RGS16 function is regulated by epidermal growth factor receptor-mediated tyrosine phosphorylation.
Galpha(i)-coupled receptor stimulation results in epidermal growth factor receptor (EGFR) phosphorylation and MAPK activation. Regulators of G protein signaling (RGS proteins) inhibit G protein-dependent signal transduction by accelerating Galpha(i) GTP hydrolysis, shortening the duration of G protein effector stimulation. RGS16 contains two conserved tyrosine residues in the RGS box, Tyr(168) and Tyr(177), which are predicted sites of phosphorylation. RGS16 underwent phosphorylation in response to m2 muscarinic receptor or EGFR stimulation in HEK 293T or COS-7 cells, which required EGFR kinase activity. Mutational analysis suggested that RGS16 was phosphorylated on both tyrosine residues (Tyr(168) Tyr(177)) after EGF stimulation. RGS16 co-immunoprecipitated with EGFR, and the interaction did not require EGFR activation. Purified EGFR phosphorylated only recombinant RGS16 wild-type or Y177F in vitro, implying that EGFR-mediated phosphorylation depended on residue Tyr(168). Phosphorylated RGS16 demonstrated enhanced GTPase accelerating (GAP) activity on Galpha(i). Mutation of Tyr(168) to phenylalanine resulted in a 30% diminution in RGS16 GAP activity but completely eliminated its ability to regulate G(i)-mediated MAPK activation or adenylyl cyclase inhibition in HEK 293T cells. In contrast, mutation of Tyr(177) to phenylalanine had no effect on RGS16 GAP activity but also abolished its regulation of G(i)-mediated signal transduction in these cells. These data suggest that tyrosine phosphorylation regulates RGS16 function and that EGFR may potentially inhibit Galpha(i)-dependent MAPK activation in a feedback loop by enhancing RGS16 activity through tyrosine phosphorylation.
Derrien
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A
Molecular Signal Transduction Section, Laboratory of Allergic Diseases, National Institutes of Health, Rockville, Maryland 20852, USA.
Druey
K M
KM
D000595:Amino Acid Sequence; D002460:Cell Line; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry / Q000502:physiology*; D020710:RGS Proteins / Q000737:chemistry / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*
D011506:Proteins; D020710:RGS Proteins; C107413:RGS16 protein; D014443:Tyrosine; D066246:ErbB Receptors