Structure/function analysis of the interaction of phosphatidylinositol 4,5-bisphosphate with actin-capping protein: implications for how capping protein binds the actin filament.
The heterodimeric actin-capping protein (CP) can be inhibited by polyphosphoinositides, which may be important for actin polymerization at membranes in cells. Here, we have identified a conserved set of basic residues on the surface of CP that are important for the interaction with phosphatidylinositol 4,5-bisphosphate (PIP(2)). Computational docking studies predicted the identity of residues involved in this interaction, and functional and physical assays with site-directed mutants of CP confirmed the prediction. The PIP(2) binding site overlaps with the more important of the two known actin-binding sites of CP. Correspondingly, we observed that loss of PIP(2) binding correlated with loss of actin binding among the mutants. Using TIRF (total internal reflection fluorescence) microscopy, we observed that PIP(2) rapidly converted capped actin filaments to a growing state, consistent with uncapping. Together, these results extend our understanding of how CP binds to the barbed end of the actin filament, and they support the idea that CP can "wobble" when bound to the barbed end solely by the C-terminal "tentacle" of its beta-subunit.
Kim
Kyoungtae
K
Department of Cell Biology and Physiology.
McCully
Michelle E
ME
Bhattacharya
Nandini
N
Butler
Boyd
B
Sept
David
D
Cooper
John A
JA
D051344:Actin Capping Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D013329:Structure-Activity Relationship
D051344:Actin Capping Proteins; D000199:Actins; D019269:Phosphatidylinositol 4,5-Diphosphate; D021122:Protein Subunits