Masaharu Terashima⁽¹⁾, Makoto Shimoyama⁽²⁾, Mikako Tsuchiya^{(3)
(1)(2)(3)}Department of Biochemistry. Shimane Medical University - Izumo. Japan

	[Biochemistry]	[Cell Biology & Cytology]

RESULTS AND DISCUSSION

ADP-ribosylation of G- and F-actins

To examine the extent of ADP-ribosylation of F-actin, polymerized actin was incubated with [³²P]NAD and ADPRT. As shown in Fig. 1, both G- and F-actins appeared as 43 kDa single bands and were apparently radiolabeled. The degree of the ADP-ribosylation of F-actin was nearly a half of that of G-actin (lanes 1 and 2). ADP-ribose incorporation into the F- and G-actins during 15 min incubation with ADPRT were 0.29 mol/mol and 0.57 mol/mol, respectively. These results are consistent with the data in which G-actin was modified at Arg28 and Arg206 on the molecule, whereas F-actin was Arg28 only ⁽⁴⁾. When ADPRT was omitted from the reaction mixture, F-actin was hardly labeled (lane 3).

ADP-ribosylation of F-actin causes its depolymerization

Next, we examined effects of ADP-ribosylation of F-actin on its polymerization state by measuring viscosity of the actin solutions. G-actin solution including 2 mM NAD was incubated with 2 mM MgCl₂for 60 min, and then, ADPRT was added to the solution. The flow time required for the solution to pass through a capillary tube was measured at each time indicated, and the specific viscosity was calculated. As shown in Fig. 2, viscosity of the solution was increased and reached to the maximum level, 0.20 at 60 min. After adding ADPRT, the viscosity was gradually decreased and the lowest viscosity 0.10 was obtained 2 hr after the addition. Prolonged incubation no longer decreased the viscosity (data not shown). These results suggest that ADP-ribosylation decreases the F-actin content. This was confirmed by assessment of contents of G- and F-forms in the actin solution with ultracentrifugation. Incubation of polymerized actin with ADPRT in the presence of NAD for 2 hr reduced F-actin content from 96% to 45% and increased monomeric G-actin content from 4% to 55%. These results indicate that ADP-ribosylation of F-actin decreases its polymerized form. Taken together with the previous observation that ADP-ribosylation of G-actin inhibits its polymerization ⁽³⁾, ADP-ribosylation may shift the equilibrium state between G- and F-actins toward G-actin.

ADP-ribosylation of actin affects its G-F equilibrium state

Actin, a highly conserved family of cytoplasmic proteins and major constituent in all eukaryotic cells, is one of the most important components of cytoskeletal architecture microfilament. Actin is involved in a wide variety of cellular processes, such as phagocytosis, secretion, cell locomotion, and the maintenance of the cell shape, besides muscle contractions. All these functions depend on the capacity of actin to polymerize and form filamentous actin, and to depolymerize to monomeric actin. In the resting state of the cell, monomeric G-actin and polymerized F-actin are in dynamic equilibrium state, which is regulated by various actin binding proteins ⁽⁶⁾. Actin filaments have two polar, non-equivalent ends for polymerization and depolymerization; one is barbed end and the other pointed end. As a result of the difference in the assembly rates at the two ends, actin monomers can cycle through the filaments from the barbed end to pointed end, thus keeping the equilibrium state (Fig. 3a) .

When G-actin is ADP-ribosylated, ADP-ribose moiety covers the pointed end of the actin at Arg206 to inhibit polymerization by a steric hindrance ^(3,4) (Fig.3b) . Mechanism of the inhibition seems different from that caused by clostridial ADP-ribosylating toxins, C.botulinumC2 and C.perfringensiota toxins, since both toxins ADP-ribosylate the barbed end of actin at Arg177 ^(7,8).

Though we demonstrated that ADP-ribosylation of actin in the pointed end at Arg206 might cause the inhibition of actin polymerization ^(3,4), the role of ADP-ribosylation of actin Arg28 which is located in the lateral surface had been remained obscure. In this study, we demonstrated that ADP-ribosylation of F-actin induces its depolymerization. The result suggests that ADP-ribosylation of Arg28 in the F-actin causes the disruption of ordered conformation of the filament, and may stimulate depolymerization of F-actin. When F-actin is ADP-ribosylated, ADP-ribose moiety attaches to the lateral surface of actin filaments at Arg28 and probably causes conformational changes in F-actin ^(4,6,9), leading to induce the depolymerization (Fig. 3c) . In this state, additional modification may occur in the pointed end on the depolymerizing G-actin which has been already modified at the lateral surface. Thus, ADP-ribosylation would facilitate actin depolymerization, through both inhibition of the polymerization and induction of depolymerization. Taken together, ADP-ribosylation of G- and F-actins may have functional roles to inhibit actin polymerization and induce actin depolymerization, respectively, and by the sum of these effects, actin equilibrium would be shifted to the G- actin-dominant state.

Cellular concentration of G-actin is much higher than the critical concentration of G-actin for actin polymerization in vitro, and the phenomenon has been ascribed to numerous actin-binding proteins ⁽⁶⁾. It has been also shown that post-translational modifications of actin including phosphorylation may be involved in the regulation of actin polymerization. We previously reported the in situ ADP-ribosylation of actin in saponin-permeabilized polymorphonuclear leukocytes and the inhibitory effect of the ADP-ribosylation on actin polymerization ^(3,4). We postulate here that ADP-ribosylation may have a role to the regulation by shifting G-F actin equilibrium toward G-actin through the modification of both G- and F-actins. Taken together with our recent study that ADP-ribosylation of tubulin, which is also a major component of cytoskeleton, was ADP-ribosylated and lost the capacity to form microtubule ⁽¹⁰⁾, ADP-ribosylation may participate in the regulation of cytoskeletal reorganization in the cells.

Discussion Board

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[ABSTRACT] [INTRODUCTION] [MATERIAL & METHODS] [RESULTS AND DISCUSSION] [FIGURES] [ACKNOWLEDGEMENTS] [REFERENCES] [Discussion Board]

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	[Biochemistry]	[Cell Biology & Cytology]

Masaharu Terashima, Makoto Shimoyama, Mikako Tsuchiya
Copyright © 1999-2000. All rights reserved.

Last update: 5/01/00