DEPHOSPHORYLATION OF PHOSPHOPEPTIDES BY CALCINEURIN (PROTEIN PHOSPHATASE 2B)

38 (6-32 residues) enzymically phosphorylated synthetic peptides have been assayed as substrates for calcineurin, a Ca2+/calmodulin-dependen t protein phosphatase (PP-2B) belonging to the family of Ser/Thr-speci fic enzymes but also active on phosphotyrosine residues. Many peptides reproduce, with suitable modifications, naturally occurring phosphoac ceptor sites. While protein phosphatases 2A and 2C are also very activ e on short phosphopeptides, an extended N-terminal stretch appears to be a necessary, albeit not sufficient, condition for an optimal dephos phorylation, comparable to that of protein substrates, of both phospho seryl and phosphotyrosyl peptides by calcineurin. This finding corrobo rates the view that higher-order structure is an important determinant for the substrate specificity of calcineurin. However, a number of sh orter peptides are also appreciably dephosphorylated by this enzyme, t heir efficiency as substrates depending on local structural features. All the peptides that are appreciably dephosphorylated by calcineurin contain basic residue(s) on the N-terminal side. A basic residue locat ed at position -3 relative to the phosphorylated residue plays a parti cularly relevant positive role in determining the dephosphorylation of short phosphopeptides. Acidic residue(s) adjacent to the C-terminal s ide of the phosphoamino acid are conversely powerful negative determin ants, preventing the dephosphorylation of otherwise suitable peptide s ubstrates. However, calcineurin displays an only moderate preference f or phosphothreonyl peptides which are conversely strikingly preferred over their phosphoseryl counterparts by the other classes of Ser/Thr-s pecific protein phosphatases. Moreover calcineurin does not perceive a s a strong negative determinant the motif Ser/Thr-Pro in peptides wher e this motif prevents dephosphorylation by the other classes of Ser/Th r protein phosphatases. Whenever tested on phosphotyrosyl peptides, ca lcineurin exhibits a specificity which is strikingly different from th at of T-cell protein tyrosine phosphatase, a bona fide protein tyrosin e phosphatase. In particular while the latter enzyme is especially act ive toward a number of phosphopeptides reproducing the phosphoacceptor sites of src products and of calmodulin whose N-terminal moieties are predominantly acidic, the artificial substrate phospho-angiotensin II , bearing an arginine residue at position -2, is far preferred by calc ineurin over all phosphotyrosyl peptides of similar size. Collectively taken these results show that the specificity of calcineurin, rather than resting on a given consensus sequence, is determined by a variety of primary and higher-order structural features conferring to it an o verall selectivity that is different from those of any other known pro tein phosphatase.