ACTIN FILAMENT CROSS-LINKING BY CHICKEN GIZZARD FILAMIN IS REGULATED BY PHOSPHORYLATION IN-VITRO

Filamin is a dimeric muscle phosphoprotein that cross-links actin fila ments. We have found that purified chicken gizzard filamin is phosphor ylated in vitro at serine residues by the Ca2+/calmodulin-dependent pr otein kinase II (CaM kinase II). Up to 0.9 mol of phosphate can be inc orporated into 1 mol of filamin dimer. Phosphorylation by CaM kinase I I increases filamin's critical actin filament gelling concentration an d diminishes the amount of actin sedimented by filamin at low G-force. The modulation of filamin function by CaM kinase II requires ATP, Ca2 +, and calmodulin, and it is abolished when CaM kinase II is inactivat ed with heat, Protein phosphatase 2A removed the phosphate added by Ca M kinase II and restored filamin's actin filament cross-linking activi ty to the untreated basal level. In cosedimentation experiments, phosp horylation reduces the binding of filamin to actin filaments. The K-d for binding of filamin to actin filaments increases similar to 2-fold, from 3.2 to 6.9 mu M, following CaM kinase II-mediated phosphorylatio n. Phosphorylation by CaM kinase II, therefore, regulates the binding of filamin to actin filaments.