Actin filament cross-linking by MARCKS: characterization of two actin-binding sites within the phosphorylation site domain

J Biol Chem. 2001 Jun 22;276(25):22351-8. doi: 10.1074/jbc.M101457200. Epub 2001 Apr 6.

Abstract

We recently identified conformational changes that occur upon phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) that preclude efficient cross-linking of actin filaments (Bubb, M. R., Lenox, R. H., and Edison, A. S. (1999) J. Biol. Chem. 274, 36472-36478). These results implied that the phosphorylation site domain of MARCKS has two actin-binding sites. We now present evidence for the existence of two actin-binding sites that not only mutually compete but also specifically compete with the actin-binding proteins thymosin beta(4) and actobindin to bind to actin. The effects of substitution of alanine for phenylalanine within a repeated hexapeptide segment suggest that the noncharged region of the domain contributes to binding affinity, but the binding affinity of peptides corresponding to each binding site has a steep dependence on salt concentration, consistent with presumed electrostatic interactions between these polycationic peptides and the polyanionic N terminus of actin. Phosphorylation decreases the site-specific affinity by no more than 0.7 kcal/mol, which is less than the effect of alanine substitution. However, phosphorylation has a much greater effect than alanine substitution on the loss of actin filament cross-linking activity. These results are consistent with the hypothesis that the compact structure resulting from conformational changes due to phosphorylation, in addition to modest decreases in site-specific affinity, explains the loss of cross-linking activity in phosphorylated MARCKS.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Actins / metabolism*
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Binding, Competitive
  • Carrier Proteins / metabolism
  • Contractile Proteins*
  • Intracellular Signaling Peptides and Proteins*
  • Membrane Proteins*
  • Microfilament Proteins / metabolism
  • Molecular Sequence Data
  • Myristoylated Alanine-Rich C Kinase Substrate
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Phosphorylation
  • Profilins
  • Proteins / chemistry
  • Proteins / metabolism*
  • Protozoan Proteins
  • Rabbits
  • Thymosin / metabolism

Substances

  • Actins
  • Carrier Proteins
  • Contractile Proteins
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Microfilament Proteins
  • Peptide Fragments
  • Profilins
  • Proteins
  • Protozoan Proteins
  • actobindin protein, Acanthamoeba
  • Myristoylated Alanine-Rich C Kinase Substrate
  • thymosin beta(4)
  • Thymosin