SB-BB

Passage 6

Second messenger cAMP activates protein kinase A (PKA), which can then activate nuclear transcription factor cAMP response-element binding protein (CREB). CREB exists as two isoforms in cells, one being CREB327 (327 residues). CREB327 is phosphorylated by glycogen synthase kinase-3 (GSK-3) at serine 115. PKA phosphorylates CREB327 at serine 119. Variants of wild-type CREB327 (CREB327^WT) that cannot be phosphorylated at serine 115 or 119 were created (CREB327¹¹⁵ and CREB327¹¹⁹, respectively).

Experiment 1

CREB327^WT and CREB327¹¹⁹ were incubated with various combinations of PKA and GSK-3 in the presence of radiolabeled ATP. SDS-PAGE gel electrophoresis was performed under reducing conditions, and radiolabeled protein bands were detected (Figure 1).

Figure 1 Phosphorylation of CREB327^WT and CREB327¹¹⁹

Experiment 2

CREB327^WT was incubated with radiolabeled ATP and either PKA, GSK-3, or sequentially with PKA then GSK-3. Isoelectric focusing and autoradiography were used to detect phosphorylation (Figure 2).

Figure 2 Isoelectric focusing of samples

Experiment 3

A specific assay uses the percent conversion of chloramphenicol (Chl) to its acetylated form (Chl-A) as an indicator of the transcriptional activity of CREB. Plasmids encoding CREB327^WT and its variants were transfected into cells, and their transcription activities were measured (endogenous CREB327 activity is not detected) (Table 1).

Table 1 Percent Conversion of Chl to Chl-A

Plasmid	Percent conversion (%)
CREB327WT	27
CREB327115	8.5
CREB327119	1.7
Control	1.6

Adapted from C. J. Fiol, J. S. Williams, C. Chou, Q. M. Wang, P. J. Roach, and O. M. Andrisani, Journal of Biological Chemistry. ©1994 The American Society for Biochemistry and Molecular Biology, Inc.

Questions

40. The method used to visualize CREB327^WT in Experiment 2 must include which step?

1. Amplify the samples by PCR before running on the gel.
2. Establish a stable pH gradient in the gel before adding samples.
3. Denature the samples so the proteins unfold into a linear chain.
4. Treat the samples with a chemical that will add an overall negative charge to each protein

41. Which conclusion about the phosphorylation of CREB327^WT is supported by the data in figures 1 and 2?

1. GSK-3 also phosphorylates CREB327^WT at residue 119.
2. GSK-3 phosphorylates CREB327^WT at a faster rate than PKA.
3. Phosphorylation of CREB327^WT by PKA occurs at more than one residue.
4. Phosphorylation of CREB327^WT by GSK-3 can only occur after phosphorylation with PKA.

42. When designing variant CREB327¹¹⁹, which of the following residues was most likely chosen as the substitute for residue 119 in CREB327^WT?

1. Tyrosine
2. Glutamate
3. Alanine
4. Threonine

43. Which statement explains how two forms of CREB can be generated in cells?

1. Pre-mRNA is transcribed from one CREB allele versus the other.
2. CREB mRNA transcripts with different combinations of exons are generated.
3. Different post-translational modifications of CREB occur in the endoplasmic reticulum.
4. Pre-mRNA of various lengths are transcribed based on different termination sequences in the CREB gene.

44. Which statement about CREB327 phosphorylation is supported by the data presented in Table 1?

1. Phosphorylation by GSK-3 has no effect on CREB327^WT activation levels.
2. PKA has a higher specificity for CREB327^WT than CREB327¹¹⁵.
3. Phosphorylation by PKA can partially activate CREB327.
4. The k_cat for the phosphorylation of CREB327 by GSK-3 is higher than the k_cat for phosphorylation by PKA.

45. Under certain conditions, PKA and GSK-3 have been shown to autophosphorylate. The control group used in Experiment 2 (Lane 1) was designed to account for this possibility. Given this, the control group most likely contained:

1. CREB327^WT with ATP but no PKA or GSK-3.
2. PKA and GSK-3 with ATP but no CREB327^WT.
3. PKA and GSK-3 with no ATP or CREB327^WT.
4. CREB327^WT with no ATP, PKA, or GSK-3.

Answers

Question 40

Solution: The correct answer is B. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is B because according to the passage, visualization of the proteins in Experiment 2 is done through isoelectric focusing, which separates proteins based on isoelectric point. To do this, a stable pH gradient must be established in the gel. It is a Reasoning about the Design and Execution of Research question because you are asked to demonstrate an understanding of how to correctly execute an experimental analysis.

Question 41

Solution: The correct answer is D. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is D because the data in Figure 1 show that phosphorylation of CREB327^WT by GSK-3 only occurs if PKA is present in the reaction and if PKA is able to phosphorylate CREB327^WT. Figure 2 shows that phosphorylation of CREB327^WT by GSK-3 occurs when CREB327^WT has been previously incubated with PKA. All of these data suggest that phosphorylation of CREB327^WT by PKA and GSK-3 occurs sequentially, with phosphorylation by PKA occurring first. It is a Data-based and Statistical Reasoning question because you are asked to make a conclusion based on experimental data.

Question 42

Solution: The correct answer is C. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.“ The answer to this question is C because alanine is similar in size to serine, cannot be phosphorylated, and will not add a positive or negative charge to the protein. It is the best choice. It is a Scientific Reasoning and Problem Solving question because you are asked to use your knowledge amino acid properties to identify an appropriate model for use in a specific experiment.

Question 43

Solution: The correct answer is B. This is a Biology question that falls under the content category “Transmission of genetic information from the gene to the protein.“ The answer to this question is B because CREB341 and CREB327 are isoforms. Different protein isoforms are synthesized from the same gene through alternative splicing, during which sections of the full transcript (both introns and exons) are spliced. Different combinations of exons can produce different protein isoforms. It is a Knowledge of Scientific Concepts and Principles question because you are asked to recall the mechanisms by which mRNA is processed in eukaryotes.

Question 44

Solution: The correct answer is C. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is C because the CREB327¹¹⁹ sample group shows transcription activation levels as low as the control, suggesting that CREB327 transcription activity is abolished if PKA cannot phosphorylate residue 119. However, in the CREB327¹¹⁵ sample group, in which GSK-3 cannot phosphorylate CREB327 but PKA still can, some activation occurs, though not as much as is observed with CREB327^WT. Thus, phosphorylation by PKA alone can activate CREB327, but only partially. It is a Data-based and Statistical Reasoning question because you are asked to intepret the results of an experiment.

Question 45

Solution: The correct answer is B. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is B because the best control against the variable of enzyme autophosphorylation would be the enzymes alone without substrate. ATP must be included so that autophosphorylation would be possible. The substrate should be excluded so that it is clear that any phosphorylation detected is due to autophosphorylation and not phosphorylation of the substrate. It is a Reasoning about the Design and Execution of Research question because you are asked to use scientific reasoning to identify the appropriate control for a given experiment.