title: Identification of the calmodulin-binding domain of recombinant calcium-independent phospholipase A2beta. implications for structure and function.
abstract: Calcium-independent phospholipase A(2) (iPLA(2)) is the major phospholipase A(2) activity in many cell types, and at least one isoform of this enzyme class is physically and functionally coupled to calmodulin (CaM) in a reversible calcium-dependent fashion. To identify the domain in recombinant iPLA(2)beta (riPLA(2)beta) underlying this interaction, multiple techniques were employed. First, we identified calcium-activated CaM induced alterations in the kinetics of proteolytic fragment generation during limited trypsinolysis (i.e. CaM footprinting). Tryptic digests of riPLA(2)beta (83 kDa) in the presence of EGTA alone, Ca(+2) alone, or EGTA and CaM together resulted in the production of a major 68-kDa protein whose kinetic rate of formation was specifically attenuated in incubations containing CaM and Ca(+2) together. Western blotting utilizing antibodies directed against either the N- or C-terminal regions of riPLA(2)beta indicated the specific protection of riPLA(2)beta by calcium-activated CaM at a cleavage site approximately 15 kDa from the C terminus. Moreover, calcium-activated calmodulin increased the kinetic rate of tryptic cleavage near the active site of riPLA(2)beta. Second, functional characterization of products from these partial tryptic digests demonstrated that approximately 90% of the 68-kDa riPLA(2)beta tryptic product (i.e. lacking the 15-kDa C-terminus) did not bind to a CaM affinity matrix in the presence of Ca(2+), although >95% of the noncleaved riPLA(2)beta as well as a 40-kDa C-terminal peptide bound tightly under these conditions. Third, when purified riPLA(2)beta was subjected to exhaustive trypsinolysis followed by ternary complex CaM affinity chromatography, a unique tryptic peptide ((694)AWSEMVGIQYFR(705)) within the 15-kDa C-terminal fragment was identified by RP-HPLC, which bound to CaM-agarose in the presence but not the absence of calcium ion. Fourth, fluorescence energy transfer experiments demonstrated that this peptide (694) bound to dansyl-calmodulin in a calcium-dependent fashion. Collectively, these results identify multiple contact points in the 15-kDa C terminus as being the major but not necessarily the only binding site responsible for the calcium-dependent regulation of iPLA(2)beta by CaM.
authors: Division of Bioorganic Chemistry and Molecular Pharmacology, Departments of Medicine, Chemistry, Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors: C M
authors: CM
authors: Jenkins
authors: M J
authors: MJ
authors: Wolf
authors: D J
authors: DJ
authors: Mancuso
authors: R W
authors: RW
authors: Gross
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D017353:Gene Deletion; D054513:Group IV Phospholipases A2; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; D014357:Trypsin; D002118:Calcium