ADDUCIN IS AN IN-VIVO SUBSTRATE FOR PROTEIN-KINASE-C - PHOSPHORYLATION IN THE MARCKS-RELATED DOMAIN INHIBITS ACTIVITY IN PROMOTING SPECTRIN-ACTIN COMPLEXES AND OCCURS IN MANY CELLS, INCLUDING DENDRITIC SPINES OF NEURONS

Adducin is a heteromeric protein with subunits containing a COOH-termi nal myristoylated alanine-rich C kinase substrate (MARCKS)-related dom ain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in alpha, beta, and gamma adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC), This repor t presents the first evidence that phosphorylation of the MARCKS-relat ed domain modifies in vitro and in vivo activities of adducin involvin g actin and spectrin, and we demonstrate that adducin is a prominent i n vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA )-activated kinases in multiple cell types, including neurons. PKC pho sphorylation of native and recombinant adducin inhibited actin capping measured using pyrene-actin polymerization and abolished activity of adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS- serine, which is the major PKC phosphorylation site in the MARCKS-rela ted domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofo ld in rat hippocampal slices, eight- to ninefold in human embryonal ki dney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fol d in human erythrocytes after treatments with PMA, but not with forsko lin, Thus, the RTPS-serine of adducin is an in vivo phosphorylation si te for PKC or other PMA-activated kinases but not for cAMP-dependent p rotein kinase in a variety of cell types. Physiological consequences o f the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type o r PKC-unphosphorylatable S716A/S726A mutant alpha adducin. The mutant alpha adducin was no longer concentrated at the cell membrane at sites of cell-cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant alpha add ucin exhibited increased levels of cytoplasmic spectrin, which was col ocalized with the mutant alpha adducin in a punctate pattern. Immunonu ofluorescence with the phosphoadducin-specific antibody revealed the R TPS-serine phosphorylation of adducin in postsynaptic areas in the dev eloping rat hippocampus, High levels of the phosphoadducin were detect ed in the dendritic spines of cultured hippocampal neurons. Spectrin a lso was a component of dendritic spines, although at distinct sites fr om the ones containing phosphoadducin. These data demonstrate that add ucin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin oc curs in dendritic spines that are believed to respond to external sign als by changes in morphology and reorganization of cytoskeletal struct ures.