The growth of many normal cells requires contact with an adhesive substratum, a requirement that is frequently abrogated in the transformed phenotype. We have explored pathways that can lead to the anchorage-independent growth of cultured Rat-i fibroblasts. PasteureUa mulocida toxin (PMT), a 146-kDa mitogenic protein, caused a striking increase in the formation of colonies (>200 jam) from single cells in soft agar. The magnitude of the effect of PMT was greater than that achieved by epidermal growth factor or platelet-derived growth factor. The toxin was extremely potent, with half-maximal and maximal effects observed at 1 and 10 pM PMT, respectively. This concentration dependence of the action of the toxin is similar to that for the stimulation of DNA synthesis in adherent cultures of the cells. Stimulation ofcolony formation could be achieved by a transient exposure of the cells to PMT and it was blocked by methylamine, indicating that the toxin enters the cells to act. Colony formation was stimulated equally by native and recombinant PMT, but a truncated version (33.5 kDa) of the recombinant toxin was ineffective. PMT antiserum blocked colony formation in response to PMT. In the Rat-1 cells, PMT stimulated the phospholipase C-mediated hydrolysis of inositolphospholipids, as indicated by the stimulation of inositol phosphate release, Ca2+ mobilization, and phosphorylation of a protein kinase C substrate. The results indicate that the deregulation of signal-transduction pathways as elicited by an intracellularly acting bacterial toxin can induce a malignant phenotype.The mechanisms of action of bacterial toxins have provided insights into the control of cellular regulatory processes, including signal transduction and cell proliferation (1-3). Pasteurella multocida toxin (PMT) has been shown to be an extremely potent and effective mitogen for fibroblast cell lines and early-passage cultures (4). The toxin is a monomeric 146-kDa protein that has been purified (5-8), cloned (9-11), and sequenced (12-14). The deduced amino acid sequence of PMT did not reveal any significant homologies with other toxins or proteins (13,14). Both native and recombinant PMT (rPMT) are mitogenic at picomolar concentrations (4). Several lines of evidence indicate that PMT enters the cells and acts intracellularly to initiate and sustain DNA synthesis. Thus, a transient exposure of the cells to the toxin was sufficient to commit them to S phase and cell division. Furthermore, early but not late addition of the lysosomotrophic agent methylamine selectively blocked the mitogenic action of rPMT (4).Prior to the stimulation of DNA synthesis, rPMT enhanced the formation of inositol phosphates (15), increased the cellular content of diacylglycerol, caused the translocation of protein kinase C, and stimulated the phosphorylation of an 80-kDa protein (16) that is a major substrate of protein kinase C (refs. 17-19, and see ref. 20). Furthermore, the binding of epidermal growth factor (EGF) to its receptor was decreased by rPMT, an action ...

Stathmin is a regulatory phosphoprotein that is a target for both cell cycle and cell surface receptor-regulated phosphorylation events. There are at least 14 isoforms of stathmin that migrate on two-dimensional electrophoresis (2-DE): two unphosphorylated, and 12 increasingly phosphorylated proteins. Following extracellular stimuli, stathmin is phosphorylated on four serines (Ser16, Ser25, Ser38, and Ser63) by several kinases, such as mitogen-activated protein (MAP), cdc2 kinase, protein kinase A, and Ca2+/calmodulin-dependent kinase-Gr. While all forms of stathmin are derived from the same protein encoded by a single mRNA, the precise nature of the post-translational modifications has not been clear. In this study we have characterized three rat brain stathmin isoforms, #1, #3 and #4, which electrophorese on 2-DE with apparent molecular weight (Mr)/isoelectric point (pI) values of 15,500/6.2, 15,000/6.1, and 15,000/6.0, respectively. The phosphorylation status of these isoforms was determined using a combination of peptide mapping, matrix-assisted laser desorption/ionization mass spectrometry and electrospray-ionization ion trap mass spectrometry. Stathmin isoform #1 was not phosphorylated, stathmin isoform #3 was phosphorylated on Ser38 only, and stathmin isoform #4 was phosphorylated on Ser38; however, the phosphorylation status of Ser63 could not be determined. In addition, three proteins which electrophorese near stathmin were identified in order to more accurately define the Mr/pI locus of this region of the 2-DE gel map. These include: phosphatidyl ethanolamine binding protein (Mr approximately 18,000/pI 6.0), synuclein forms 2 and 3 (Mr approximately 14,000/pI 5.4), and synuclein form 2 (Mr approximately 15,000/pI 5.0).

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Pasteurella multocida toxin (PMT) is a potent mitogen for Swiss 3T3 fibroblasts and cytotoxic to embryonic bovine lung cells. Site-directed mutagenesis was used to investigate the functional signiticance of a three amino acid motif in PMT that is present in five other bacterial protein toxins which exhibit ADP-ribosyl transferase activity. Crude lysates of mutant clones were fully cytotoxic for embryonic bovine lung cells. Purified mutant toxin was also as effective at stimulating inositol phosphate turnover and nucleic acid synthesis as wild type toxin. We conclude that this motif has no functional signiticance in P~teurella multocida toxin.

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