Identification of two calcineurin B-binding proteins: Tubulin and heat shock protein 60

Calcineurin (CaN) is a Ca⁺⁺/calmodulin-dependent protein phosphatase with two subunits: a catalytic subunit (CaNA) and a regulatory subunit (CaNB). With four Ca⁺⁺-binding sites and a sequence homology to calmodulin, CaNB has been defined as the regulatory subunit for CaNA. However, we have shown that mitochondrial expression of CaNB far exceeds that of CaNA. To investigate the role of this excess CaNB, we have generated glutathione-S-transferase-CaNB (GST-CaNB) fusion protein and demonstrated that the fusion protein predominantly bound to alpha-tubulin, a 57 kDa protein in bovine brain extracts, and heat shock protein 60 (Hsp60) in bovine kidney extracts. Their Ca⁺⁺-dependent interactions with CaNB were verified by immunoprecipitation. The binding of CaNB could be demonstrated with purified alpha/beta tubulins and Hsp60, but not GroEL, a bacterial Hsp60 analog. The interaction of CaNB and Hsp60 was not disrupted by the incubation with Hsp10, ATP and Mg⁺⁺, suggesting that CaNB was not associated with Hsp60 as a misfolded substrate, and may serve as a regulatory protein. Thus, CaNB may play other regulatory roles in Ca⁺⁺-dependent events in addition to its interaction with CaNA, and may be important for Ca⁺⁺-dependent processes in mitochondria.