Glutamate substitutions at a PKA consensus site are consistent with inactivation of calpain by phosphorylation.

Regulation of calpain by phosphorylation has often been suggested, but has proved difficult to detect. Calpains extracted from mammalian tissue are reported to contain 2-4 mol phosphate/mol of enzyme distributed over multiple sites, but phosphate groups are not detectable in the X-ray structures of recombinant calpain. Some serine and threonine residues in the large subunit of rat m-calpain were converted to aspartic or glutamic acid residues, at sites suggested by previous studies, to assess the probable effects of phosphate groups on the enzyme. Expression of the mutant calpains in Escherichia coli, and their heat stabilities, did not differ from those of the wild-type enzyme. m-Calpains with the mutations Ser50Asp, Ser50Glu, Ser67Glu, and Thr70Glu had the same specific activity and Ca(2+) requirement as the wild-type enzyme. In contrast, Ser369Asp-, Ser369Glu-, and Thr370Glu-m-calpain were inactive. This result is consistent with the recent report that phosphorylation at position 369 or 370 in vivo reduced m-calpain activation.