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Cell cycle-regulated phosphorylation of p21-activated kinase 1
Debra A Thiel1, Melissa K Reeder, Amanda Pfaff
1Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Abstract:
Mammalian p21-activated kinase 1 (Pak1) is a highly conserved effector for the small GTPases Cdc42 and Rac1. In lower eukaryotes, Pak1 homologs are regulated during the cell cycle by phosphorylation. Here, we show that Pak1 is phosphorylated during mitosis in mammalian fibroblasts. This phosphorylation occurs at a single site, Thr 212, within a domain that is unique to Pak1. Cdc2 phosphorylates Pak1 at the identical site in vitro, and inhibition of Cdc2 abolishes Pak1 mitotic phosphorylation in vivo, indicating that Cdc2 is the kinase responsible for phosphorylating Pak1 in mitotic cells. Expression of a Pak1 mutant in which Thr 212 is replaced with a phosphomimic (aspartic acid) has marked effects on the rate and extent of postmitotic spreading of fibroblasts. The mitotic phosphorylation of Pak1 does not alter the basal or Rac-stimulated activity of this kinase, but it does affect the coimmunoprecipitation of at least three proteins with Pak1. These findings are the first to implicate a mammalian Pak in cell cycle regulation and suggest that Pakl, as a result of phosphorylation by Cdc2, alters its association with binding partners and/or substrates that are relevant to the morphologic changes associated with cell division.
Insights
Mammalian p21-activated kinase 1 (Pak1) undergoes mitotic phosphorylation by Cdc2 at Thr 212. This phosphorylation impacts fibroblast morphology post-division by altering Pak1
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mammalian p21-activated kinase 1 (Pak1) is a key effector of Cdc42 and Rac1 small GTPases.
- Pak1 homologs in lower eukaryotes are regulated by cell cycle-dependent phosphorylation.
- The role of Pak1 phosphorylation in mammalian cell cycle regulation remains largely unexplored.
Purpose of the Study:
- To investigate the cell cycle-dependent regulation of mammalian Pak1.
- To identify the specific site and kinase responsible for Pak1 phosphorylation during mitosis.
- To elucidate the functional consequences of Pak1 mitotic phosphorylation on cell behavior.
Main Methods:
- Phosphorylation site mapping in mitotic mammalian fibroblasts.
- In vitro kinase assays using Cdc2 and Pak1.
- Site-directed mutagenesis to create phosphomimic Pak1 mutants.
- Analysis of fibroblast spreading rates and co-immunoprecipitation assays.
Main Results:
- Pak1 is phosphorylated at a single site, Thr 212, during mitosis in mammalian cells.
- Cdc2 directly phosphorylates Pak1 at Thr 212 in vitro and is responsible for its mitotic phosphorylation in vivo.
- A phosphomimetic mutation at Thr 212 significantly alters postmitotic fibroblast spreading and affects Pak1's association with binding partners.
Conclusions:
- Mammalian Pak1 is a novel cell cycle-regulated protein, phosphorylated by Cdc2 during mitosis.
- Mitotic phosphorylation of Pak1 at Thr 212 influences cell morphology and dynamics post-division.
- Pak1's altered interactions with binding partners upon phosphorylation suggest a role in mediating cell division-related morphologic changes.