategy to clone a truncated LANP (residues 129–247) into the NdeI-SmaI sites of pGBK-T7. Cell Culture, Transfection, Expression Constructs, and Immunofluorescence—Clones derived from the pACT2 yeast two-hybrid screen were subcloned into the mammalian expression vector pCMV-HA (Clontech). The construction of the mammalian tet-responsive expression plasmids (pMT5tet and pMT17tet) containing the heavy chain and full-length clones of rat MAP1B tagged at their C-terminal ends to the myc epitope has been described (17Togel M. Wiche G. Propst F. J. Cell Biol. 1998; 143: 695-707Crossref PubMed Scopus (135) Google Scholar). The nuclear localization signal mutants of LANP were constructed by a PCR based mutagenesis strategy so as to alter residues 236 and 237 to alanine residues (from lysine and argine respectively) followed by subcloning into pCMV-myc (Clontech). Tissue culture cells were obtained from American Type Tissue Collection (Manassas, VA). Neuro2a was used as a prototypical neuronal cell line. Differentiation of neuro2a cells was performed by growing cells in serum-free medium (Opti-MEM, Invitrogen) containing 0.3 mm dibutyryl cAMP (18De Girolamo L.A. Billett E.E. Hargreaves A.J. J. Neurochem. 2000; 75: 133-140Crossref PubMed Scopus (20) Google Scholar). For experiments on non-neuronal cells, COS-7 cells or BHK-21 cells (a kind gift of Dr. O. Skalli, University of Illinois) were used. To quantify neuritogenesis, cells were counted as having extended neurites if they exhibited at least one process longer than two cell bodies in length. Transfections were performed on coverslips using the LipofectAMINE Plus reagent (Invitrogen). Cells were fixed 48 h post-transfection before being processed for immunofluorescence (17Togel M. Wiche G. Propst F. J. Cell Biol. 1998; 143: 695-707Crossref PubMed Scopus (135) Google Scholar). All images were captured by either light or confocal laser-scanning microscopy (Zeiss). Images were manipulated using Adobe Photoshop 5.0. Antibodies—Anti-LANP antibody (antibody 3118) was generated by immunizing goat with bacterially expressed full-length LANP as a glutathione S-transferase fusion protein expressed and purified after subcloning into the bacterial expression vector pGEX5X3 (Amersham Biosciences). Anti-MAP1B C-20 antibody recognizes the light chain of MAP1B (Santa Cruz Biotechnology). The following antibodies to epitope tags were used: anti-myc epitope, clone 9E10 (Sigma); anti-FLAG, clone m2 or polyclonal F7425 (Sigma); and anti-HA, clone HA.11 (Covance); dilutions of 1:100 for immunofluorescence and 1:1000 for Western blotting. Co-immunoprecipitation—Cells were transfected at ∼80% confluence on 150-mm dishes using 50 μg of DNA and LipofectAMINE Plus reagent (Invitrogen). The light chain of MAP1B was expressed as an HA-tagged fusion using the vector pCMV-HA; LANP was expressed as a FLAG-tagged fusion in pFLAG CMV-2. Two days post-transfection cells were washed twice with phosphate-buffered saline and then lysed in 3 ml of lysis buffer: phosphate-buffered saline, 0.5% Nonidet P-40, 5 mm EDTA, and protease inhibitors (Complete, Roche Applied Science) using the protocol recommended in the antibodies protocol guide using the relevant antibody or nonspecific immunoglobulins as controls (Clontech). Anti-HA immunoprecipitation (IP) was performed with anti-HA beads (Sigma); anti-LANP IP was performed by protein G beads coupled to FLAG M2 and 3118. LANP Is a Nucleo-cytoplasmic Shuttling Protein That Translocates from the Nucleus to the Cytoplasm upon Differentiation—LANP has been described as both a nuclear and a cytoplasmic protein (1Matilla A. Koshy B. Cummings C.J. Isobe T. Orr H.T. Zoghbi H.Y. Nature. 