Actin filament cross-linking by MARCKS: characterization of two actin-binding sites within the phosphorylation site domain.
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.
Yarmola
E G
EG
Departments of Medicine and Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA.
Edison
A S
AS
Lenox
R H
RH
Bubb
M R
MR
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism; D003285:Contractile Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D051304:Profilins; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D015800:Protozoan Proteins; D011817:Rabbits; D013947:Thymosin / Q000378:metabolism; D000099201:Thymosin beta(4)
D000199:Actins; D002352:Carrier Proteins; D003285:Contractile Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010446:Peptide Fragments; D051304:Profilins; D011506:Proteins; D015800:Protozoan Proteins; D013947:Thymosin; D000099201:Thymosin beta(4); C050869:actobindin protein, Acanthamoeba