Identification of a new microtubule-interacting protein Mip-90.
The interaction of different protein systems with microtubules is a critical step in the cellular function of these organelles. The family of microtube-associated proteins (MAPs) together with a set of motor proteins such as kinesin, cytosolic dynein and dynamin are among the most clear examples of microtubule-interacting proteins. In addition, an increasing number of recently discovered proteins have been shown to interact with microtubules, even though they do not remain associated after cycles of assembly and disassembly. By using affinity columns of agarose derivatized with peptides from the C-terminal regulatory domain on tubulin, we found a 90 kDa protein that interacts with tubulin and microtubules. This protein, here designated as Mip-90, was isolated from neuroblastoma N2A and HeLa cells. It was also identified in high-speed supernatants of the neuroblastoma N-115, and non-neuronal cell lines NIH 3T3, Huh-7, HTB-145 and SW-13 vim+. Mip-90 was able to specifically bind to affinity columns of the agarose-bound beta-II(422-434) and beta-II(434-443) tubulin peptides, containing the sequences of MAP binding domains on beta-II-tubulin. Specific antibodies to Mip-90 along with an anti-beta-tubulin antibody used in double immunofluorescence experiments revealed a striking colocalization of this protein with the microtubule network. Nocodazole-treated cells showed significant changes in Mip-90 distribution as correlated to disruption of the microtubule cytoskeleton. On the other hand, Mip-90 colocalized with microtubule bundles with a perinuclear distribution in HeLa cells treated with taxol. The binding of Mip-90 to microtubules was confirmed by cosedimentation experiments. This protein also exhibited a strong affinity for a calmodulin-agarose affinity matrix, and a preparation of Mip-90 isolated by this affinity procedure was able to promote in vitro tubulin assembly into microtubules. The capacity of Mip-90 to interact with microtubules and with calmodulin suggested functional similarities to tau proteins. However, Western blot analysis using a polyclonal antibody against this protein revealed no cross-reactivity of Mip-90 with tau components. In addition, the 90 kDa protein is a thermosensitive protein. On the other hand, site-directed antibodies that recognize a repetitive binding domain on tau, MAP-2 and MAP-4 failed to react with Mip-90. The studies suggest that Mip-90, a microtubule-interacting protein incorporates into microtubules in vitro, and may play a role in modulating microtubule assembly and organization in non-neuronal cells, thus contributing to the regulation of the dynamics of the cytoskeletal network.
González
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International Center for Cancer and Developmental Biology (ICC), Santiago/Chile.
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Maccioni
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D000595:Amino Acid Sequence; D002147:Calmodulin / Q000378:metabolism; D002846:Chromatography, Affinity; D003429:Cross Reactions; D003593:Cytoplasm / Q000737:chemistry; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009447:Neuroblastoma; D015739:Nocodazole / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism; D014407:Tumor Cells, Cultured; D016875:tau Proteins / Q000378:metabolism
D002147:Calmodulin; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D014404:Tubulin; D016875:tau Proteins; D017239:Paclitaxel; D015739:Nocodazole