1997; 389: 974-978Crossref PubMed Scopus (231) Google Scholar, 16Ulitzur N. Rancaño C. Pfeffer S.R. J. Biol. Chem. 1997; 272: 30577-30582Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 19Brennan C.M. Gallouzi I.E. Steitz J.A. J. Cell Biol. 2000; 151: 1-14Crossref PubMed Scopus (313) Google Scholar). Brennan et al. (19Brennan C.M. Gallouzi I.E. Steitz J.A. J. Cell Biol. 2000; 151: 1-14Crossref PubMed Scopus (313) Google Scholar, 20Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1741) Google Scholar) had earlier shown than in non-neuronal HeLa cells, LANP is a nucleocytoplasmic shuttling protein that interacts with the nuclear export factor CRM1, presumably via its leucine-rich domains, two of which are consensus sequences for the leptomycin-sensitive nuclear export signals, seen in rev and other shuttling proteins. Although it is not possible to mutate these putative nuclear export signal regions without affecting the conserved leucine-rich domains, the defining feature of the family of LRR proteins (6Kobe B. Deisenhofer J. Curr. Opin. Struct. Biol. 1995; 5: 409-416Crossref PubMed Scopus (322) Google Scholar), the existence of these export signals seems likely based on the ability of leptomycin B to inhibit shuttling of LANP. LANP also bears a putative nuclear localization signal (NLS) in its C-terminal acidic domain. This is a four-amino acid stretch of basic residues seen in LANP spanning residues 234–237 (lysine-arginine-lysinearginine). In a mouse LANP-like protein, this sequence when tagged to green fluorescent protein is sufficient to induce the nuclear localization of green fluorescent protein suggesting that this stretch of amino acids can function as an NLS (21Matsubae M. Kurihara T. Tachibana T. Imamoto N. Yoneda Y. FEBS Lett. 2000; 468: 171-175Crossref PubMed Scopus (15) Google Scholar). To test whether this sequence is indeed the NLS for LANP and whether these residues are necessary for the nuclear localization of LANP, we mutated the second lysine and arginine residues of the this quartet to alanine residues. This mutant form of LANP (henceforth called LANP-KRAA) remains mainly cytoplasmic even when co-expressed with wild type LANP (that remains predominantly nuclear) (Fig. 1). This experiment also demonstrates the inability of mutant LANP to piggy-back on its wild type counterpart and enter the nucleus. Such a scenario might have been expected based on the ability of LANP to self-associate existing as dimers and trimers as has been reported previously (16Ulitzur N. Rancaño C. Pfeffer S.R. J. Biol. Chem. 1997; 272: 30577-30582Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Since LANP is a developmentally regulated protein in neuronal cells, with maximum abundance in the early postnatal life, we sought to determine whether its localization is altered depending on the process of differentiation, as has been observed for other proteins expressed at high levels during development (22Wakamatsu Y. Watanabe Y. Shimono A. Kondoh H. Neuron. 1993; 10: 1-9Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 23Carlock L. Vo T. Lorincz M. Walker P.D. Bessert D. Wisniewski D. Dunbar J.C. Brain Res. Mol. Brain Res. 1996; 42: 202-212Crossref PubMed Scopus (12) Google Scholar). In undifferentiated neuro2a cells, LANP typically is nuclear with little cytoplasmic staining. Upon differentiation by dbcAMP in the presence of low serum (18De Girolamo L.A. Billett E.E. Hargreaves A.J. J. Neurochem. 2000; 75: 133-140Crossref PubMed Scopus (20) Google Scholar), LANP tends to be diffuse cytoplasmic, particularly in those cells with the most extensive neurites (Fig. 2, top panel). In addition to staining endogenous LANP with an LANP specific antibody, we also transfected epitope-tagged LANP into neuro2a cells so as to follow localization by an antibody specific to the epitope tag to rule out the possibility of nonspecific staining. Immunofluorescence microscopy once again revealed a dramatic alteration in LANP localization from the nucleus to the cytoplasm, often to the extent that the nucleus became completely devoid of LANP staining (Fig. 2, bottom panel). Approximately 80% of undifferentiated cells showed nuclear staining, while upon differentiation roughly the same percentage showed a cytoplasmic staining pattern for LANP. This translocation suggests that LANP shifts from the nucleus to the cytoplasm during neuritogenesis, where its cytoplasmic function(s) may be more critical. LANP Interacts with MAP1B—To further delineate the neuronal properties of LANP, we decided to search for LANP interacting proteins expressed in neurons. To this end we performed a yeast two-hybrid screen using a mouse brain library and full-length LANP