ADP‐ribosylation of actins by arginine‐specific ADP‐ribosyltransferase purified from chicken heterophils

Abstract

We reported the purification and characterization of an arginine-specific ADP-ribosyltransferase and acceptor protein p33 in granules of chicken peripheral polymorphonuclear leukocytes (heterophils) [Mishima, K., Terashima, M., Obara, S.,In the present study, we obtained evidence that chicken non-muscle Ply-actin, skeletal muscle a-actin and smooth-muscle y-actin were ADP ribosylated by the heterophil ADP-ribosyltransferase. The stoichiometry of ADP -ribose incorporation into these actins was 3.2 mol, 1.0 mol and 2.0 mol ADP -ribose/mol of Ply-actin, a-actin and y-actin, respectively. The optimal pH for the ADP ribosylation was at pH 8.5, with the respective actin. K,,, values for NAD were calculated to be 30 pM with B/y-actin, 35 pM with a-actin and 20 pM with y-actin. The K , values for the actin isoforms were 15 pM for j/y-actin, 2.5 pM for a-actin and 10 pM for y-actin. ADP ribosylation of actin inhibited its capacity to polymerize, as determined by the increase in fluoresence intensity with N-(1 -pyrenyl)iodoacetamide-labelled actin. Filamentous actin (F-actin) polymerized with the respective actin isoform was also ADP ribosylated, although the extent of the modification of F-actin was lower than that of globular actin (G-actin). In situ ADP ribosylation of Ply-actin was evidenced with chicken peripheral heterophils permeabilized with saponin. Thus, the endogenous ADP ribosylation of actin in the heterophils may be involved in the cellular processes such as phagocytosis, secretion and migration.

Cysteine‐specific ADP‐ribosylation of actin

“…Our data are contradictory to the findings of Clancy et al [54] who found a clear nitric oxidedependent increase in the ADP-ribosylation of actin in the lysate of neutrophils. Furthermore, the cysteine-specific ADP-ribosylation of actin reported here is quite different from the arginine-specific ADP-ribosylation of actin which is catalyzed by a transferase from chicken heterophils [29]. This enzyme modifies all actin isoforms, resulting in inhibition of polymerization.…”

“…Recently, it has been reported that mammalian actin is the substrate of eukaryotic ADP-ribosyltransferases. For example the arginine-specific ADP-ribosyltransferase purified from chicken heterophils was shown to modify actin [29]. Several mammalian ADP-ribosyltransferases from brain can ADP-ribosylate purified actin [30].…”

ADP-Ribosylation of Tubulin by Chicken NAD-Arginine ADP-Ribosyltransferase Suppresses Microtubule Formation.

“…During the search of target proteins for ADP-ribosylation in the brain, we found that two cytosolic proteins, tubulin and actin, were modified by ADPRT. Here we show that ADP-ribosylation of tubulin inhibits its assembly , as was seen in the case of actin (3,4), and that the inhibition is due to ADP-ribosylation of the sites required for tubulin assembly. substrate for the arginine-specific ADPRT in bovine brain cytosol.…”

Nitric oxide blocks the cell cycle of mouse macrophage‐like cells in the early G2+M phase

“…The mechanism is thought to be that the myosin light chain (MLC) is phosphorylated by MLC-kinase in a calmodulindependent manner, inducing the dissociation of myosin and actin filaments [17,18]. In addition, it has been shown in vitro that actin is ADP-ribosylated by nitric oxide and the ADP-ribosylated actin is unable to polymerize [19,20]. These observations and the data in the present report suggest that endogenous nitric oxide acts on actin filaments and blocks the cell cycle in the early G,+M phase.…”