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...
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| Veröffentlicht in: | Biochemical and biophysical research communications 2006-12, Vol.351 (4), p.1018-1023 |
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| container_title | Biochemical and biophysical research communications |
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| creator | Lock, Peter I, Stacey T.T. Straffon, Andrew F.L. Schieb, Heinke Hovens, Christopher M. Stylli, Stanley S. |
| description | 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. |
| doi_str_mv | 10.1016/j.bbrc.2006.10.150 |
| format | Article |
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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.</description><identifier>ISSN: 0006-291X</identifier><identifier>EISSN: 1090-2104</identifier><identifier>DOI: 10.1016/j.bbrc.2006.10.150</identifier><identifier>PMID: 17094949</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Cbl ; 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 ; Proto-Oncogene Proteins c-cbl - metabolism ; Repressor Proteins - metabolism ; RNA Interference ; Spred-2 ; Tyrosine - metabolism ; Ubiquitin - metabolism ; Ubiquitination ; Vanadates - pharmacology</subject><ispartof>Biochemical and biophysical research communications, 2006-12, Vol.351 (4), p.1018-1023</ispartof><rights>2006 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c385t-7a1dce896601cd2bcb82a7ca7c4507195f5db99b196a442495d8dfc8fe72f5973</citedby><cites>FETCH-LOGICAL-c385t-7a1dce896601cd2bcb82a7ca7c4507195f5db99b196a442495d8dfc8fe72f5973</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.bbrc.2006.10.150$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17094949$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lock, Peter</creatorcontrib><creatorcontrib>I, Stacey T.T.</creatorcontrib><creatorcontrib>Straffon, Andrew F.L.</creatorcontrib><creatorcontrib>Schieb, Heinke</creatorcontrib><creatorcontrib>Hovens, Christopher M.</creatorcontrib><creatorcontrib>Stylli, Stanley S.</creatorcontrib><title>Spred-2 steady-state levels are regulated by phosphorylation and Cbl-mediated ubiquitination</title><title>Biochemical and biophysical research communications</title><addtitle>Biochem Biophys Res Commun</addtitle><description>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.</description><subject>Cbl</subject><subject>Cells, Cultured</subject><subject>Epidermal Growth Factor - pharmacology</subject><subject>Humans</subject><subject>Intracellular Signaling Peptides and Proteins - metabolism</subject><subject>Membrane Proteins</subject><subject>Phosphorylation</subject><subject>Protein Kinase Inhibitors - pharmacology</subject><subject>Protein Tyrosine Phosphatases - antagonists & inhibitors</subject><subject>Proto-Oncogene Proteins c-cbl - antagonists & inhibitors</subject><subject>Proto-Oncogene Proteins c-cbl - genetics</subject><subject>Proto-Oncogene Proteins c-cbl - metabolism</subject><subject>Repressor Proteins - metabolism</subject><subject>RNA Interference</subject><subject>Spred-2</subject><subject>Tyrosine - metabolism</subject><subject>Ubiquitin - metabolism</subject><subject>Ubiquitination</subject><subject>Vanadates - pharmacology</subject><issn>0006-291X</issn><issn>1090-2104</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkE1LAzEQhoMotlb_gAfZk7etk3SzuwEvUvyCggcVPAghH7Oast1tk91C_73pB3hTkjDwzjMTeAi5pDCmQPOb-Vhrb8YMIB9vMw5HZEhBQMooZMdkCLGTMkE_BuQshDkApVkuTsmAFiCyeIbk83Xp0aYsCR0qu0lDpzpMalxjHRLlMfH41dcxs4neJMvvNsTnNzFxbZOoxiZTXacLtG7H9Nqtete5Ztc_JyeVqgNeHOqIvD_cv02f0tnL4_P0bpaaScm7tFDUGixFngM1lmmjS6YKE2_GoaCCV9xqITQVucoylgluS1uZssKCVVwUkxG53u9d-nbVY-jkwgWDda0abPsg85JykRXsXzD-BeUkLyPI9qDxbQgeK7n0bqH8RlKQW_lyLrfy5Vb-LuMQh64O23sdjfyOHGxH4HYPRLm4duhlMA4bE-15NJ20rftr_w8JOJau</recordid><startdate>20061229</startdate><enddate>20061229</enddate><creator>Lock, Peter</creator><creator>I, Stacey T.T.</creator><creator>Straffon, Andrew F.L.</creator><creator>Schieb, Heinke</creator><creator>Hovens, Christopher M.</creator><creator>Stylli, Stanley S.</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>20061229</creationdate><title>Spred-2 steady-state levels are regulated by phosphorylation and Cbl-mediated ubiquitination</title><author>Lock, Peter ; I, Stacey T.T. ; Straffon, Andrew F.L. ; Schieb, Heinke ; Hovens, Christopher M. ; Stylli, Stanley S.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c385t-7a1dce896601cd2bcb82a7ca7c4507195f5db99b196a442495d8dfc8fe72f5973</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Cbl</topic><topic>Cells, Cultured</topic><topic>Epidermal Growth Factor - pharmacology</topic><topic>Humans</topic><topic>Intracellular Signaling Peptides and Proteins - metabolism</topic><topic>Membrane Proteins</topic><topic>Phosphorylation</topic><topic>Protein Kinase Inhibitors - pharmacology</topic><topic>Protein Tyrosine Phosphatases - antagonists & inhibitors</topic><topic>Proto-Oncogene Proteins c-cbl - antagonists & inhibitors</topic><topic>Proto-Oncogene Proteins c-cbl - genetics</topic><topic>Proto-Oncogene Proteins c-cbl - metabolism</topic><topic>Repressor Proteins - metabolism</topic><topic>RNA Interference</topic><topic>Spred-2</topic><topic>Tyrosine - metabolism</topic><topic>Ubiquitin - metabolism</topic><topic>Ubiquitination</topic><topic>Vanadates - pharmacology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lock, Peter</creatorcontrib><creatorcontrib>I, Stacey T.T.</creatorcontrib><creatorcontrib>Straffon, Andrew F.L.</creatorcontrib><creatorcontrib>Schieb, Heinke</creatorcontrib><creatorcontrib>Hovens, Christopher M.</creatorcontrib><creatorcontrib>Stylli, Stanley S.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemical and biophysical research communications</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lock, Peter</au><au>I, Stacey T.T.</au><au>Straffon, Andrew F.L.</au><au>Schieb, Heinke</au><au>Hovens, Christopher M.</au><au>Stylli, Stanley S.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Spred-2 steady-state levels are regulated by phosphorylation and Cbl-mediated ubiquitination</atitle><jtitle>Biochemical and biophysical research communications</jtitle><addtitle>Biochem Biophys Res Commun</addtitle><date>2006-12-29</date><risdate>2006</risdate><volume>351</volume><issue>4</issue><spage>1018</spage><epage>1023</epage><pages>1018-1023</pages><issn>0006-291X</issn><eissn>1090-2104</eissn><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. 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| ispartof | Biochemical and biophysical research communications, 2006-12, Vol.351 (4), p.1018-1023 |
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| subjects | Cbl 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 Proto-Oncogene Proteins c-cbl - metabolism Repressor Proteins - metabolism RNA Interference Spred-2 Tyrosine - metabolism Ubiquitin - metabolism Ubiquitination Vanadates - pharmacology |
| title | Spred-2 steady-state levels are regulated by phosphorylation and Cbl-mediated ubiquitination |
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