as bait. We identified MAP1B as a potential interacting partner. Three representative library clones of MAP1B that were fished out by the two-hybrid screening are shown in the schematic in relationship to the sequence of the rat MAP1B construct (Fig 3A). We also pulled out several clones corresponding to the C-terminal domain of MAP1A. These interacting clones were in general short. This result was not surprising in view of the fact that MAP1B and A share extensive similarity in their primary structure (80% identity in their last 100 amino acids). Intriguingly we also identified a tau isoform (GenBank™ accession number U12916) as an interacting bait. Since LANP had been demonstrated to interact with tau in biochemical assays we did not pursue the interaction of LANP with tau (15Ulitzur N. Humbert M. Pfeffer S.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5084-5089Crossref PubMed Scopus (51) Google Scholar, 16Ulitzur N. Rancaño C. Pfeffer S.R. J. Biol. Chem. 1997; 272: 30577-30582Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 24Itin C. Ulitzur N. Muhlbauer B. Pfeffer S.R. Mol. Biol. Cell. 1999; 10: 2191-2197Crossref PubMed Scopus (57) Google Scholar). Of our microtubule-associated interactors, we decided to pursue the MAP1B interaction further, as it would indicate that LANP has the intriguing property of binding to all classes of structural microtubule-associated proteins. MAP1B, along with MAP1A, constitute the family of large molecular weight microtubule-associated proteins. Both these proteins have shared domains in addition to the C-terminal tail, some of which contain canonical repeats (Lys-Lys-Glu-X) involved in binding to microtubules (25Noble M. Lewis S.A. Cowan N.J. J. Cell Biol. 1989; 109: 3367-3376Crossref PubMed Scopus (262) Google Scholar, 26Zauner W. Kratz J. Staunton J. Feick P. Wiche G. Eur. J. Cell Biol. 1992; 57: 66-74PubMed Google Scholar). The proteins are also processed similarly, undergoing proteolytic cleavage of a single precursor to produce a light chain and a heavy chain (27Hammarback J.A. Obar R.A. Hughes S.M. Vallee R.B. Neuron. 1991; 7: 129-139Abstract Full Text PDF PubMed Scopus (93) Google Scholar, 28Langkopf A. Hammarback J.A. Muller R. Vallee R.B. Garner C.C. J. Biol. Chem. 1992; 267: 16561-16566Abstract Full Text PDF PubMed Google Scholar). In the case of MAP1B the heavy chain is 243 kDa, while the light chain is 27 kDa; the cleavage site has been narrowed to within 40 residues of a proline-rich hydrophobic domain of the full-length MAP1B (29Togel M. Eichinger R. Wiche G. Propst F. FEBS Lett. 1999; 451: 15-18Crossref PubMed Scopus (16) Google Scholar). One of our clones (clone 3-87; 242 residues) corresponded almost exactly to the complete light chain of MAP1B, beginning four residues downstream from the predicted cleavage event. This clone was the strongest interactor in our yeast two-hybrid screen, interacting more robustly with LANP than the other MAP1B clones picked up in the yeast two-hybrid that were either longer (clone 3-75, 559 residues) or shorter (clone 3-3, 168 residues) than the light chain. This suggests that LANP binds to the light chain per se (Fig. 3A). Since we were keen to determine which domain of LANP mediates the interaction of LANP with MAP1B, we generated N- and C-terminal deletions of LANP. Using yeast two-hybrid assays we were able to demonstrate that the acidic C-terminal domain of LANP interacted more robustly with the light chain of MAP1B than did the N-terminal domain of LANP, the domain bearing the LRRs (Fig. 3B). Incidentally, it is the C-terminal domain that bears the nuclear localization signal. It should be mentioned in the context of discussing the domain structure of LANP that it is the N-terminal region, i.e. the leucine-rich region, that is responsible for the interaction of LANP with ataxin-1 (1Matilla A. Koshy B. Cummings C.J. Isobe T. Orr H.T. Zoghbi H.Y. Nature. 