Microtubule-associated Protein Light Chain 3B Research Articles

Interaction of microtubule‐associated protein 1B light chain (MAP1B‐LC1) and p53 represses transcriptional activity of p53

The tumor suppressor and transcription factor p53 is a key modulator of cellular stress responses, and can trigger apoptosis in many cell types including neurons. In this study, we have shown that Microtubule‐associated protein 1B (MAP1B) light chain interacts with tumor suppressor p53. MAP1B is one of the major cytoskeletal proteins in the developing nervous system and essential in forming axons during elongation. We also demonstrate that both p53 and MAP1B‐LC1 interact in the nucleus in HEK 293 cells. Indeed, we show that the MAP1B‐LC1 negatively regulates p53‐dependent transcriptional activity of a reporter containing the p21 promoter. Consequently, MAP1B light chain binds with p53 and their interaction leads to the inhibition of doxorubicin‐induced apoptosis in HEK 293 cells. Furthermore, these examinations might be taken into consideration when knock‐down of MAP1B‐LC1 is used as a cancer therapeutic strategy to enhance p53's apoptotic activity in chemotherapy.

Rare Missense and Synonymous Variants in UBE1 Are Associated with X-Linked Infantile Spinal Muscular Atrophy

X-linked infantile spinal muscular atrophy (XL-SMA) is an X-linked disorder presenting with the clinical features hypotonia, areflexia, and multiple congenital contractures (arthrogryposis) associated with loss of anterior horn cells and infantile death. To identify the XL-SMA disease gene, we performed large-scale mutation analysis in genes located between markers DXS8080 and DXS7132 (Xp11.3-Xq11.1). This resulted in detection of three rare novel variants in exon 15 of UBE1 that segregate with disease: two missense mutations (c.1617 G-->T, p.Met539Ile; c.1639 A-->G, p.Ser547Gly) present each in one XL-SMA family, and one synonymous C-->T substitution (c.1731 C-->T, p.Asn577Asn) identified in another three unrelated families. Absence of the missense mutations was demonstrated for 3550 and absence of the synonymous mutation was shown in 7914 control X chromosomes; therefore, these results yielded statistical significant evidence for the association of the synonymous substitution and the two missense mutations with XL-SMA (p = 2.416 x 10(-10), p = 0.001815). We also demonstrated that the synonymous C-->T substitution leads to significant reduction of UBE1 expression and alters the methylation pattern of exon 15, implying a plausible role of this DNA element in developmental UBE1 expression in humans. Our observations indicate first that XL-SMA is part of a growing list of neurodegenerative disorders associated with defects in the ubiquitin-proteasome pathway and second that synonymous C-->T transitions might have the potential to affect gene expression.

Microtubule-Associated Protein 1B-Light Chain 1 Enhances Activation of Rap1 by Exchange Protein Activated by Cyclic AMP but Not Intracellular Targeting

We have previously demonstrated that EPAC1 interacts with light chain (LC) 2 of microtubule-associated protein (MAP) 1A. In the present study, we investigated whether the structurally related LC1 of MAP1B also interacts with EPAC1. We demonstrate that LC1 copurifies with EPAC1 from extracts of PC-12 cells, using cyclic AMP-agarose. Using recombinant LC1 and LC2 in pull-down and solid phase binding assays, we demonstrate direct interaction with a glutathione S-transferase-fusion of the cyclic AMP-binding (CAMP) domain of EPAC1. We also tested whether LC1 directed intracellular targeting of EPAC1 through its interaction with the CAMP domain. EPAC1 was found be in the soluble and particulate, nuclear/perinuclear fractions of cells. We found that the catalytic (CAT) domain of EPAC1, and not the CAMP domain, was responsible for recruitment to the nuclear/perinuclear fraction of cells. The targeting sequence responsible was located between amino acids 764 and 838 of EPAC1. Overexpresssion of an isolated CAT domain in COS1 cells was found to displace endogenous EPAC1 from the nuclear/perinuclear fraction, thereby inhibiting EPAC-activated Rap1 in this compartment. In contrast, LC1 was not able to compete for the binding of EPAC1 to this fraction. LC1, however, was able to enhance interaction of EPAC1 with cyclic AMP and heightened the ability of EPAC to activate Rap1. Antibody disruption of EPAC1/LC1 interaction in PC-12 cells ablated the ability of cyclic AMP to activate Rap1. LC1 is therefore not involved in intracellular targeting of EPAC1, but it is rather a molecular chaperone of EPAC activity toward Rap1.

