The Ca2+/calmodulin (CaM)-dependent protein phosphatase calcineurin (CaN) is a critical component of several Ca2+-dependent signalling pathways (Rusnak & Mertz, 2000). CaN is involved in the regulation of T cell activation, long-term depression of postsynaptic potential, cardiac and skeletal muscle hypertrophy, synaptic vesicle recycling, transcription factors, and ion channels (Rusnak & Mertz, 2000).
CaN is a heterodimer of a catalytic A subunit (CnA) and a Ca2+-binding B subunit. Two genes encode the CnAα and CnAβ isoforms. The amino acid sequences of CnAα and CnAβ are 81 % identical. The most striking difference between the catalytic subunit isoforms is the 12 prolines within the first 23 amino acid residues of CnAβ. Both catalytic subunits are present in all tissues examined, although variations in the ratio of CnAα:CnAβ have been noted within and between tissues (Jiang et al. 1997). The physiological significance of the two CaN catalytic subunits is unknown. It has been proposed that the two isoforms may exhibit substrate preferences and may also be selectively targeted to distinct subcellular locations.
To investigate differences in substrate selectivity between the α and β catalytic subunit isoforms, we are investigating the enzymatic characteristics of CaN heterodimers composed of either CnAα or CnAβ. Human or rat brain CnAα or CnAβ was expressed in the baculovirus/Sf21 cell system with the Ca2+-binding B subunit, purified by CaM-Sepharose chromatography, and used in in vitro phosphatase assays (Perrino et al. 1995). The dephosphorylation of PO4-RII peptide and PO4-DARPP32 peptide by CaN heterodimers composed of either CnAα or CnAβ was compared by investigating Ca2+/CaM activation kinetics and by determining the Km and Vmax values of the two CaN heterodimers for each substrate. CaN containing CnAα has higher Vmax values and lower Km values than CaN containing CnAβ toward both substrates. The Vmax values of CaN containing CnAα toward PO4-RII and PO4-DARPP32 are 4.1 and 0.667 µmol min-1 mg-1, respectively, while Vmax values of 2.5 and 0.454 µmol min-1 mg-1, respectively, were obtained with CaN containing CnAβ. The Km values of CaN containing CnAα toward PO4-RII and PO4-DARPP32 are 43 and 19 µM, respectively, while Km values of 100 and 53 µM, respectively, were obtained with CaN containing CnAβ (n = 2 for each CaN heterodimer and each substrate, replicates = 2). At each CaM:CaN molar ratio (0.25:1, 0.5:1, 1:1, 2:1, 4:1) CaN containing CnAα had higher levels of activity toward both substrates than CaN containing CnAβ (n = 2 for each CaN heterodimer and each substrate, replicates = 2).
These results are the first indication of differences in phosphatase activity between the CaN catalytic subunit isoforms towards different substrates. These findings suggest that the CnAα:CnAβ, and CaN:substrate ratios may determine the levels of CaN phosphatase activity toward specific substrates within a tissue and at specific subcellular locations.We thank Michael M. Lai and Solomon H. Snyder (The Johns Hopkins University School of Medicine) for the generous gift of the CnAβ cDNA.
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Rusnak F. & Mertz, P. (2000). Physiol. Rev. 4, 1483-1521.