Spred-2 steady-state levels are regulated by phosphorylation and Cbl-mediated ubiquitination.

Abstract

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.

• Authors: Peter Lock, Stacey T T I, Andrew F L Straffon, Heinke Schieb, Christopher M Hovens, Stanley S Stylli
• Journal: Biochemical and biophysical research communications (Biochem Biophys Res Commun) Vol. 351 Issue 4 Pg. 1018-23 (Dec 29 2006) ISSN: 0006-291X [Print] United States
• PMID: 17094949 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Adaptor Proteins, Signal Transducing
• Intracellular Signaling Peptides and Proteins
• Membrane Proteins
• Protein Kinase Inhibitors
• Repressor Proteins
• SPRED1 protein, human
• SPRED2 protein, human
• SPRED3 protein, human
• Ubiquitin
• pervanadate
• Vanadates
• Tyrosine
• Epidermal Growth Factor
• Proto-Oncogene Proteins c-cbl
• Protein Tyrosine Phosphatases
• CBL protein, human

Topics

• Adaptor Proteins, Signal Transducing
• Cells, Cultured
• Epidermal Growth Factor (pharmacology)
• Humans
• Intracellular Signaling Peptides and Proteins (metabolism)
• Membrane Proteins
• Phosphorylation
• Protein Kinase Inhibitors (pharmacology)
• Protein Tyrosine Phosphatases (antagonists & inhibitors)
• Proto-Oncogene Proteins c-cbl (antagonists & inhibitors, genetics, metabolism)
• RNA Interference
• Repressor Proteins (metabolism)
• Tyrosine (metabolism)
• Ubiquitin (metabolism)
• Vanadates (pharmacology)