A quantitative proteomic view of cofilin phosphorylation and its modulation using the LIM kinase inhibitor Pyr1

LIM kinases are located at a strategic crossroad, downstream of several signaling pathways and upstream of effectors such as microtubules and the actin cytoskeleton.. Cofilin is the only LIM kinases substrate that is well described to date, and its phosphorylation on serine 3 by LIM Kinases controls cofilin actin-severing activity. Consequently, LIM Kinases inhibition leads to actin cytoskeleton disorganization and blockade of cell motility, which makes this strategy attractive in anticancer strategies. We have used proteomic approaches to investigate quantitatively and in detail the phosphorylation status of cofilin in myeloid tumor cell lines of murine and human origin. Our results show that under standard conditions, only a relatively small fraction (10 to 30% depending on the cell line) of cofilin is phosphorylated (including serine 3 phosphorylation). In addition, after a pharmacological inhibition of LIM kinases, a residual cofilin phosphorylation is observed on serine 3. Interestingly, this 2D gel based proteomic study identified new phosphorylation sites on cofilin, such as threonine 63, tyrosine 82 and serine 108.