1997; 389: 974-978Crossref PubMed Scopus (231) Google Scholar). LANP Interacts with the Light Chain of MAP1B in Vivo— Since the majority of the MAP1B fragments isolated by the yeast two-hybrid screen correspond to the C-terminal domain of the MAP1B precursor that eventually becomes the light chain, we sought to test the idea that the interaction of LANP is specific to the light chain of MAP1B. We first performed immunfluorescence studies to look at the subcellular localization of LANP in the presence of the MAP1B light chain (Fig. 4). When transfected alone, LANP localized predominantly to the nucleus in ∼80% of the cells. In the small percentage of COS-7 cells in which LANP is cytoplasmic, the staining is either diffuse or slightly vesicular, as was reported for CHO cells (Fig. 4A) (16Ulitzur N. Rancaño C. Pfeffer S.R. J. Biol. Chem. 1997; 272: 30577-30582Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Incidentally, in our own studies using CHO cells, we found that LANP (transfected or endogenous) displays a predominant nuclear staining in addition to the lower intensity cytoplasmic staining (data not shown). Discrepancy with earlier findings might relate to the fact that the antibody used by Ulitzur et al. (16Ulitzur N. Rancaño C. Pfeffer S.R. J. Biol. Chem. 1997; 272: 30577-30582Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) recognizes only a subset of LANP in cells based on post-translational modifications (for instance phosphorylation). When co-tranfected with the MAP1B light chain, however, LANP relocated to the cytoplasm in ∼80% of COS-7 cells. This relocation to the cytoplasm was dramatic and in most of the cells LANP tends to be diffuse and cytoplasmic. However, in a few cells the LANP distribution was clearly fibrillar, with both MAP1B and LANP, co-localizing in a perinuclear filamentous pattern. This pattern reflects the bundled and collapsed microtubule networks described during earlier studies on its role in organizing tubulin networks (Ref. 17Togel M. Wiche G. Propst F. J. Cell Biol. 1998; 143: 695-707Crossref PubMed Scopus (135) Google Scholar and our own data not shown using anti-tubulin antibodies) (Fig. 4). This suggests not only that the light chain of MAP1B interacts with LANP, but that LANP can bind at least to some extent with the microtubule-bound pool of the MAP1B light chain. In undifferentiated neuro2a cells this relocation to the cytoplasm is even more dramatic with more than 90% showing a cytoplasmic distribution of LANP when MAP1B light chain is overexpressed, reminiscent of the translocation of LANP seen during the process of differentiation. We next sought co-IP evidence. In Fig. 4H we demonstrated that we can co-immunoprecipitate the MAP1B light chain using antibodies targeted to LANP and also in the reverse direction, i.e. co-IP LANP when using antibodies directed against MAP1B light chain. The interaction between the two is thus fairly robust. Finally, to determine whether the preference of LANP for the light chain holds in cells, we used tagged constructs of MAP1B corresponding to the full-length precursor and the heavy chain of rat MAP1B (∼95% identity to mouse MAP1B) (Fig. 5 schematic) and transfected cells with FLAG LANP along side either full-length MAP1B or just with its heavy chain. We discovered that, much like the light chain of MAP1B, full-length MAP1B also causes a dramatic shift of staining from the nucleus to the cytoplasm, with a majority of cells (close to 70%) showing a cytoplasmic staining (Fig. 5). This was to be expected, since full-length MAP1B is cleaved into its heavy and light chain in both neuronal and non-neuronal cells (17 and data not shown). In contrast, expressing the heavy chain alone causes a less dramatic transition from nuclear to cytoplasmic staining, although it did increase the total number of cells showing a cytoplasmic staining from ∼20% to 40%. We speculate that this increase might be due to alterations in the dynamics of the endogenous light chain caused by the abundant quantities of newly introduced heavy chain, although it is possible that there is an interaction albeit less robust with the heavy chain as well. This is conceivable since the heavy chain contains a microtubule binding domain consisting of several KKEX and KKEE motifs, thought to contribute basic properties to the heavy chain and that could also potentially interact with the acidic tail domain of LANP (25Noble M. Lewis S.A. Cowan N.J. J. Cell Biol. 