title: Dephosphorylation of phosphopeptides by calcineurin (protein phosphatase 2B).
abstract: 38 (6-32 residues) enzymically phosphorylated synthetic peptides have been assayed as substrates for calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (PP-2B) belonging to the family of Ser/Thr-specific enzymes but also active on phosphotyrosine residues. Many peptides reproduce, with suitable modifications, naturally occurring phosphoacceptor sites. While protein phosphatases 2A and 2C are also very active on short phosphopeptides, an extended N-terminal stretch appears to be a necessary, albeit not sufficient, condition for an optimal dephosphorylation, comparable to that of protein substrates, of both phosphoseryl and phosphotyrosyl peptides by calcineurin. This finding corroborates the view that higher-order structure is an important determinant for the substrate specificity of calcineurin. However, a number of shorter peptides are also appreciably dephosphorylated by this enzyme, their 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 located at position -3 relative to the phosphorylated residue plays a particularly relevant positive role in determining the dephosphorylation of short phosphopeptides. Acidic residue(s) adjacent to the C-terminal side of the phosphoamino acid are conversely powerful negative determinants, preventing the dephosphorylation of otherwise suitable peptide substrates. However, calcineurin displays an only moderate preference for phosphothreonyl peptides which are conversely strikingly preferred over their phosphoseryl counterparts by the other classes of Ser/Thr-specific protein phosphatases. Moreover calcineurin does not perceive as a strong negative determinant the motif Ser/Thr-Pro in peptides where this motif prevents dephosphorylation by the other classes of Ser/Thr protein phosphatases. Whenever tested on phosphotyrosyl peptides, calcineurin exhibits a specificity which is strikingly different from that of T-cell protein tyrosine phosphatase, a bona fide protein tyrosine phosphatase. In particular while the latter enzyme is especially active 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 calcineurin 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 overall selectivity that is different from those of any other known protein phosphatase.
authors.affiliation: Dipartimento di Chimica Biologica, Università di Padova, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Donella-Deana
authors.forename: M H
authors.initials: MH
authors.lastname: Krinks
authors.forename: M
authors.initials: M
authors.lastname: Ruzzene
authors.forename: C
authors.initials: C
authors.lastname: Klee
authors.forename: L A
authors.initials: LA
authors.lastname: Pinna
mesh_terms: D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D047388:Casein Kinases; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010768:Phosphoserine / Q000378:metabolism; D019000:Phosphotyrosine; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D002148:Calmodulin-Binding Proteins; D010748:Phosphopeptides; D000804:Angiotensin II; D010768:Phosphoserine; D019000:Phosphotyrosine; D014443:Tyrosine; D011494:Protein Kinases; D047388:Casein Kinases; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases