Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes.
We have observed that stimulation of human natural killer cells with dibutyryl cAMP (Bt2cAMP) reproduced the effects of ADP ribosylation of the GTP binding protein RhoA by Clostridium botulinum C3 transferase: both agents induced similar morphological changes, inhibited cell motility and blocked the cytolytic function. We demonstrate here that cAMP-dependent protein kinase A (PKA) phosphorylates RhoA in its C-terminal region, on serine residue 188. This phosphorylation does not affect the ability of recombinant RhoA to bind guanine nucleotides, nor does it modify its intrinsic GTPase activity. However, treatment of cells with Bt2cAMP results in the translocation of membrane-associated RhoA towards the cytosol. Experiments using purified membrane preparations indicated that Rho-GDP dissociation inhibitor, which can complex phosphorylated RhoA in its GTP-bound state, was the effector of this translocation. Taken together, these data suggest that PKA phosphorylation of RhoA is a central event in mediating the cellular effects of cAMP, and support the existence of an alternative pathway for terminating RhoA signalling whereby GTP-bound RhoA, when phosphorylated, could be separated from its putative effector(s) independently of its GTP/GDP cycling.
Lang
P
P
INSERM, Chatenay-Malabry, France.
Gesbert
F
F
Delespine-Carmagnat
M
M
Stancou
R
R
Pouchelet
M
M
Bertoglio
J
J
D036002:ADP Ribose Transferases / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D001693:Biological Transport, Active / Q000187:drug effects; D001905:Botulinum Toxins; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein
D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D003994:Bucladesine; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D017868:Cyclic AMP-Dependent Protein Kinases; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein