Activation of Pyk2 by stress signals and coupling with JNK signaling pathway

Science

Volumn 273, Issue 5276, 1996, Pages 792-794

  Tokiwa, G ^a   Dikic, I ^a   Lev, S ^b   Schlessinger, J ^a  

^a NEW YORK UNIVERSITY MEDICAL CENTER   (United States)

^b SUGEN INC   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CYTOKINE; GUANINE NUCLEOTIDE BINDING PROTEIN; ONCOPROTEIN; PROTEIN TYROSINE KINASE; TUMOR NECROSIS FACTOR ALPHA;

ANIMAL CELL; ARTICLE; CELL STRAIN HL 60; CONTROLLED STUDY; ENZYME ACTIVATION; HUMAN; HUMAN CELL; NONHUMAN; ONCOGENE C JUN; OSMOLARITY; OSMOTIC STRESS; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION; RAT; SIGNAL TRANSDUCTION; ULTRAVIOLET RADIATION;

ANIMALIA;

EID: 0029770721     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5276.792     Document Type: Article

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19. Cell lysis, Pyk2 immunoprecipitations, and immunoblotting were carried out as previously described (11). Polyclonal antibodies to amino acids 684 to 1009 of human Pyk2 cDNA (anti-Pyk2) expressed as a GST fusion protein in pGEX-2T (Pharmacia Biotech) and phosphotyrosine antibodies were used. Equal amounts of total cell lysate (500 to 700 μg) as determined by Bradford assay (Bio-Rad) were subjected to immunoprecipitation.
20. 32P]adenosine triphosphate was added, and the phosphorylation of the probe was analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. JNK activity was determined by quantitation of the amount of GST-c-Jun(1-79) phosphorylation with a Molecular Dynamics Phosphorlmager and ImageQuant software (Molecular Dynamics).
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22. 293T cells grown on 10-cm plates were transiently transfected [lipofectamine reagent (Gibco BRL)] with various amounts (5, 10, or 15 μg) of expression vector alone (pRK5); 5 or 10 μg of Pyk2 expression vector together with various amounts of pRK5 (5 or 10 μg); or 5 μg of Pyk2 together with 10 or 20 μg of a dominant-negative mutant of Pyk2 (PKM). COS and PC-12 cells on 10-cm plates were transiently transfected with 10 or 20 μg of pRK5 and 10 or 20 μ-g of PKM. After 48 hours, the cells were treated as described (11, 13).
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28. We thank M. Karin, A. Lin, A. Abo, J. Vilcek, and E. Skolnik for plasmids and reagents; S. Earp and members of the Schlessinger lab for their helpful discussions; M. Cleary and G. Rodrigues for comments on the manuscript; and J. Small for dedicated secretarial assistance. This work was supported by a grant from SUGEN (J.S.).