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 -