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.
Howard Hughes Medical Institute and Departments of Cell Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Y
Y
Matsuoka
X
X
Li
V
V
Bennett
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