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Q59 - Mapping p35 Phosphorylation Sites with a Phospho-Tag Gel

Theoretical B Real exam question - full text reproduced under IBO's CC BY-NC-SA 4.0 license
Cell Signaling & Communication

Phosphorylation is a major post-translational modification widely used in the regulation of many cellular processes. A method to determine the phosphorylation status of proteins is to run an electrophoresis in a modified gel with a chemical group containing metal ions (M) that can reversibly bind phosphates and thus affects migration of phosphorylated proteins.

Figure Q.59.A Phospho-tag polyacrylamide gel

This technique was used to study the phosphorylation of protein p35. Three mutant forms of this protein were generated: a serine to alanine substitution in position 8 (S8A); a threonine to alanine substitution in position 138 (T138A) and both amino acid substitutions (2A). Note that serine and threonine can be phosphorylated while alanine cannot. Then two yeast strains with normal (wt) or inactive cyclin-dependent kinase 5 (Cdk5) (kn) were transformed with either the wild type version of p35 gene (wt) or one of the three mutant forms. Cell lysate of the eight resulting strains was loaded on a Phospho-tag gel. The proteins from the gel were transferred by western- blot to a membrane that was treated with anti-p35 antibodies. The result is shown below.

Figure Q.59.B Immunoblotting with anti-p35. The arrow indicates the direction of migration. p35 bands are named M1, M2, L1, L2, L3, and L4. L4 band corresponds to the completely non-phosphorylated form of p35.

Figure 1. Figure 1.

Figure 2. Figure 2.

Using the information and data, determine which of the statements are true or which are false.

A. Protein p35 has only two phosphorylation sites: serine 8 and threonine 138.
B. Protein p35 can be phosphorylated by a protein kinase different from Cdk5.
C. In strain Cdk5-wt p35-S8A only a few p35 molecules are phosphorylated at T138.
D. Phosphate groups attached to S8 are more accessible to phosphate binding groups of the Phospho-tag gel than phosphate groups attached to T138.
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Question reproduced from IBO 2016, Theoretical Paper B, licensed under CC BY-NC-SA 4.0 - attributed to the International Biology Olympiad. Open the full exam PDF · Community solutions (unofficial)