Adducin regulation. Definition of the calmodulin-binding domain and sites of phosphorylation by protein kinases A and C

J Biol Chem. 1996 Oct 11;271(41):25157-66. doi: 10.1074/jbc.271.41.25157.

Abstract

Adducin promotes association of spectrin with actin and caps the fast growing end of actin filaments. Adducin contains N-terminal core, neck, and C-terminal tail domains, is a substrate for protein kinases A (PKA) and C (PKC), and binds to Ca2+/calmodulin. Ser-726 and Ser-713 in the C-terminal MARCKS-related domains of alpha- and beta-adducin, respectively, were identified as the major phosphorylation sites common for PKA and PKC. PKA, in addition, phosphorylated alpha-adducin at Ser-408, -436, and -481 in the neck domain. Phosphorylation by PKA, but not PKC, reduced the affinity of adducin for spectrin-F-actin complexes as well as the activity of adducin in promoting binding of spectrin to F-actin. The myristoylated alanine-rich protein kinase C substrate-related domain of beta-adducin was identified as the dominant Ca2+-dependent calmodulin-binding site. Calmodulin-binding was inhibited by phosphorylation of beta-adducin and of a MARCKS-related domain peptide by PKA and PKC. Calmodulin in turn inhibited the rate, but not the extent, of phosphorylation of beta-adducin, but not alpha-adducin, by PKA and that of each subunit by PKC. These findings suggest a complex reciprocal relationship between regulation of adducin function by calmodulin binding and phosphorylation by PKA and PKC.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Calmodulin / metabolism*
  • Calmodulin-Binding Proteins / chemistry
  • Calmodulin-Binding Proteins / metabolism*
  • Chromatography, High Pressure Liquid
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Cytoskeletal Proteins / metabolism
  • Erythrocytes / metabolism
  • Humans
  • Intracellular Signaling Peptides and Proteins*
  • Kinetics
  • Membrane Proteins*
  • Molecular Sequence Data
  • Myristoylated Alanine-Rich C Kinase Substrate
  • Peptide Fragments / chemical synthesis
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Phosphopeptides / chemistry
  • Phosphopeptides / isolation & purification
  • Phosphorylation
  • Phosphoserine / analysis
  • Phosphothreonine / analysis
  • Phosphotyrosine / analysis
  • Protein Kinase C / metabolism*
  • Proteins / chemistry
  • Proteins / metabolism

Substances

  • Calmodulin
  • Calmodulin-Binding Proteins
  • Cytoskeletal Proteins
  • Intracellular Signaling Peptides and Proteins
  • MARCKS protein, human
  • Membrane Proteins
  • Peptide Fragments
  • Phosphopeptides
  • Proteins
  • adducin
  • Phosphothreonine
  • Myristoylated Alanine-Rich C Kinase Substrate
  • Phosphoserine
  • Phosphotyrosine
  • Cyclic AMP-Dependent Protein Kinases
  • Protein Kinase C