Spred-2 steady-state levels are regulated by phosphorylation and Cbl-mediated ubiquitination.
Spred proteins modulate growth factor receptor signaling by inhibiting the Ras-MAPK cascade. Here, we show that Spred-1, Spred-2, and Spred-3 are ubiquitinated in HEK293T cells stimulated with epidermal growth factor (EGF) or pervanadate. Spred-2 tyrosines Y228 and/or Y231 in the Kit binding domain were identified as putative phosphorylation site(s) critical for Spred-2 ubiquitination. Depletion of Cbl and Cbl-b E3 ubiquitin ligases by RNA interference, or overexpression of a Cbl dominant inhibitory mutant (Cbl-N), inhibited Spred-2 ubiquitination, while conversely, wild type Cbl enhanced Spred-2 ubiquitination. Interaction of Spred-2 with Cbl-N was detectable by co-immunoprecipitation and required the Cbl SH2 domain and Spred-2 Y228 and Y231 residues. Studies on endogenous Spred-2 in ME4405 melanoma cells showed that pervanadate induced Spred-2 ubiquitination and a marked reduction in Spred-2 steady-state levels that was partially blocked by the proteasomal inhibitor, MG-132. These results suggest a role for Spred-2 tyrosine phosphorylation and ubiquitination in controlling Spred-2 expression levels.
Lock
Peter
P
Cell Signaling Laboratory, Department of Surgery, University of Melbourne, Royal Melbourne Hospital, Parkville 3050, Australia. petelock@unimelb.edu.au
I
Stacey T T
ST
Straffon
Andrew F L
AF
Schieb
Heinke
H
Hovens
Christopher M
CM
Stylli
Stanley S
SS
D048868:Adaptor Proteins, Signal Transducing; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D050721:Proto-Oncogene Proteins c-cbl / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012097:Repressor Proteins / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D047428:Protein Kinase Inhibitors; D012097:Repressor Proteins; C504812:SPRED1 protein, human; C500760:SPRED2 protein, human; C511416:SPRED3 protein, human; D025801:Ubiquitin; C076715:pervanadate; D014638:Vanadates; D014443:Tyrosine; D004815:Epidermal Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D017027:Protein Tyrosine Phosphatases; C067419:CBL protein, human