Abstract
Insulin exerts many of its metabolic actions via the canonical phosphatidylinositide 3 kinase (PI3K)/Akt pathway, leading to phosphorylation and 14-3-3 binding of key metabolic targets. We previously identified a GTPase-activating protein (GAP) for Rac1 called RhoGAP22 as an insulin-responsive 14-3-3 binding protein. Insulin increased 14-3-3 binding to RhoGAP22 fourfold, and this effect was PI3K dependent. We identified two insulin-responsive 14-3-3 binding sites (pSer(16) and pSer(395)) within RhoGAP22, and mutagenesis studies revealed a complex interplay between the phosphorylation at these two sites. Mutating Ser(16) to alanine blocked 14-3-3 binding to RhoGAP22 in vivo, and phosphorylation at Ser(16) was mediated by the kinase Akt. Overexpression of a mutant RhoGAP22 that was unable to bind 14-3-3 reduced cell motility in NIH-3T3 fibroblasts, and this effect was dependent on a functional GAP domain. Mutation of the catalytic arginine of the GAP domain of RhoGAP22 potentiated growth factor-stimulated Rac1 GTP loading. We propose that insulin and possibly growth factors such as platelet-derived growth factor may play a novel role in regulating cell migration and motility via the Akt-dependent phosphorylation of RhoGAP22, leading to modulation of Rac1 activity.
Original language | English |
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Pages (from-to) | 4789-4800 |
Number of pages | 12 |
Journal | Molecular and Cellular Biology |
Volume | 31 |
Issue number | 23 |
DOIs | |
Publication status | Published - Dec 2011 |
Keywords
- Cell Movement
- Animals
- 14-3-3 Proteins
- GTPase-Activating Proteins
- Humans
- Catalytic Domain
- Proto-Oncogene Proteins c-akt
- Mice
- Amino Acid Sequence
- Protein Binding
- Insulin
- Binding Sites
- Rats
- Gene Knockdown Techniques
- Phosphorylation
- Conserved Sequence
- Molecular Sequence Data
- RNA Interference
- Protein Structure, Tertiary
- Amino Acid Substitution
- Cell Line
- Cricetinae