A Conserved Docking Surface on Calcineurin Mediates Interaction with Substrates and Immunosuppressants

Abstract

SUMMARY The phosphatase calcineurin, target of the immunosuppressants cyclosporin A and FK506, dephosphorylates NFAT transcription factors to promote immune activation and development of the vascular and nervous systems. NFAT interacts with calcineurin through distinct binding motifs: the PxIXIT and LxVP sites. While many calcineurin substrates contain PxIxIT motifs, the generality of LxVP-mediated interactions is unclear. We define critical residues in the LxVP motif, and demonstrate its binding to a hydrophobic pocket at the interface of the two calcineurin subunits. Mutations in this region disrupt binding of mammalian calcineurin to NFATc1, and interaction of yeast calcineurin with substrates including Rcn1, which contains an LxVP motif. These mutations also interfere with calcineurin-immunosuppressant binding, and an LxVP-based peptide competes with immunosuppressant-immunophilin complexes for binding to calcineurin. These studies suggest that LxVP-type sites are a common feature of calcineurin substrates and that immunosuppressant-immunophilin complexes inhibit calcineurin by interfering with this mode of substrate recognition.

Cooperative autoinhibition and multi-level activation mechanisms of calcineurin

“…Notably, Tyr418 in CN V418Y/F419L , which is immediately N-terminal to the LxVP motif, fits into a deep pocket formed by the loops connecting EF hands 1 and 2 and EF hands 3 and 4 of CNB. This is consistent with the experimental observations that many LxVP motifs are immediately preceded by an aromatic residue (Phe or Tyr) [23], which may act as a binding strength enhancer.…”

“…The substrates and regulators of CN contain at least one of the two conserved CN-binding motifs, PxIxIT and LxVP, which interact with two distinct docking sites on CN [3,[20][21][22][23]. Recently, Grigoriu et al [24] reported the first high-resolution structure of CN α bound to a physiological binding partner, the protein inhibitor A238L from African swine fever virus.…”

Opposing Action of Casein Kinase 1 and Calcineurin in Nucleo-cytoplasmic Shuttling of Mammalian Translation Initiation Factor eIF6

“…For example, nuclear accumulation of NFAT family of transcription factors in vertebrates and the stress-responsive transcription factor Crz1p in yeast is regulated by dephosphorylation mediated by the calcium/calmodulin-dependent calcineurin phosphatase (21)(22)(23)(24)(25)(26)(27)(28). Indeed, examination of the amino acid sequence of eIF6 shows that, in addition to a conserved docking motif for CK1 (FXXXF, where X is any amino acid) (29) at amino acid positions 34 -38, and well-characterized CK1 phosphorylation sites at Ser-174 and Ser-175, the protein also possesses a binding motif LXVP for the Ca 2ϩ -regulated protein phosphatase calcineurin (30). This putative calcineurin binding motif FIGURE 2.…”

“…In addition to the PXIXIT binding motif, a second calcineurin binding motif with a moderately conserved consensus sequence, LXVP lies near the C terminus of the NFAT regulatory region (33). Calcineurin binds to this site at a hydrophobic pocket formed at the interface of its two subunits A and B (30). This hydrophobic pocket formed at the interface of the two calcineurin subunits is targeted by the immunosuppressant-immunophilin complexes (e.g.…”

“…This hydrophobic pocket formed at the interface of the two calcineurin subunits is targeted by the immunosuppressant-immunophilin complexes (e.g. cyclosporin A (CsA)-cyclophilin or FK506-FK binding protein 12) (30,33). Indeed, immunosuppressants CsA and FK506 have been widely used as specific potent inhibitors of calcineurin-mediated dephosphorylation reaction in vivo (23)(24)(25)(26)(27)(28).…”