Identification of RhoGAP22 as an Akt-dependent regulator of cell motility in response to insulin

TY - JOUR

T1 - Identification of RhoGAP22 as an Akt-dependent regulator of cell motility in response to insulin

AU - Rowland, Alexander F.

AU - Larance, Mark

AU - Hughes, William E.

AU - James, David E.

PY - 2011/12

Y1 - 2011/12

N2 - 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.

AB - 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.

KW - Cell Movement

KW - Animals

KW - 14-3-3 Proteins

KW - GTPase-Activating Proteins

KW - Humans

KW - Catalytic Domain

KW - Proto-Oncogene Proteins c-akt

KW - Mice

KW - Amino Acid Sequence

KW - Protein Binding

KW - Insulin

KW - Binding Sites

KW - Rats

KW - Gene Knockdown Techniques

KW - Phosphorylation

KW - Conserved Sequence

KW - Molecular Sequence Data

KW - RNA Interference

KW - Protein Structure, Tertiary

KW - Amino Acid Substitution

KW - Cell Line

KW - Cricetinae

U2 - 10.1128/MCB.05583-11

DO - 10.1128/MCB.05583-11

M3 - Article

C2 - 21969604

SN - 1098-5549

VL - 31

SP - 4789

EP - 4800

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

IS - 23

ER -