A key serine for the GTPase-activating protein function of regulator of G protein signaling proteins is not a general target for 14-3-3 interactions.
Mammalian regulator of G protein signaling (RGS) proteins are highly conserved within the RGS domain. Of amino acids that are universal, a serine residue at the C terminus of this domain has been described as the binding site in RGS7 for 14-3-3 proteins. However, studies with the related RGS3 indicate that the site of interaction is not within the RGS domain. We confirm that the interaction of RGS3 with 14-3-3tau and 14-3-3zeta requires Ser264 and not the RGS domain and show both that mutation of the conserved RGS domain serine, Ser496 in RGS3, to either alanine or aspartate does not prevent binding of 14-3-3 proteins and that 14-3-3 proteins do not inhibit GTPase-activating protein (GAP) activity against receptor-activated Galpha(o1). However, mutation of Ser496 does directly impair the action of RGS3 as a GAP against receptor-activated Galpha(o1). We mutated the equivalent serine residue in the family B/R4 RGS proteins RGS1 and RGS16. Using two distinct assay formats, conversion to aspartate virtually abolished GAP activity, whereas conversion to alanine decreased potency 20-fold. Neither alteration modulated interactions with 14-3-3tau or 14-3-3zeta, but the 14-3-3 proteins did not modulate the GAP activity of the wild-type or mutant RGS proteins. Although interactions between 14-3-3 proteins and many RGS proteins can be observed, this does not involve this conserved serine and does not inherently modify GAP function.
Ward
Richard J
RJ
Molecular Pharmacolofy Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
Milligan
Graeme
G
D048948:14-3-3 Proteins / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D005136:Eye Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D019204:GTP-Binding Proteins; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051379:Mice; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012694:Serine / Q000378:metabolism*; D014162:Transfection
D048948:14-3-3 Proteins; D005136:Eye Proteins; D020690:GTPase-Activating Proteins; D020710:RGS Proteins; C107413:RGS16 protein; C485898:RGS3 protein, human; C492039:RGS7 protein, human; D012694:Serine; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go