Gigaxonin-controlled degradation of MAP1B light chain is critical to neuronal survival

Giant axonal neuropathy (GAN) is a devastating sensory and motor neuropathy caused by mutations in the GAN gene, which encodes the ubiquitously expressed protein gigaxonin. Cytopathological features of GAN include axonal degeneration, with accumulation and aggregation of cytoskeletal components. Little is currently known about the molecular mechanisms underlying this recessive disorder. Here we show that gigaxonin controls protein degradation, and is essential for neuronal function and survival. We present evidence that gigaxonin binds to the ubiquitin-activating enzyme E1 through its amino-terminal BTB domain, while the carboxy-terminal kelch repeat domain interacts directly with the light chain (LC) of microtubule-associated protein 1B (MAP1B). Overexpression of gigaxonin leads to enhanced degradation of MAP1B-LC, which can be antagonized by proteasome inhibitors. Ablation of gigaxonin causes a substantial accumulation of MAP1B-LC in GAN-null neurons. Moreover, we show that overexpression of MAP1B in wild-type cortical neurons leads to cell death characteristic of GAN-null neurons, whereas reducing MAP1B levels significantly improves the survival rate of null neurons. Our results identify gigaxonin as a ubiquitin scaffolding protein that controls MAP1B-LC degradation, and provide insight into the molecular mechanisms underlying human neurodegenerative disorders.

Regulation of microtubule‐associated protein 1B (MAP1B) subunit composition

The MAP1B and MAP1A genes each produce an mRNA that encodes a polyprotein. When cleaved, each polyprotein yields a single heavy chain and a single light chain, which become noncovalently associated. In previous work, it was found that the MAP1B light chains and heavy chains exist in a 2:1 ratio. Through use of quantitative immunoblot techniques, this finding was further examined in the developing rat brain. MAP1B heavy chain (HC) and light chain (LC1), as well as the light chain of MAP1A (LC2), were prepared in purified form for use as standards and/or immunogens for generation of antibodies for immunoblotting. Brain homogenates and microtubule-enriched fractions from developing rats were assayed for MAP1B HC and LC1 content. Results indicated that postnatal rat brain homogenates contain LC1 in a 6:1 to 8:1 molar ratio to the MAP1B HC. Purified microtubules also contain LC1 in excess of MAP1B HC, but at a ratio of 2:1. We propose that most of the excess LC1 in homogenates is either insoluble or not bound to microtubules. The findings raise the possibility of a function for the “excess” LC1 that does not require association with MAP1 HC and/or microtubules. Given a synthetic mechanism that produces MAP1B HC and LC1 in a 1:1 ratio at both transcription and translation steps, we propose that the “excess” LC1 in brain homogenates is a consequence of LC1 having a greater half-life than the MAP1B HC. Consistently with this hypothesis, a major pool of MAP1B HC is rapidly degraded after blocking protein synthesis with cycloheximide, whereas LC1 levels remain constant even after 24 hr of cycloheximide treatment. J. Neurosci. Res. 62:56–64, 2000. © 2000 Wiley-Liss, Inc.

Regulation of microtubule-associated protein 1B (MAP1B) subunit composition.

