|Yeoh, S., Pope, B., Mannherz, H. G. &
Weeds, A. (2002) Determining the Differences in Actin Binding by Human
ADF and Cofilin. J.Mol.Biol. 315, 911-925.
|The actin-depolymerizing factor (ADF)/cofilin family of
proteins play an essential role in actin dynamics and cytoskeletal
re-organization. Human tissues express two isoforms in the same cells,
ADF and cofilin, and these two proteins are more than 70% identical in
amino acid sequence. We show that ADF is a much more potent
actin-depolymerizing agent than cofilin: the maximum level of
depolymerization at pH 8 by ADF is about 20 microM compared to 5 microM
for cofilin, but little depolymerization occurs at pH 6.5 with either
protein. However, we find little difference between the two proteins in
their binding to filaments, their severing activities or their
activation of subunit release from the pointed ends of filaments.
Likewise, they show no significant differences in their affinities for
monomeric actin: both bind 15-fold more tightly to actin.ADP than to
actin.ATP. Complexes between actin.ADP and ADF or cofilin associate with
both barbed and pointed ends of filaments at similar rates (close to
those of actin.ATP and much higher than those of actin.ADP). This
explains why high concentrations of both proteins reverse the activation
of subunit release at pointed ends. The major difference between the two
proteins is that the nucleating activity of cofilin-actin.ADP complexes
is twice that of ADF-actin.ADP complexes and this, in turn, is twice
that of actin.ATP alone. It is this weaker nucleating potential of ADF-actin.ADP
that accounts for the much higher steady-state depolymerizing activity.
The pH-sensitivity is due to the nucleating activity of complexes being
greater at pH 6.5 than at pH 8. Sequence analysis of mammalian and avian
isoforms shows a consistent pattern of charge differences in regions of
the protein associated with F-actin-binding that may account for the
differences in activity between ADF and cofilin.