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J. Cell Biol.,
Volume 142, Number 2, July 27, 1998 485-497
Howard Hughes Medical Institute and Departments of Cell Biology and Biochemistry, Duke University Medical Center,
Durham, North Carolina 27710
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 
, 
, and 
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 adducin. The mutant 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 adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant 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.
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