Abstract
Phostensin, a protein phosphatase 1 F-actin cytoskeleton-targeting subunit encoded by KIAA1949, consists of 165 amino acids and caps the pointed ends of actin filaments. Sequence alignment analyses suggest that the C-terminal region of phostensin, spanning residues 129 to 155, contains a consensus actin-binding motif. Here, we have verified the existence of an actin-binding motif in the C-terminal domain of phostensin using colocalization, F-actin co-sedimentation and single filament binding assays. Our data indicate that the N-terminal region of phostensin (1–129) cannot bind to actin filaments and cannot retard the pointed end elongation of gelsolin-actin seeds. Furthermore, the C-terminal region of phostensin (125–165) multiply bind to the sides of actin filaments and lacks the ability to block the pointed end elongation, suggesting that the actin-binding motif is located in the C-terminal region of the phostensin. Further analyses indicate that phostensin binding to the pointed end of actin filament requires N-terminal residues 35 to 51. These results suggest that phostensin might fold into a rigid structure, allowing the N-terminus to sterically hinder the binding of C-terminus to the sides of actin filament, thus rendering phostensin binding to the pointed ends of actin filaments.
Highlights
Phostensin, a protein phosphatase 1 F-actin cytoskeleton targeting subunit, encoded by KIAA1949, consists of 165 amino acids with a consensus protein phosphatase 1 (PP1)-binding motif located at residues 91–94 (K91ISF94) [1]
Sequence analyses suggest that the C-terminal domain, spanning phostensin residues 129 to 155, contains an actin-binding motif with conserved amino acids in a leucine-rich array (Figure 1), which is often found in actin-binding proteins [10]
Phostensin consists of 165 amino acids, and the consensus actin-binding motif is located at the C-terminal region between residues 129 to 155
Summary
Phostensin, a protein phosphatase 1 F-actin cytoskeleton targeting subunit, encoded by KIAA1949, consists of 165 amino acids with a consensus protein phosphatase 1 (PP1)-binding motif located at residues 91–94 (K91ISF94) [1]. Phostensin is a ubiquitous protein in mature human leukocytes and leukemia cell lines [5] It is mainly concentrated at the cell periphery and colocalizes with actin filaments and protein phosphatase 1 (PP1). Several types of actin-binding proteins (ABPs) have been found to bind to different sites on actin (including monomeric G-actin), actin filament caps and filament sides. We find that phostensin, an F-actin pointed end-capping protein, contains a consensus actin-binding motif, located in the C-terminal domain. Phostensin mutants, which lack the N-terminal domain, bind to the sides of actin filaments, suggesting that the N-terminal region of phostensin is responsible for capping the pointed ends of actin filaments
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