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-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain 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 report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of adducin involving actin and spectrin, and we demonstrate that adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation 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-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in human embryonal kidney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or 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 adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant alpha adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody revealed the RTPS-serine phosphorylation of adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin were detected in the dendritic spines of cultured hippocampal neurons. Spectrin also was a component of dendritic spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures.
Matsuoka
Y
Y
Howard Hughes Medical Institute and Departments of Cell Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Li
X
X
Bennett
V
V
D000199:Actins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D003712:Dendrites / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009154:Mutation; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D013049:Spectrin / Q000378:metabolism; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000099291:Adducins
D000199:Actins; D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D011506:Proteins; D013049:Spectrin; D013755:Tetradecanoylphorbol Acetate; D000099291:Adducins; C000619710:MARCKS protein, human; C000619712:Marcks protein, rat