Tyrosine phosphorylation of Sprouty proteins regulates their ability to inhibit growth factor signaling: a dual feedback loop.
Sprouty proteins are recently identified receptor tyrosine kinase (RTK) inhibitors potentially involved in many developmental processes. Here, we report that Sprouty proteins become tyrosine phosphorylated after growth factor treatment. We identified Tyr55 as a key residue for Sprouty2 phosphorylation and showed that phosphorylation was required for Sprouty2 to inhibit RTK signaling, because a mutant Sprouty2 lacking Tyr55 augmented signaling. We found that tyrosine phosphorylation of Sprouty2 affected neither its subcellular localization nor its interaction with Grb2, FRS2/SNT, or other Sprouty proteins. In contrast, Sprouty2 tyrosine phosphorylation was necessary for its binding to the Src homology 2-like domain of c-Cbl after fibroblast growth factor (FGF) stimulation. To determine whether c-Cbl was required for Sprouty2-dependent cellular events, Sprouty2 was introduced into c-Cbl-wild-type and -null fibroblasts. Sprouty2 efficiently inhibited FGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 in c-Cbl-null fibroblasts, thus indicating that the FGF-dependent binding of c-Cbl to Sprouty2 was dispensable for its inhibitory activity. However, c-Cbl mediates polyubiquitylation/proteasomal degradation of Sprouty2 in response to FGF. Last, using Src-family pharmacological inhibitors and dominant-negative Src, we showed that a Src-like kinase was required for tyrosine phosphorylation of Sprouty2 by growth factors. Thus, these data highlight a novel negative and positive regulatory loop that allows for the controlled, homeostatic inhibition of RTK signaling.
Division of Hematology/Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.
Jacqueline M
JM
Mason
Debra J
DJ
Morrison
Bhramdeo
B
Bassit
Manjari
M
Dimri
Hamid
H
Band
Jonathan D
JD
Licht
Isabelle
I
Gross
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D005346:Fibroblast Growth Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology; D051380:GRB2 Adaptor Protein; D047908:Intracellular Signaling Peptides and Proteins; D042541:Intracellular Space / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D041681:NIH 3T3 Cells; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C488187:FRS2alpha protein, mouse; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; C425468:Spry1 protein, mouse; D014443:Tyrosine; D005346:Fibroblast Growth Factors; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D020794:Receptor Protein-Tyrosine Kinases; D019061:src-Family Kinases; D017346:Protein Serine-Threonine Kinases; C118389:Spry2 protein, mouse; D048052:Mitogen-Activated Protein Kinase 3; C493849:Cbl protein, mouse