Serine phosphorylation negatively regulates RhoA in vivo

TY - JOUR

T1 - Serine phosphorylation negatively regulates RhoA in vivo

AU - Ellerbroek, Shawn M

AU - Wennerberg, Krister

AU - Burridge, Keith

PY - 2003/5/23

Y1 - 2003/5/23

N2 - Previous work indicates that RhoA phosphorylation on Ser188 by cAMP or cGMP-dependent kinases inhibits its activity. However, these studies lacked the possibility to directly study phosphorylated RhoA activity in vivo. Therefore, we created RhoA proteins containing phosphomimetic residues in place of the cAMP/cGMP-dependent kinase phosphorylation site. RhoA phosphorylation or phosphomimetic substitution did not affect Rho guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity in vitro but promoted binding to the Rho guanine-dissociation inhibitor as measured by exchange factor competition assays. The in vitro similarities between RhoA phosphomimetic proteins and phosphorylated RhoA allowed us to study function of phosphorylated RhoA in vivo. RhoA phosphomimetic proteins display depressed GTP loading when transiently expressed in NIH 3T3 cells. Stable-expressing RhoA and RhoA(S188A) clones spread significantly slower than mock-transfected or RhoA(S188E) clones. RhoA(S188A) clones were protected from the morphological effects of a cAMP agonist, whereas phosphomimetic clones exhibit stress fiber disassembly similar to control cells. Together, these data provide in vivo evidence that addition of a charged group to Ser188 upon phosphorylation negatively regulates RhoA activity and indicates that this occurs through enhanced Rho guanine-dissociation inhibitor interaction rather than direct perturbation of guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity.

AB - Previous work indicates that RhoA phosphorylation on Ser188 by cAMP or cGMP-dependent kinases inhibits its activity. However, these studies lacked the possibility to directly study phosphorylated RhoA activity in vivo. Therefore, we created RhoA proteins containing phosphomimetic residues in place of the cAMP/cGMP-dependent kinase phosphorylation site. RhoA phosphorylation or phosphomimetic substitution did not affect Rho guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity in vitro but promoted binding to the Rho guanine-dissociation inhibitor as measured by exchange factor competition assays. The in vitro similarities between RhoA phosphomimetic proteins and phosphorylated RhoA allowed us to study function of phosphorylated RhoA in vivo. RhoA phosphomimetic proteins display depressed GTP loading when transiently expressed in NIH 3T3 cells. Stable-expressing RhoA and RhoA(S188A) clones spread significantly slower than mock-transfected or RhoA(S188E) clones. RhoA(S188A) clones were protected from the morphological effects of a cAMP agonist, whereas phosphomimetic clones exhibit stress fiber disassembly similar to control cells. Together, these data provide in vivo evidence that addition of a charged group to Ser188 upon phosphorylation negatively regulates RhoA activity and indicates that this occurs through enhanced Rho guanine-dissociation inhibitor interaction rather than direct perturbation of guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity.

KW - 3T3 Cells

KW - Alkyl and Aryl Transferases/metabolism

KW - Animals

KW - Colforsin/pharmacology

KW - Cyclic AMP-Dependent Protein Kinases/metabolism

KW - GTP Phosphohydrolases/metabolism

KW - GTPase-Activating Proteins/metabolism

KW - Gene Expression

KW - Glutathione Transferase/genetics

KW - Guanine Nucleotide Dissociation Inhibitors/metabolism

KW - Guanine Nucleotide Exchange Factors/metabolism

KW - Guanosine Triphosphate/metabolism

KW - Mice

KW - Mutagenesis

KW - Phosphorylation

KW - Recombinant Fusion Proteins

KW - Serine/metabolism

KW - Transfection

KW - rho-Specific Guanine Nucleotide Dissociation Inhibitors

KW - rhoA GTP-Binding Protein/chemistry

U2 - 10.1074/jbc.M213066200

DO - 10.1074/jbc.M213066200

M3 - Journal article

C2 - 12654918

SN - 0021-9258

VL - 278

SP - 19023

EP - 19031

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 21

ER -