2002 by The International Union of Biochemistry and Molecular Biology

Printed in U.S.A.

BIOCHEMISTRY

MOLECULAR BIOLOGY EDUCATION

Vol. 30, No. 1, pp. 6263, 2002

AND

Problem-based Learning
The Mechanism of Action of Anthrax Toxin Lethal Factor*
Received for publication, January 10, 2002
Jozsef Szeberenyi
From the Department of Medical Biology, School of Medicine, University of Pecs, H-7624 Pecs,
Szigeti 12, Hungary
Terms to be familiar with before you start to solve the test: signal transduction, anthrax, Bacillus anthracis,
RasH protein, Western blotting, extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase
(MAPK), MAPK/ERK kinase (MEK), SDS-polyacrylamide gel electrophoresis, proteolytic cleavage,
phosphorylation, ubiqitination, glycosylation, growth factor signaling, stress-activated protein kinases.

Anthrax, an infectious disease caused by Bacillus anthracis, is a life-threatening, often lethal disease. The main
cause of death is anthrax toxin, a product of the bacterium
consisting of several protein components. One of the components is a channel-forming protein that makes penetration of the membrane by the other polypeptides possible.
A component of the toxin called lethal factor then kills the
cell. The experiments described in this test deal with the
effect of lethal factor on signal transduction processes.
Descriptions of the experiments are presented in the figure
legends. Study Figs. 13, and solve the multiple-choice
questions.

FIG. 2. Normal (wild-type) and mutant MEK protein in which

the first seven amino acids had been deleted (7) were incubated in vitro in the presence (samples 2 and 4) or in the
absence of lethal factor (samples 1 and 3) and then analyzed
by Western blotting using anti-MEK(CT). The arrow on the right
side indicates the direction of electrophoresis.

FIG. 3. Mixtures containing normal (LF) or mutant lethal factor (E687C), normal (wt) or mutant MEK (7) and ERK enzyme
were incubated in the combinations shown in the figure, in
the presence of myelin basic protein (MBP) and [-32P]ATP.
Samples were subjected to SDS-polyacrylamide gel electrophoresis. The figure shows the region of the autoradiogram displaying myelin basic protein.

FIG. 1. Mouse fibroblasts expressing an activated RasH

protein were cultured without treatment (C, samples 1, 4,
and 7) or in the presence of lethal factor (LF, samples 2, 5,
and 8) or a lethal factor carrying a point mutation (E687C,
samples 3, 6, and 9). After incubations of various durations
cytoplasmic cell extracts were prepared, and Western blotting
was performed using anti-ERK, anti-pERK (antibody against
phosphorylated ERK), anti-MEK(CT) (antibody against the Cterminal region of MEK), and anti-MEK (NT) (antibody against
the N-terminal region of MEK) antibodies. Arrows on the right
side of the figure indicate the direction of electrophoresis in the
SDS-polyacrylamide gels.

C. Cleaved off the N-terminal region of MEK

D. Inhibited the initiation of MEK synthesis
E. Inhibited the termination of MEK synthesis

2. ____ What changes of myelin basic protein can be

studied under these conditions?
A. Its phosphorylation
B. Its dephosphorylation
C. Its degradation
D. Its ubiquitination
E. Its glycosylation

FIVE-CHOICE COMPLETION

Select the one best answer.

1. ____ Explain the different behavior of anti-MEK(CT)
and anti-MEK(NT) antibodies toward samples 2, 5, and 8.
The lethal factor did which of the following?

EXPERIMENT ANALYSIS

The following statements are related to the information

presented in the description of the experiment. Based on
the information given, select one of the following.

A. Caused complete degradation of MEK

B. Cut MEK into several smaller fragments

A. The statement is supported by the information given

B. The statement is contradicted by the the information given

To whom correspondence should be addressed. E-mail:

joszef.szeberenyi@aok.pte.hu.

62

This paper is available on line at http://www.bambed.org

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C. The statement is neither supported nor contradicted by the
information given

3. ____ The C-terminal region of MEK is required for

growth factor signaling.
4. ____ The N-terminal region of MEK is required for
growth factor signaling.
5. ____ Anthrax lethal factor has proteolytic activity.
6. ____ Anthrax lethal factor causes the phosphorylation
of ERK enzymes.
7. ____ Anthrax lethal factor causes the ubiquitination of
ERK enzymes.
8. ____ MEK phosphoryIates myelin basic protein.
9. ____ Myelin basic protein is the substrate of ERK
enzymes.
10. ____ Myelin basic protein is cleaved by anthrax lethal
factor.
Based on these experiments, what is the mechanism of
the cytocidal action of anthrax lethal factor?
FIVE-CHOICE COMPLETION

Fill in the missing terms by selecting the one best

answer.
11. ____ Anthrax lethal factor __________ the __________
protein thereby blocking signal transduction mediated by
__________.
A. Cleaves, MEK, ERK enzymes
B. Cleaves, MEK, growth factors
C. Phosphorylates, MEK, stress-responsive kinases
D. Cleaves, ERK, MEK enzymes
E. Phosphorylates, ERK, MEK enzymes
MULTIPLE COMPLETION

One or more of the following statements are correct.

Select one.
A. 1, 2, and 3 are correct
B. 1 and 3 are correct
C. 2 and 4 are correct
D. Only 4 is correct
E. All four are correct

12. ____ Based on the mechanism of action of anthrax

lethal toxin described in this test, what kind of drug could
be effective against infection by B. anthracis?

1.
2.
3.
4.

An
An
An
An

1.
2.
3.
4.
5.
6.

C
A
C
A
A
B

agent
agent
agent
agent

inhibiting the entrance of lethal factor into the cell

inhibiting the nuclear translocation of lethal factor
inducing overexpression of MEK
inducing overexpression of ERK
CORRECT ANSWERS

7. B
8. B
9. A
10. C
11. A
12. B
EXPLANATIONS

The major mitogenic signal transduction mechanism

stimulated by growth factors in fibroblasts is the tyrosine
kinase receptor/Ras/Raf/MEK/ERK pathway. This experiment indicates that anthrax lethal factor kills the cells by
interfering with this signaling route.
The main target of lethal factor is the MEK enzyme; its
N-terminal region is proteolytically removed by the toxin
increasing the eletrophoretic mobility of the rest of the
protein. (Ubiquitination or glycosylation of the enzyme
would have resulted in decreased mobility in the Western
blot experiments; MCQ 1, C; MCQ 5, A.) This leads to
reduced phosphorylation (see Fig. 1 and Fig. 2) and activation (as measured by myelin basic protein phosphorylation; see Fig. 3) of ERK (MCQ 2, A; MCQ 9, A; MCQ 11,
A). A set of carefully designed in vitro experiments indicates that the N terminus of MEK is essential for signaling
to ERK (MCQ 3, C; MCQ 4, A) and that the only target of
lethal factor in this system is MEK (MCQ 6, B; MCQ 7, B;
MCQ 8, B; MCQ 10, C; MCQ 11, A). Identification of the
cellular targets of B. anthracis infections opens possibilities to design rational therapeutic approaches against this
deadly microbe (MCQ 12, B).
The test was based on Duesbery, N. S., Webb, C. P.,
Leppla, S. H., Gordon, V. M., Klimpel, K. R., Copeland,
T. D., Ahm, N. G., Oskarsson, M. K., Fukasawa, K., Paull,
K. D., Vande Woude, G. F. (1998) Proteolytic inactivation of
MAP kinase kinase by anthrax lethal factor, Science 280,
734 737.