Table 3.1: Effect of phosphatase inhibitors on phosphorylation of PAK and cofilin.
Calcineurin Inhibitors PP1/PP2A Inhibitors
Protein steady-state NMDA steady-state NMDA
phospho-cofilin increased no change highly increased no change phospho-PAK1-3 increased no change highly increased ?
To confirm the activation of actin-regulatory pathways by NMDA, we measured phosphorylation of PAK isoforms 1–3 and phospho-cofilin. We observed a hundred- fold increase in basal phospho-PAK1-3 when PP1 and PP2A are inhibited with okadaic acid. This observation is consistent with prior demonstrations of increases in phospho-PAK in neutrophils in response to okadaic acid (Westphal et al. 1999; Zhan et al. 2003, but see Chan et al. 2008). The present findings also further support the hypothesis that PAK1-3 does not phosphorylate cofilin in spines.
Due to the increased phosphorylation of PAK1-3 when PP1 and PP2A are in- hibited, we were unable to definitively assess the effect of adding NMDA. Measure- ments of the increased phosphorylation signal have an error margin larger than the magnitude of the NMDA-dependent increase observed when PP1 and PP2A are not inhibited. Thus, it is difficult to observe an NMDA-dependent increase in the signal.
An alternate consideration concerns the total amount of PAK that can be phospho- rylated. If PAK is maximally phosphorylated when PP1 and PP2A are inhibited, there may be no further response to NMDA.
Although we did not identify the phosphatase involved in the NMDA-dependent dephosphorylation of cofilin, this evidence indicates that the most promiscuous phos-
phatases are not likely candidates. We also confirmed the presence of two cofilin- specific phosphatases in the system.
As discussed in the introduction, a dominant negative form of PAK can rescue cytoskeletal and behavioral deficits in a mouse model of Fragile X syndrome. However, our work shows that the PAK signaling observed in most other systems is altered in neurons, as a hundred-fold increase in phospho-PAK does not increase phospho-cofilin levels in neurons. Understanding neuron-specific regulation of PAK and cofilin is critical for implementation of treatments that will target PAK and cofilin regulation.
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Chapter 4
Arc protein interactions
Holly Beale, Mary Kennedy