IN-VITRO GENERATION OF AN ACTIVE CALMODULIN-INDEPENDENT PHOSPHODIESTERASE FROM BRAIN CALMODULIN-DEPENDENT PHOSPHODIESTERASE (PDE1A2) BY M-CALPAIN

In the present study we have shown that bovine brain 60-kDa calmodulin -dependent cyclic nucleotide phosphodiesterase isozyme (CaMPDE - PDE1A 2) is proteolyzed by a Ca2+-dependent cysteine protease, m-calpain. Th e proteolysis of PDE1A2 by m-calpain results in its conversion to a to tally calmodulin (CaM)independent form accompanied by degradation of P DE1A2 into a 45-kDa catalytic fragment and a 15-kDa fragment. The acti vity of PDE1A2 is unaffected by the presence or absence of CaM during cleavage, suggesting that the interaction between CaM and PDE1A2 does not alter substrate recognition by calpain. Furthermore, we provide ev idence, based on the studies df CaM overlay and phosphorylation, that the cleavage site is not present either in the CaM-binding domain or p hosphorylation site. N-terminal sequence analysis of the 45-kDa fragme nt indicated that cleavage occurs between residues (126)Gln and (127)A la, and eliminates the CaM-dependent activity of carboxy termini PDE1A 2. The present findings suggest that limited proteolysis in the brain through calpains could be an alternate mechanism for activating CaMPDE (s) and for regulating intracellular levels of cAMP. (C) 1998 Academic Press.