Abstract
Compared with kinases, the role of protein phosphatases in regulating biological functions is less well understood. Here we show that alpha4, a non-catalytic subunit of the protein phosphatase 2A, plays a major role in the control of cell spreading, migration, and cytoskeletal architecture. Fibroblasts lacking alpha4 were impaired in their ability to spread and migrate compared with wild-type cells, whereas enforced expression of alpha4 promoted cell spreading and migration. These effects were not restricted to fibroblasts. Using a T cell-specific alpha4 transgenic mouse model, increased alpha4 expression was found to increase lymphocyte motility and chemotaxis. Elevated alpha4 expression results in an increase in the GTP-bound state of Rac1, and GTP-bound Rac1 was dramatically reduced in alpha4-deficient cells. A constitutively active mutant of Rac1 rescued the defects of cell spreading and migration caused by alpha4 deletion, while inhibition of Rac1 blocked the ability of alpha4 to promote cell migration. Together, these data define a novel role for the protein phosphatase 2A regulatory subunit alpha4 in the regulation of cell spreading and migration.
Highlights
It has been shown that several protein phosphatases are involved in the regulation of cell migration and spreading, such as phosphatase and tesin homolog [7], Src homology 2-containing protein tyrosine phosphatase-2 [8], protein tyrosine phosphatase ␣ [9], and protein phosphatase 2A (PP2A)3 [10], indicating that protein phosphatases could play an important role in the control of cell motility
Tap42, is a PP2A regulatory subunit that functions in target of rapamycin (TOR)-dependent nutrient sensing [18]
The results demonstrate that the protein phosphatase 2A binding partner ␣4 is a non-redundant regulator of cell spreading and migration
Summary
Cell Lines and DNA Constructs—MEFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml of penicillin, and 100 g/ml of streptomycin. ␣4flox-immortalized MEFs stably expressing Bcl-xL were generated and infected as previously described [19]. For retroviral MIGRI-GFP-Creinfected cells, non-migrated cells were removed using a cotton swab and the transwell chambers were analyzed by fluorescent microscopy using a microscope (Eclipse E300; Nikon) equipped with a 10ϫ objective. In this way, only fluorescent (green) cells that had migrated were visualized and counted. The total abundance of Rac was determined by immunoblot analysis of cell lysates
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