The MAP1B and MAP1A genes each produce an mRNA that encodes a polyprotein. When cleaved, each polyprotein yields a single heavy chain and a single light chain, which become noncovalently associated. In previous work, it was found that the MAP1B light chains and heavy chains exist in a 2:1 ratio. Through use of quantitative immunoblot techniques, this finding was further examined in the developing rat brain. MAP1B heavy chain (HC) and light chain (LC1), as well as the light chain of MAP1A (LC2), were prepared in purified form for use as standards and/or immunogens for generation of antibodies for immunoblotting. Brain homogenates and microtubule-enriched fractions from developing rats were assayed for MAP1B HC and LC1 content. Results indicated that postnatal rat brain homogenates contain LC1 in a 6:1 to 8:1 molar ratio to the MAP1B HC. Purified microtubules also contain LC1 in excess of MAP1B HC, but at a ratio of 2:1. We propose that most of the excess LC1 in homogenates is either insoluble or not bound to microtubules. The findings raise the possibility of a function for the "excess" LC1 that does not require association with MAP1 HC and/or microtubules. Given a synthetic mechanism that produces MAP1B HC and LC1 in a 1:1 ratio at both transcription and translation steps, we propose that the "excess" LC1 in brain homogenates is a consequence of LC1 having a greater half-life than the MAP1B HC. Consistently with this hypothesis, a major pool of MAP1B HC is rapidly degraded after blocking protein synthesis with cycloheximide, whereas LC1 levels remain constant even after 24 hr of cycloheximide treatment.

Regulation of microtubule-associated protein 1B (MAP1B) subunit composition

The MAP1B and MAP1A genes each produce an mRNA that encodes a polyprotein. When cleaved, each polyprotein yields a single heavy chain and a single light chain, which become noncovalently associated. In previous work, it was found that the MAP1B light chains and heavy chains exist in a 2:1 ratio. Through use of quantitative immunoblot techniques, this finding was further examined in the developing rat brain. MAP1B heavy chain (HC) and light chain (LC1), as well as the light chain of MAP1A (LC2), were prepared in purified form for use as standards and/or immunogens for generation of antibodies for immunoblotting. Brain homogenates and microtubule-enriched fractions from developing rats were assayed for MAP1B HC and LC1 content. Results indicated that postnatal rat brain homogenates contain LC1 in a 6:1 to 8:1 molar ratio to the MAP1B HC. Purified microtubules also contain LC1 in excess of MAP1B HC, but at a ratio of 2:1. We propose that most of the excess LC1 in homogenates is either insoluble or not bound to microtubules. The findings raise the possibility of a function for the "excess" LC1 that does not require association with MAP1 HC and/or microtubules. Given a synthetic mechanism that produces MAP1B HC and LC1 in a 1:1 ratio at both transcription and translation steps, we propose that the "excess" LC1 in brain homogenates is a consequence of LC1 having a greater half-life than the MAP1B HC. Consistently with this hypothesis, a major pool of MAP1B HC is rapidly degraded after blocking protein synthesis with cycloheximide, whereas LC1 levels remain constant even after 24 hr of cycloheximide treatment.

Novel features of the light chain of microtubule-associated protein MAP1B: microtubule stabilization, self interaction, actin filament binding, and regulation by the heavy chain.

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 revealed moderate effects on microtubule stability. Here we have analyzed the effects of the MAP1B light chain in the absence or presence of the heavy chain by immunofluorescence microscopy of transiently transfected cells. Distinct from all other MAPs, the MAP1B light chain-induced formation of stable but apparently flexible microtubules resistant to the effects of nocodazole and taxol. Light chain activity was inhibited by the heavy chain. In addition, the light chain was found to harbor an actin filament binding domain in its COOH terminus. By coimmunoprecipitation experiments using epitope-tagged fragments of MAP1B we showed that light chains can dimerize or oligomerize. Furthermore, we localized the domains for heavy chain-light chain interaction to regions containing sequences homologous to MAP1A. Our 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.