Poly(ADP-ribose) Modulates the Properties of MARCKS Proteins

Abstract

In mammalian cells, the formation of DNA strand breaks is accompanied by synthesis of poly(ADP-ribose). This nucleic acid-like homopolymer may modulate protein functions by covalent and/or noncovalent interactions. Here we show that poly(ADP-ribose) binds strongly to the proteins of the myristoylated alanine-rich C kinase substrate (MARCKS) family, MARCKS and MARCKS-related protein (also MacMARCKS or F52). MARCKS proteins are myristoylated proteins associated with membranes and the actin cytoskeleton. As targets for both protein kinase C (PKC) and calmodulin (CaM), MARCKS proteins are thought to mediate cross-talk between these two signal transduction pathways. Dot blot assays show that poly(ADP-ribose) binds to MARCKS proteins at the highly basic effector domain. Complex formation between MARCKS-related protein and CaM as well as phosphorylation of MARCKS-related protein by the catalytic subunit of PKC are strongly inhibited by equimolar amounts of poly(ADP-ribose), suggesting a high affinity of poly(ADP-ribose) for MARCKS-related protein. Binding of MARCKS-related protein to membranes is also inhibited by poly(ADP-ribose). Finally, poly(ADP-ribose) efficiently reverses the actin-filament bundling activity of a peptide corresponding to the effector domain and inhibits the formation of actin filaments in vitro. Our results suggest that MARCKS proteins and actin could be targets of the poly(ADP-ribose) DNA damage signal pathway.

Poly(ADP-ribose) Contributes to an Association between Poly(ADP-ribose) Polymerase-1 and Xeroderma Pigmentosum Complementation Group A in Nucleotide Excision Repair

“…The best characterized PAR-binding motif contains ϳ20 -25 amino acids with an N-terminal basic amino acid cluster followed by hydrophobic residues interspersed with basic amino acids. This motif has been identified in several of the core histones, p53, cyclin-dependent kinase inhibitor 1 (p21), XRCC1, and other proteins including XPA (14,37,38). The presence of a PAR-binding motif in XPA was predicted by sequence alignments that identified the 20 -25-amino acid motif in the C terminus of XPA.…”

Poly(ADP-ribose) Binds to Specific Domains in DNA Damage Checkpoint Proteins

“…A putative calmodulin-binding domain can be found in all members of the NOS family. By analogy to the MARCKS protein family, calmodulin-iNOS interactions could be regulated by PAR polymers (11).…”

“…Using site-directed mutagenesis, we have previously demonstrated that this domain is both necessary and sufficient for noncovalent PAR binding to MARCKS proteins (11). A series of mutated effector peptides was generated, dotblotted onto nitrocellulose membranes, and examined for PAR binding (polymer blot analysis) under high stringency (1 M NaCl) conditions (Fig.…”

Modulation of DNMT1 activity by ADP-ribose polymers

“…Recent studies show the presence of specific poly(ADPribose)-binding motifs in a group of proteins (Schmitz et al, 1998;Pleschke et al, 2000). The typical motif contains two conserved regions: (i) a cluster rich in positive residues and (ii) the consensus patternhxbxhhbbhhb-, where h indicates residues with hydrophobic side chains, b stands for a preference for basic amino-acid residues and x is any amino-acid residue.…”

“…This very strong interaction (Panzeter et al, 1992) introduces, therefore, a structural and functional modification of these proteins, supporting a role for ADP-ribose polymers as 'molecular adaptors'. The interaction between the polymers and the modified proteins is nonionic in nature since it does not depend on the basic charge of proteins; rather, it depends on the presence of a particular amino-acid motif, which represents the consensus domain for the noncovalent link with ADPribose polymers (Schmitz et al, 1998;Pleschke et al, 2000).…”