Calcineurin (CaN), a Ca++/calmodulin (CaM)-dependent protein phosphatase (PP2B), has two subunits: a 61 kDa catalytic A subunit (CaNA) and a 19 kDa regulatory B subunit (CaNB) with four Ca++-binding sites. To determine if CaNB has protein ligands other than CaNA, an affinity column was prepared with a glutathione-S-transferase-CaNB fusion protein. A 57 kDa protein from extracts of bovine brain and a 60 kDa protein from bovine kidney were the predominant proteins that specifically bound to CaNB in a Ca++-dependent manner. Western blotting indicated that the proteins were not CaNA. Protein sequencing revealed that the 57 kDa CaNB-binding protein from brain was alpha-tubulin and the 60 kDa protein from kidney was heat shock protein 60 (HSP60). The Ca++-dependence of these interactions with CaNB were further confirmed using anti-CaNB immunoaffinity columns. Although CaNB specifically associated with purified alpha-tubulin, beta-tubulin and HSP60, GroEL, a bacterial HSP60 analog, did not bind to CaNB. The association of CaNB with HSP60 was not disrupted by incubation with heat shock protein 10 (HSP10), ATP and Mg++, suggesting that CaNB may serve a regulatory role in the activity of HSP60 and not reflect its association as a misfolded substrate with HSP60 functioning as the molecular chaperon. Finally, the concentration of CaNB in tissues is in excess of CaNA, a result consistent with other Ca++-dependent regulatory roles in addition to its effect on the catalytic function of CaNA.
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology