Calmodulin binds and stabilizes the regulatory enzyme, CTP: phosphocholine cytidylyltransferase.
CTP:phosphocholine cytidylyltransferase (CCTalpha) is a proteolytically sensitive enzyme essential for production of phosphatidylcholine, the major phospholipid of animal cell membranes. The molecular signals that govern CCTalpha protein stability are unknown. An NH(2)-terminal PEST sequence within CCTalpha did not serve as a degradation signal for the proteinase, calpain. Calmodulin (CaM) stabilized CCTalpha from calpain proteolysis. Adenoviral gene transfer of CaM in cells protected CCTalpha, whereas CaM small interfering RNA accentuated CCTalpha degradation by calpains. CaM bound CCTalpha as revealed by fluorescence resonance energy transfer and two-hybrid analysis. Mapping and site-directed mutagenesis of CCTalpha uncovered a motif (LQERVDKVK) harboring a vital recognition site, Gln(243), whereby CaM directly binds to the enzyme. Mutagenesis of CCTalpha 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 CCTalpha using a structurally similar molecular signature that profoundly affects CCTalpha levels. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTalpha that stabilizes the enzyme under proinflammatory stress.
Chen
Bill B
BB
Department of Biochemistry, University of Iowa, Iowa City, Iowa, 52242.
Mallampalli
Rama K
RK
Department of Biochemistry, University of Iowa, Iowa City, Iowa, 52242; Department of Internal Medicine, University of Iowa, Iowa City, Iowa, 52242; Department of Veterans Affairs Medical Center and the Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242. Electronic address: ramamallampalli@uiowa.edu.
D000256:Adenoviridae / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry* / Q000502:physiology*; D002154:Calpain / Q000737:chemistry; D019916:Choline-Phosphate Cytidylyltransferase / Q000737:chemistry / Q000502:physiology*; D018014:Gene Transfer Techniques; D005982:Glutathione Transferase / Q000378:metabolism; D008074:Lipoproteins / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D034741:RNA, Small Interfering / Q000378:metabolism; D020798:Two-Hybrid System Techniques