We have identified a novel protein, protein phosphatase I F-actin cytoskeleton targeting subunit (phostensin). This protein is encoded by KIAA1949 and was found to associate with protein phosphatase I (PPI) in the yeast two-hybrid assay, co-immunoprecipitation, and GST pull-down assay. Northern blot analysis revealed that phostensin mRNA was predominantly distributed in leukocytes and spleen, and phostensin protein was present in crude extracts of human peripheral leukocytes. Immunofluorescence microscopic analysis revealed that the phostensin/PPI complex was conspicuously localized with the actin cytoskeleton at the cell periphery in Madin-Darby canine kidney (MDCK) epithelial cells. Taken together, our data shows that phostensin targets PPI to F-actin cytoskeleton. The phostensin/PPI complex may play a vital role in modulation of actin rearrangements. (c) 2007 Elsevier Inc. All rights reserved.