ERK5 is a MAP kinase that is activated upon phosphorylation by MEK5. MEK5 binds with MEKK2 when co-expressed. HEK293 cells were transfected with plasmid encoding ERK5, along with plasmids encoding either MEK5 alone, or MEKK2 alone, or both MEKK2 and MEK5, or both MEKK2 and MEK5AA(MEK5 mutant). Lysates of transfected cells were analyzed by Western blotting using anti-ERS5 antibody as shown below:

From the data in the figure above, the following conclusions were drawn.

A. Full activation of ERK5 requires both MEKK2 and MEK5.

B. Phosphorylation with MEKK2 alone suggests that it can activate ERK5 without MEK5.

C. Difference in the levels of phosphorylation with MEKK2 alone and MEKK2 + MEK5 is due to more phosphorylation at the same site.

D. Phosphorylation with only MEKK2 transfection suggests that it might be associating with endogenous MEK5 to get partially activated, leading to ERK5 phosphorylation to some extent.

E. MEK5AA might be a dominant-negative mutant of MEK5 which prevents signalling through active, endogenous MEK5.

Which of the following options represents the combination of all correct statements?

Correct option is D

ERK5 is a MAP kinase activated via phosphorylation by MEK5. The western blot shows different levels of phospho-ERK5 under various transfection conditions.

Key Observations from the Blot:

• MEK5 alone does not strongly activate ERK5.
• MEKK2 alone results in some ERK5 phosphorylation.
• MEKK2 + MEK5 leads to stronger phosphorylation, indicating full activation.
• MEKK2 + MEK5AA (mutant) shows a reduced phosphorylation level, suggesting MEK5AA inhibits ERK5 activation.

Analysis of Answer Choices:

1. A - Full activation of ERK5 requires both MEKK2 and MEK5 (Correct )

   • The strongest phospho-ERK5 band appears when both MEKK2 and MEK5 are present.
   • MEKK2 alone leads to weak phosphorylation, suggesting it may require MEK5 for full activation.
   • Thus, full activation of ERK5 requires both kinases.

2. B - Phosphorylation with MEKK2 alone suggests that it can activate ERK5 without MEK5 (Incorrect )

   • The phosphorylation observed in the MEKK2-only lane might be due to endogenous MEK5, not direct activation of ERK5 by MEKK2.
   • Since MEKK2 likely acts upstream of MEK5, rather than directly on ERK5, this interpretation is misleading.

3. C - Difference in phosphorylation levels with MEKK2 alone and MEKK2 + MEK5 is due to more phosphorylation at the same site (Incorrect )

   • The difference could be due to synergistic effects rather than phosphorylation at the same site.
   • The presence of two kinases working together (MEKK2 and MEK5) could lead to a more efficient cascade activation rather than simply more phosphorylation at a single site.

4. D - Phosphorylation with only MEKK2 transfection suggests it might be associating with endogenous MEK5 (Correct )

   • The presence of phospho-ERK5 in MEKK2-only transfection suggests endogenous MEK5 might be present in HEK293 cells.
   • This could explain why some ERK5 activation is observed even without MEK5 overexpression.

5. E - MEK5AA might be a dominant-negative mutant of MEK5 that prevents signaling through active, endogenous MEK5 (Correct )

   • The reduced phosphorylation signal in the MEKK2 + MEK5AA lane suggests that MEK5AA blocks normal ERK5 activation.
   • This supports the idea that MEK5AA is a dominant-negative mutant, preventing endogenous MEK5 from functioning properly.