Chapter 16. Identifying the MAP Kinase Signaling Pathway Components
Introduction
Analyze the Data 16-1: Identifying the MAP Kinase Signaling Pathway Components
G. Johnson and colleagues have analyzed the MAP kinase cascade in which the MEK kinase MEKK2 participates in mammalian cells. By a yeast two-hybrid screen (see Chapter 9), MEKK2 was found to bind MEK5, which can phosphorylate a MAP kinase. To elucidate the signaling pathway transduced by MEKK2 in vivo, the following studies were performed in human embryonic kidney (HEK293) cells in culture.
a. HEK293 cells were transfected with a plasmid encoding recombinant, tagged MEKK2, along with a plasmid encoding MEK5 or a control vector that did not encode a protein (mock). Recombinant MEK5 was precipitated from the cell extract by absorption to a specific antibody. The immunoprecipitated material was then resolved by polyacrylamide gel electrophoresis, transferred to a membrane, and examined by Western blotting with an antibody that recognized tagged MEKK2. The results are shown in part (a) of the figure below. What information about this MAP kinase cascade do we learn from this experiment? Do the data in part (a) of the figure prove that MEKK2 activates MEK5, or vice versa?
This experiment reveals that MEK5 and MEKK2 co-localize within a complex because immunoprecipitation with a MEK5 antibody also precipitates MEKK2. However, these data give no evidence as to whether MEKK2 activates MEK5 or MEK5 activates MEKK2.
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