MP1: A MEK binding partner that enhances enzymatic activation of the MAP kinase cascade

Science

Volumn 281, Issue 5383, 1998, Pages 1668-1671

  Schaeffer, Hans J ^a   Catling, Andrew D ^a   Eblen, Scott T ^a   Collier, Lara S ^a,b   Krauss, Anke ^a,c   Weber, Michael J ^a  

^a UNIVERSITY OF VIRGINIA   (United States)

^b STANFORD UNIVERSITY SCHOOL OF MEDICINE   (United States)

^c EUROPEAN MOLECULAR BIOLOGY LABORATORY   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

MITOGEN ACTIVATED PROTEIN KINASE; TRANSCRIPTION FACTOR; TRANSCRIPTION FACTOR ELK 1; UNCLASSIFIED DRUG;

AMINO ACID SEQUENCE; APOPTOSIS; ARTICLE; CELL DIFFERENTIATION; ENZYME ACTIVATION; ENZYME REGULATION; GROWTH REGULATION; NONHUMAN; NUCLEOTIDE SEQUENCE; PRIORITY JOURNAL; PROTEIN EXPRESSION; PROTEIN PROTEIN INTERACTION; SACCHAROMYCES CEREVISIAE; SIGNAL TRANSDUCTION;

ANIMALS; CA(2+)-CALMODULIN DEPENDENT PROTEIN KINASE; CARRIER PROTEINS; CELL LINE; DNA-BINDING PROTEINS; ENZYME ACTIVATION; ETS-DOMAIN PROTEIN ELK-1; MAP KINASE KINASE 1; MAP KINASE KINASE 2; MITOGEN-ACTIVATED PROTEIN KINASE 1; MITOGEN-ACTIVATED PROTEIN KINASE 3; MITOGEN-ACTIVATED PROTEIN KINASE KINASES; MITOGEN-ACTIVATED PROTEIN KINASES; MOLECULAR SEQUENCE DATA; PHOSPHORYLATION; PROTEIN-SERINE-THREONINE KINASES; PROTEIN-TYROSINE KINASES; PROTO-ONCOGENE PROTEINS; PROTO-ONCOGENE PROTEINS C-RAF; RECOMBINANT FUSION PROTEINS; SIGNAL TRANSDUCTION; TRANS-ACTIVATION (GENETICS); TRANSCRIPTION FACTORS; TRANSFECTION;

EID: 0032508538     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.281.5383.1668     Document Type: Article

References (16)

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9. 6 clones of a mouse lymphoma library were screened, and seven independent clones coding for MP1 were isolated. MP1 clones with additional 5′ sequence were isolated from the same library by plaque hybridization. MP1 failed to interact with various control proteins including Lamin, p53, SNF1, Ras, c-Raf1, and the Gal4 DNA-binding domain alone. The cDNA of the longest clone isolated had an open reading frame of 372 nucleotides and coded for a protein with a predicted molecular size of 13.5 kD. The putative initiation codon is flanked by a consensus Kozak sequence. An in-frame stop codon 5′ of the predicted start site excludes the possibility of a longer open reading frame.
10. 6 clones of a mouse lymphoma library were screened, and seven independent clones coding for MP1 were isolated. MP1 clones with additional 5′ sequence were isolated from the same library by plaque hybridization. MP1 failed to interact with various control proteins including Lamin, p53, SNF1, Ras, c-Raf1, and the Gal4 DNA-binding domain alone. The cDNA of the longest clone isolated had an open reading frame of 372 nucleotides and coded for a protein with a predicted molecular size of 13.5 kD. The putative initiation codon is flanked by a consensus Kozak sequence. An in-frame stop codon 5′ of the predicted start site excludes the possibility of a longer open reading frame.
11. H. J. Schaeffer, A. D. Catling, S. T. Eblen, L. S. Collier, A. Krauss, M. J. Weber, data not shown.
12. 6-tagged MP1 was purified from baculovirus-infected Sf21 cells. GST-B-Raf was purified from baculovirus-infected Sf9 cells by elution from glutathione-Sepharose. The preparation contained a mixture of GST-B-Raf and 14-3-3 proteins and was essentially free of other contaminating proteins, as judged by Coomassie blue staining. Biochemical assays to assess the role of MP1 in MEK1 phosphorylation and activation in vitro used reaction conditions essentially as described (3).
13. 7 0.2 mM sodium orthovanadate, and protease inhibitors] 24 hours after transfection. Clarified extracts were incubated for 2 hours with 20 μg of anti-FLAG affinity resin (M2, Kodak) at 4°C. MEK-MP1 coimmunoprecipitates were washed four times with FLAG lysis buffer. ERK-MP1 coimmunoprecipitates were washed twice with FLAG lysis buffer and twice with a phosphate-buffered saline solution containing 0.5 M NaCl.
14. T. Jelinek et al., Mol. Cell. Biol. 14, 8212 (1994).
15. M. Fukuda, Y. Gotoh, E. Nishida, EMBO J. 16, 1901 (1997).
16. We thank W. Kolch for B-Raf baculovirus; C. Der for HA-ERK constructs; R. Maurer for reporter constructs; numerous members of the Parsons-Weber-Parsons groups for valuable discussions; S. Parsons, T. Parsons, D. Brautigan, and T. Sturgill for their comments on the manuscript; and S. Elledge for two-hybrid plasmids, a cDNA library, and help in establishing the two-hybrid screen. Supported by NIH grants CA39076 and GM47332 (M.J.W.) and a predoctoral fellowship of the Studienstiftung des Deutschen Volkes (H.J.S.).