ADP-ribosylation of microtubule proteins as catalyzed by cholera toxin.

Abstract

Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8‐3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5′‐AMP, respectively labeled with the corresponding isotope. Two‐dimensional separation by isoelectric focusing and SDS‐PAGE of labeled and native tubulin revealed that labeling occurs at least in four different isotubulins. The isoelectric point of the labeled isotubulins was slightly lower than that of native purified tubulin. This shift in mobility is probably due to additional negative charges involved with the incorporation of ADP‐ribosyl residues into the tubulin subunits. SDS‐PAGE of peptides derived from [32P]ADP‐ribosylated alpha and beta tubulin subunits by Staphylococcus aureus protease cleavage showed a peptide pattern identical with that of native tubulin. Microtubule‐associated proteins (MAP1 and MAP2) of high molecular weight were also shown to undergo ADP‐ribosylation. Incubation of permeated rat neuroblastoma cells in the presence of [32P]NAD and cholera toxin results in the labeling of only a few cell proteins of which tubulin is one of the major substrates.

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

“…These conditions were comparable to those used in earlier ADP-ribosylation studies with cholera and pertussis toxins as the ADP-ribosyltransferases (Kaslow et al, 1981;Amir-Zaltsman et al, 1982;Hawkins & Browning, 1982). Electrophoresis and subsequent autoradiography of an SDS-PAGE gel of the reaction products ( Figure 1) demonstrated strong radiolabeling of two protein species of molecular mass approximately 50-55 kDa and a high molecular weight protein species of approximately 300K.…”

Modulation of the hepatic α1‐adrenoceptor responsiveness by colchicine: dissociation of free cytosolic Ca2+‐dependent and independent responses

“…On the other hand tubulin is, like the G-proteins, a 1 Author for correspondence. substrate for pertussis or cholera toxin catalysed ADP-ribosylation (Amir-Zaltsman et al, 1982;Wang et al, 1990) and seems to control cellular Ca2" fluxes (Lim et al, 1985). Based on these and other (Taylor et al, 1973;Omann et al, 1987) observations that suggest the implication of the cytoskeleton in the signal transduction mechanism of plasma membrane receptors, we found it interesting to determine the role of the cytoskeleton on the hepatic al-adrenoceptor signalling pathway.…”

ADP-ribosylation of the Mr 83,000 stress-inducible and glucose-regulated protein in avian and mammalian cells: modulation by heat shock and glucose starvation.

“…The reaction is usually thought to be catalyzed and enhanced by toxins such as cholera or diphtheria toxin, and, in the cases examined, a specific enzymatic or regulatory function is known to be inhibited by the covalent attachment of ADP-ribose to the protein acceptors (1)(2)(3)(4)(5). Re-cently, several structural proteins have been shown to be modified by ADP-ribosylation as well as a response to cholera or diphtheria toxins in vitro (6,7). Most of these modifications have been characterized and shown to occur only in vitro by utilizing NAD' as an ADP-ribose donor.…”