Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases

Science

Volumn 271, Issue 5250, 1996, Pages 822-825

  Rawlings, David J ^a   Scharenberg, Andrew M ^b,c   Park, Hyunsun ^a   Wahl, Matthew I ^a   Lin, Siqi ^b,c   Kato, Roberta M ^a   Fluckiger, Anne Catherine ^a   Witte, Owen N ^a   Kinet, Jean Pierre ^b,c  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b BETH ISRAEL DEACONESS MEDICAL CENTER   (United States)

^c HARVARD MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

B LYMPHOCYTE ANTIGEN; CELL RECEPTOR; LYMPHOCYTE RECEPTOR; PROTEIN TYROSINE KINASE; TYROSINE;

ANIMAL CELL; ARTICLE; AUTOPHOSPHORYLATION; B LYMPHOCYTE ACTIVATION; CONTROLLED STUDY; CROSS LINKING; ENZYME ACTIVATION; MOUSE; NONHUMAN; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION; PROTEIN PROTEIN INTERACTION; SIGNAL TRANSDUCTION;

ANIMALIA;

EID: 0030038840     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5250.822     Document Type: Article

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45. Recombinant vaccinia expressing murine LYNA were constituted as described (14). Wild-type and mutant murine BTK complementary DNA constructs generated by site-directed or polymerase chain reaction (PCR) mutagenesis (1, 22) were subcloned into the pSC-65 vaccinia recombination plasmid. BTK recombinant viruses and a virus with the pSC-65 plasmid alone (control) were selected with standard techniques [P L Earl. N Cooper, B Moss, Current Protocols in Molecular Biology (Green and Wiley-Interscience, New York, 1987)].
46. The EBV-transformed XLA B cell line MB (D J. Rawlings and O N Witte, unpublished data), which was BTK-deficient, was infected in RPMI with 10% calf serum with 8 plaque-forming units (PFU) per cell of the indicated viruses (Fig. 1A) for 14 hours.
47. BTK immunoprecipitations and protein immunoblotting were performed asdescribed (1) [D C. Saffran et al., N. Engl. J Med. 330, 1488 (1994)] with an affinity-purified BTK antiserum. The monoclonal phosphotyrosine antibody 4G10 or 4G10-Biotin (1.0 μg/ ml, Upstate) and horseradish peroxidase (HRP)-conjugated sheep secondary antibody to mouse or strepavidin-HRP conjugate (Amersham) were used, respectively, for antiphosphotyrosine immunoblots as recommended by each manufacturer.
48. 2 flasks 18 hours before infection. The monolayer was washed with Dulbecco's modified Eagle's medium with 0.1% bovine serum albumin and 25 mM Hepes, and then overlaid with 20 ml of the same medium containing 5 to 7 PFU per cell of the indicated viruses (Fig. 1B). Single infections contained an additional 5 to 7 PFU per cell of control virus. Cells were placed at 4°C for 30 min to synchronize the infection and then grown for 6 hours at 37°C. Cells were lysed in 150 mM NaCl, 200 mM sodium borate (pH 8.0). 5 mM EDTA, 5 mM NaF, 5 μM leupeptin, 10 μM pepstatin, 10 μM aprotinin, and 1 mM sodium vanadate. Lysates were spun at 14,000 rpm for 20 to 30 min in an Eppendorf microfuge and the supernatant was subjected to the indicated immunoprecipitations. Coexpression of BTK and LYN in NIH 3T3 cells produced results identical to those obtained with 3T3E cells (13)
49. -32P]ATP, boiled in sample buffer, separated with 10% SDS-PAGE, and subjected to antiphosphotyrosine immunoblotting and autoradiography
50. The antibody to LYN, immunoprecipitation, in vitro kinase assay, and blotting protocols were as described (13, 14).
51. We constructed the LYN(K275G) mutant by using two-step overlap PCR to change bases 908 and 909 of the wild-type murine LYN cDNA from AA to GG, resulting in substitution of Gly for Lys at position 275. The presence of the desired mutation and the absence of other mutations was confirmed by dideoxy chain termination sequencing.
52. 4 cpm, unstimulated and stimulated, respectively) Equal amounts of BTK from unactivated and activated cells were digested on nitrocellulose with trypsin; tyrosinephosphorylated peptides were enriched with antiphosphotyrosine affinity chrornatography and subsequently separated by two-dimensional thin-layer electrophoresis and visualized by autoradiography (32)
53. We thank S. Chakrabarti and B. Moss for the pSC-65 vaccinia recombination plasmid, H. Ochs for the XLA B cell line. A. Satterthwaite and S. Smale for critical reading of the manuscript, and J. Shimaoka for manuscript preparation. Supported by an NIH Physician Scientist Award (AR01912) and NIH grant AR36834 (D J.R.), a Pediatric Scientist Development Award (A.M.S.), NIH training grant CA09120-20 (H.P.), the UCLA Intercampus Medical Genetics Program (GM08243; M.I.W ), the Human Frontier Science Program (A.-C.F.), and the Howard Hughes Medical Institute (O.N.W.)