1989; 109: 3367-3376Crossref PubMed Scopus (262) Google Scholar). LANP Modulates the Functions of MAP1B in Neuritogenesis—Recent evidence suggests that the ratio of light chain to heavy chain is under strict developmental control, mediated by differential proteolysis and clearance of these fragments to keep the light and heavy chains in a 6:1 to 8:1 molar ratio (30Mei X. Sweatt A.J. Hammarback J.A. J. Neurosci. Res. 2000; 62: 56-64Crossref PubMed Scopus (2) Google Scholar). We, therefore, tested whether different fragments of MAP1B might have differential effects on neurite outgrowth in neuro2a cells and whether the binding of LANP to the light chain could thereby affect neuritogenesis. Full-length MAP1B, the light chain and the heavy chain each, had a different effect on the ability of neuro2a cells to extend neurites (Fig. 6). Although full-length MAP1B has been previously shown to have only modest effects on microtubule stability in non-neuronal cells (31Takemura R. Okabe S. Umeyama T. Kanai Y. Cowan N.J. Hirokawa N. J. Cell Sci. 1992; 103: 953-964Crossref PubMed Google Scholar, 32Goold R.G. Owen R. Gordon-Weeks P.R. J. Cell Sci. 1999; 112: 3373-3384Crossref PubMed Google Scholar), it dramatically altered the morphology of transfected neuro2a cells. These cells became rounded (more than 80%) and were incapable of spreading out or extending neurites even after stimulation with dbcAMP, a potent inducer of neuritogenesis (up to 4 days after induction). Neither the light chain nor the heavy chain alone had such a significant effects on neuritic morphology (Fig. 6), although we noticed that the heavy chain had a greater inhibitory effect on neuritogenesis when compared with the light chain (80 and 60% of cells transfected with the light or heavy chain, respectively, were able to extend neurites). Thus, it appears that both the light chain and the heavy chain are presumably required to stabilize the microtubule cytoskeleton at the expense of neuritogenesis. This inhibitory effect on neuritogenesis by full-length MAP1B now allowed us to test whether LANP modulates the ability of full-length MAP1B to suppress neurite formation induced by dbcAMP (Fig. 7). Co-transfecting LANP with full-length MAP1B significantly increased the ability of the cells to form neurites. On the other hand, LANP did not affect the ability of cells to express neurites when transfected with either the heavy chain or light chain alone, suggesting that the alleviation on the inhibition of neuritogenesis is because of its interaction with the MAP1B complex. Although it is clear that SCA1 is caused by an expansion of glutamine repeats in the disease-causing protein ataxin-1, precisely how this repeat expansion translates into neurotoxic events is still an unanswered question. The experiments in this study were driven by the rationale that understanding the interactions and functions of proteins that may be sequestered by ataxin-1 might point to possible candidate toxic scenarios. LANP is one such ataxin-1 interacting protein that interacts more strongly with mutant ataxin-1 than its wild type counterpart, is expressed at higher levels in neurons that tend to be affected in SCA1, and has been shown to be redistributed into ataxin-1 inclusions (1Matilla A. Koshy B. Cummings C.J. Isobe T. Orr H.T. Zoghbi H.Y. Nature. 1997; 389: 974-978Crossref PubMed Scopus (231) Google Scholar). We therefore sought to identify the neuronal properties of LANP as a first step toward understanding its possible role in SCA1 pathogenesis. We found that, during neuronal differentiation, LANP translocates from the nucleus to the cytoplasm, where at least one of its interacting proteins is the light chain of MAP1B. Moreover LANP influences the modulatory role of MAP1B on neurite exte" @default.
• W2000999799 created "2016-06-24" @default.
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• W2000999799 date "2003-09-01" @default.
• W2000999799 modified "2023-10-03" @default.
• W2000999799 title "Mapmodulin/Leucine-rich Acidic Nuclear Protein Binds the Light Chain of Microtubule-associated Protein 1B and Modulates Neuritogenesis" @default.
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