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.
Subject(s)
Proto-Oncogene Proteins c-cbl/metabolism , Repressor Proteins/metabolism , Ubiquitin/metabolism , 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 , Proto-Oncogene Proteins c-cbl/genetics , RNA Interference , Tyrosine/metabolism , Vanadates/pharmacologyABSTRACT
BACKGROUND/AIMS: The developed liver is able to tightly control cellular proliferation, rapidly switching from quiescence to growth in response to specific stimuli. This suggests that growth inhibitors may be involved in the control of liver growth. We analyzed the role of the Spred-family of growth inhibitors in the liver. METHODS: We screened human EST databases for Spred-related sequences. Clones were isolated, sequenced, epitope-tagged and expressed. Subcellular localization of clones were determined and their effects on cellular signaling pathways analysed using specific antibodies. Cell cycle progression assays and protein interaction studies were initiated. Organ distribution of transcripts and their expression throughout liver development and in primary hepatocytes were recorded. RESULTS: We have identified a new, liver-restricted protein, Eve-3, containing a single Ena Vasp homology (EVH1) domain that can potently block activation of the Ras/MAPK pathway. Eve-3 is specific in inhibiting the Ras/MAPK pathway. Eve-3 can block serum-mediated cell cycle progression and its expression is highly regulated during liver development. CONCLUSIONS: The liver is the only organ that can regulate its growth and mass. Eve-3 may act as an inhibitor of proliferation pathways in the mature liver and be involved in modulating the unique regenerative capacity of this organ.
Subject(s)
Growth Inhibitors/physiology , Liver/chemistry , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Repressor Proteins/physiology , ras Proteins/antagonists & inhibitors , Amino Acid Sequence , Animals , Cell Cycle/physiology , Cell Differentiation/physiology , Cell Line , Cell Proliferation , Cells, Cultured , DNA-Binding Proteins/analysis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Gene Expression Regulation , Genetic Vectors/analysis , Genetic Vectors/genetics , Growth Inhibitors/analysis , Growth Inhibitors/genetics , Hepatocytes/chemistry , Hepatocytes/cytology , Hepatocytes/physiology , Humans , Liver/growth & development , Liver/physiology , Male , Molecular Sequence Data , Protein Structure, Tertiary/physiology , Rats , Rats, Sprague-Dawley , Repressor Proteins/analysis , Repressor Proteins/genetics , TransfectionABSTRACT
Sprouty and Spred {Sprouty-related EVH1 [Ena/VASP (vasodilator-stimulated phosphoprotein) homology 1] domain} proteins have been identified as antagonists of growth factor signalling pathways. We show here that Spred-1 and Spred-2 appear to have distinct mechanisms whereby they induce their effects, as the Sprouty domain of Spred-1 is not required to block MAPK (mitogen-activated protein kinase) activation, while that of Spred-2 is required. Similarly, deletion of the C-terminal Sprouty domain of Spred-1 does not affect cell-cycle progression of G(0)-synchronized cells through to S-phase following growth factor stimulation, while the Sprouty domain is required for Spred-2 function. We also demonstrate that the inhibitory function of Spred proteins is restricted to the Ras/MAPK pathway, that tyrosine phosphorylation is not required for this function, and that the Sprouty domain mediates heterodimer formation of Spred proteins. Growth-factor-mediated activation of the small GTPases, Ras and Rap1, was able to be regulated by Spred-1 and Spred-2, without affecting receptor activation. Taken together, these results highlight the potential for different functional roles of the Sprouty domain within the Spred family of proteins, suggesting that Spred proteins may use different mechanisms to induce inhibition of the MAPK pathway.
