Calmodulin binds and stabilizes the regulatory enzyme, CTP:Phosphocholine cytidylyltransferase

CTP: phosphocholine cytidylyltransferase (CCT alpha) is a proteolytically sensitive enzyme essential for production of phosphatidylcholine, the major phospholipid of animal cell membranes. The molecular signals that govern CCT alpha protein stability are unknown. An NH2-terminal PEST sequence within CCT alpha did not serve as a degradation signal for the proteinase, calpain. Calmodulin (CaM) stabilized CCT alpha from calpain proteolysis. Adenoviral gene transfer of CaM in cells protected CCT alpha, whereas CaM small interfering RNA accentuated CCT alpha degradation by calpains. CaM bound CCT alpha as revealed by fluorescence resonance energy transfer and two-hybrid analysis. Mapping and site-directed mutagenesis of CCT alpha uncovered a motif (LQERVDKVK) harboring a vital recognition site, Gln(243), whereby CaM directly binds to the enzyme. Mutagenesis of CCT alpha Gln(243) not only resulted in loss of CaM binding but also led to complete calpain resistance in vitro and in vivo. Thus, calpains and CaM both access CCT alpha using a structurally similar molecular signature that profoundly affects CCT alpha levels. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCT alpha that stabilizes the enzyme under proinflammatory stress.