title: The proline-rich N-terminal sequence of calcineurin Abeta determines substrate binding.
abstract: Three different genes of catalytic subunit A of the Ca(2+)-dependent serine/threonine protein phosphatase calcineurin (CaN) are encoded in the human genome forming heterodimers with regulatory subunit B. Even though physiological roles of CaN have been investigated extensively, less is known about the specific functions of the different catalytic isoforms. In this study, all human CaN holoenzymes containing either the alpha, beta, or gamma isoform of the catalytic subunit (CaN alpha, beta, or gamma, respectively) were expressed for the first time. Comparative kinetic analysis of the dephosphorylation of five specific CaN substrates provided evidence that the distinct isoforms of the catalytic subunit confer substrate specificities to the holoenzymes. CaN alpha dephosphorylates the transcription factor Elk-1 with 7- and 2-fold higher catalytic efficiencies than the beta and gamma isoforms, respectively. CaN gamma exhibits the highest k(cat)/K(m) value for DARPP-32, whereas the catalytic efficiencies for the dephosphorylation of NFAT and RII peptide were 3- and 5-fold lower, respectively, when compared with the other isoforms. Elk-1 and NFAT reporter gene activity measurements revealed even more pronounced substrate preferences of CaNA isoforms. Moreover, kinetic analysis demonstrated that CaN beta exhibits for all tested protein substrates the lowest K(m) values. Enzymatic characterization of the CaN beta(P14G/P18G) variant as well as the N-terminal truncated form CaN beta(22-524) revealed that the proline-rich sequence of CaN beta is involved in substrate recognition. CaN beta(22-524) exhibits an at least 4-fold decreased substrate affinity and a 5-fold increased turnover number. Since this study demonstrates that all CaN isoforms display the same cytoplasmic subcellular distribution and are expressed in each tested cell line, differences in substrate specificities may determine specific physiological functions of the distinct isoforms.
authors.affiliation: Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle/Saale, Germany.
authors.forename: Susann
authors.initials: S
authors.lastname: Kilka
authors.forename: Frank
authors.initials: F
authors.lastname: Erdmann
authors.forename: Alexander
authors.initials: A
authors.lastname: Migdoll
authors.forename: Gunter
authors.initials: G
authors.lastname: Fischer
authors.forename: Matthias
authors.initials: M
authors.lastname: Weiwad
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D019703:Calcineurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019169:Jurkat Cells; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011392:Proline / Q000235:genetics; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D050807:ets-Domain Protein Elk-1 / Q000235:genetics / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D050778:NFATC Transcription Factors; D011993:Recombinant Fusion Proteins; D050807:ets-Domain Protein Elk-1; D011392:Proline; D008156:Luciferases; D019703:Calcineurin