Identification of the binding region of basic calponin on alpha and beta tubulins.
Calponin is a basic smooth muscle protein capable of binding to actin, calmodulin, tropomyosin, and phospholipids. We have found that the basic calponin interacted with brain tubulin under polymerized and unpolymerized conditions in vitro [Fujii, T., Hiromori, T., Hamamoto, M., and Suzuki, T. (1997) J. Biochem. 122, 344-351]. We examined the calponin-binding site on the tubulin molecule by sedimentation, limited digestion, chemical-cross linking, immunoblotting, and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometric (DE MALDI-TOF) analyses. Calponin interacts with both the alpha and beta tubulins and only slightly with the tyrosinated and acetylated form of alpha tubulin. The binding of calponin to microtubules was blocked by adding poly(L-aspartic acid) (PLAA) or MAP2. After digestion of microtubule proteins with subtilisin, the amount of calponin binding to alphabetas microtubules was reduced compared to native microtubules, but no further reduction was observed in the case of alphasbetas microtubules. The chemical cross-linked products of calponin and synthesized peptides (KDYEEVGVDSVEGE; alpha-KE) derived from the C-terminal region of alpha tubulin and (YQQYQDATADEQG; beta-YG) and (GEFEEEGEEDEA; beta-GA) from that of beta tubulin were detected by mass spectrometry. One kind of calponin-peptide complex was formed in the presence of alpha-KE or beta-YG, while five complexes (calponin:peptide = 1:1-5) were generated in the presence of beta-GA. Peptides alpha-KE and beta-GA inhibited the binding of calponin to tubulin produced by EDC in a concentration-dependent manner. These findings suggest that basic calponin interacts with both tubulin subunits and that their C-terminal regions, which also contain the binding sites of MAP2, tau, and kinesin, may be involved in calponin-binding.
Fujii
T
T
Department of Imagination Science (Kansei Engineering), Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, 386-8567, Japan. fujiit1@giptc.shinshu-u.ac.jp
Koizumi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002645:Chickens; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D013552:Swine; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D000096985:Calponins
D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D010455:Peptides; D014404:Tubulin; D000096985:Calponins; C017645:polyaspartate