The tyrosine kinase negative regulator c-Cbl as a RING-type, E2- dependent ubiquitin-protein ligase

Science

Volumn 286, Issue 5438, 1999, Pages 309-312

  Joazeiro, Claudio A P ^a   Wing, Simon S ^b   Huang, Han Kuei ^a   Leverson, Joel D ^a   Hunter, Tony ^a   Liu, Yun Cai ^c  

^a SALK INSTITUTE FOR BIOLOGICAL STUDIES   (United States)

^b MCGILL UNIVERSITY   (Canada)

^c LA JOLLA INSTITUTE FOR ALLERGY AND IMMUNOLOGY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

LIGASE; PROTEIN TYROSINE KINASE; TYROSINE KINASE RECEPTOR;

ARTICLE; CAENORHABDITIS ELEGANS; DROSOPHILA MELANOGASTER; ENZYME ACTIVITY; ENZYME ANALYSIS; LIGATION; NONHUMAN; PRIORITY JOURNAL; PROTEIN DEGRADATION; PROTEIN DOMAIN; SEQUENCE ANALYSIS; SEQUENCE HOMOLOGY; SIGNAL TRANSDUCTION;

AMINO ACID SEQUENCE; CELL LINE; HUMANS; LIGASES; MOLECULAR SEQUENCE DATA; PHOSPHOTYROSINE; POINT MUTATION; PROTO-ONCOGENE PROTEINS; PROTO-ONCOGENE PROTEINS C-CBL; RECEPTOR PROTEIN-TYROSINE KINASES; RECEPTOR, PLATELET-DERIVED GROWTH FACTOR BETA; RECOMBINANT FUSION PROTEINS; SEQUENCE ALIGNMENT; SIGNAL TRANSDUCTION; SRC HOMOLOGY DOMAINS; UBIQUITIN-CONJUGATING ENZYMES; UBIQUITIN-PROTEIN LIGASES; UBIQUITINS;

CAENORHABDITIS ELEGANS; DROSOPHILA MELANOGASTER; MELANOGASTER;

EID: 0033536637     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5438.309     Document Type: Article

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18. DNA fragments encoding the RING finger were amplified by polymerase chain reaction (PCR) of c-Cbl WT and 70Z/3 cDNA with primers CAAGACCATAT-CAAAGTGACCCAG and CTATGCTCCTTGCCTCAACAG. PCR products were subcloned first into pCRII, verified by sequencing, and then subcloned into the Eco RI site of pGEX-4T2. Point mutations were generated with QuikChange (Stratagene) with pGEX-4T2-RING as template. pGEX-cbl 1-480 (encoding GST-SH2-RING) was a gift from L. Samelson. GST fusion proteins were produced in E. coli BL21 (DE3) by induction with 0.4 mM isopropyl-β-D-thiogalactopyranoside for 3 hours at 30°C Cells were lysed in 50 mM tris-HCl (pH 8), 120 mM NaCl, 1 mM dithiothreitol (DTT), and protease inhibitors. Proteins bound to glutathione-Sepharose beads were eluted with lysis buffer containing 20 mM glutathione and dialyzed against lysis buffer containing 50 mM NaCl and 10% glycerol.
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41. n-Rβ). This measure is an underestimate, because oligoubiquitinated precursor Rβ (160 kD) cannot be resolved from unmodified mature Rβ (190 kD), and oligoubiquitinated and unmodified mature Rβ bands are not resolved in 7.5% acrytamide gels.
42. We thank M. Nakao, L. Samelson, F. Yamao, and A. Kumar for reagents; D. Koepp for advice; A. Altman, W. Langdon, G. A. Kassavetis, J. Noel. M. Verdecia, and the Hunter laboratory for discussions; E. P. Geiduschek for comments on the manuscript; C. Elly, J. Meisenhelder, S. Simon, and A. Young for help, and F. Orzalesi for encouragement. This work was supported by Fellowship 2-41-98 from the American Cancer Society, California Division (C.A.P.J.), by Medical Research Council of Canada grant MT12121 (S.S.W.), by Fellowship DRG1531 from the Damon Runyon-Walter Winchell Foundation (H.-k.H.), by American Cancer Society grant PF9922801CCG and NIH grant T32CA09523 (J.D.L.), by grant R01 DK56558 from NIH (Y.-C.L.), and by U.S. Public Health Service grants CA39780 and CA82683 from the National Cancer Institute (T.H.). S.S.W. is a recipient of a Clinician-Scientist Award from the Medical Research Council of Canada; T.H. is a Frank and Else Schilling American Cancer Society Professor.