Figure 4.

Limk1 phosphorylation sites are required for actin filament stabilization. HeLa cells were transfected with (A) WT FLAG–Limk1 or (B–D) the indicated mutants and treated for 24 h without or with PKI (10 μM) to inhibit PKA-mediated phosphorylation. Cells were studied by using immunofluorescence for FLAG and F-actin (phalloidin). Note that WT Limk1 causes actin filament formation only in the absence of PKI, whereas the S596D phosphomimetic mutation causes actin bundling regardless of the activity of PKA. The serine-to-alanine mutants S323A and S596A cause minimal changes in the actin cytoskeleton. PKA, protein kinase A; PKI, PKA inhibitor; WT, wild type.