Phosphorylation of the calpains

The calpains are phosphorylated and phosphorylation affects their activities, but no attempts have been made to determine which calpain resides are phosphorylated. Ca2+ requirements of the calpains are much higher in in vitro assays than would ever be encountered in living cells, and cells must contain a mechanism to reduce this requirement; the crystallographic structure of m‐calpain suggests that phosphorylation at selected residues in either domain IIa or IIb of the calpains may lower their Ca2+ requirement. We have used anti‐phospho‐Tyr, anti‐phospho‐Ser, and antiphospho‐Thr antibodies and mass spectrometry to characterize phosphorylation of calpains purified from bovine skeletal muscle or human placenta. Anti‐phospho antibodies show that both μ‐ and m‐calpain have at least 6 phosphorylated sites; 2 each phospho‐Tyr, phospho‐Ser, and phospho‐Thr. The N‐terminal part of the 80‐kDa subunit from residues 6‐266 (μ‐calpain) or residues 9‐265 (m‐calpain) have at least 1 each phospho‐Tyr, phospho‐Ser, and phospho‐Thr. The C‐terminal part of the 80‐kDa subunit from residues 245 to 714 (μ‐calpain) or from residues 265‐700 (m‐calpain) also have at least 1 phospho‐Tyr, 1 phospho‐Ser, and 1 phospho‐Thr. Mass spectrometry has thus far identified 4 phosphorylated sites in the 80‐kDa subunit of μ‐calpain (only 1 in the N‐terminal 266 residues), 8 phosphorylated sites in the 80kDa subunit of m‐calpain (5 in the N‐terminal 265 residues), 1 phosphorylated site in the small subunit of μ‐calpain, and 3 phosphorylated sites in the small subunit of m‐calpain. Four of the sites identified are in domains IIa or IIb. Supported by NIH and MDA