ABSTRACT
Phosphoinositide 3-kinase γ (PI3Kγ) has been implicated in a variety of cellular signaling processes. It is a multifunctional enzyme with lipid and protein kinase activity that also acts as a scaffold protein. Although it is well known that membrane recruitment is essential for the phosphorylation of phosphoinositides, the cellular localization of PI3Kγ as a protein kinase remains unclear. It has merely been described that PI3Kγ protein kinase activity leading to MAPK activation seems to be restricted to a cytosolic localization. Here, we demonstrate that a hybrid-PI3Kγ having protein kinase, but not lipid kinase activity shows a similar cellular distribution with a high membrane association and comparable liposome binding behavior to wild-type PI3Kγ. Binding of PI3Kγ to liposomes mimicking the natural plasma membrane slightly stimulates autophosphorylation of PI3Kγ. However, liposomes containing an unphysiologically high amount of PI inhibit autophosphorylation of PI3Kγ. Finally, PI3Kγ bound to membrane fragments does not show autophosphorylation which is possibly due to protein-protein-interactions at the plasma membrane. This indicates that not only MAPK activation, but PI3Kγ protein kinase activity in general is localized in the cytosol.
Subject(s)
Cell Membrane/enzymology , Cytosol/enzymology , Liposomes/metabolism , Phosphatidylinositol 3-Kinases/metabolism , HEK293 Cells , Humans , Liposomes/chemistry , Phospholipids/metabolism , TOR Serine-Threonine Kinases/metabolismABSTRACT
Phosphoinositide 3-kinase gamma is a multifunctional enzyme with lipid and protein kinase activities that also acts as a scaffold protein in many diverse signalling processes. The enzyme contains five different domains, but their individual contributions to membrane binding are not fully understood. Here, using in vitro liposome binding assays of individual domains and deletion constructs of human phosphoinositide 3-kinase gamma, we show that each domain is capable of binding anionic phospholipids to varying degrees, depending on the charge of the anionic substrate. Moreover, with the exception of the C2-domain, deletion of any single protein domain results in a complete loss of kinase activity toward both lipids and proteins.
Subject(s)
Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Phospholipids/metabolism , Amino Acid Sequence , Animals , Anions/chemistry , Anions/metabolism , Binding Sites , Class Ib Phosphatidylinositol 3-Kinase , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Liposomes/chemistry , Liposomes/metabolism , Molecular Sequence Data , Phosphatidylinositol 3-Kinases/genetics , Phospholipids/chemistry , Protein Binding , Protein Structure, TertiaryABSTRACT
A merocrine released protein (named 115k protein) was highly enriched from the secretion of the rat coagulating gland. The protein has a molecular mass of 115 kDa as calculated by SDS-PAGE under reducing conditions. Furthermore, the 115 kDa protein is glycosylated, and carries Man, GlcNAc, Gal, Fuc and sialic acid residues. For identification, N-terminal amino acid and nucleotide sequence analyses were performed. The sequences obtained showed 86 to 100% identity with human and mouse IgGFc binding proteins. The functional capacity of IgG binding of the 115 kDa protein was shown by overlay experiments, indicating its membership in the IgG binding protein family.
