1. Describe the steps that occur when the hormone epinephrine causes the activation of protein kinase A. (8 pts)
Hormone binds receptor. Receptor changes configuration and binds to the G stimulatory protein. The alpha subunit of the G stimulatory protein replaces GDP with GTP. This causes the alpha subunit to be released from the beta/gamma subunits. The alpha subunit binds adenylyl cyclase activating it to catalyze the formation of cAMP. Alpha subunit hydrolyzes GTP to GDP, disengages from the cyclase and reassociates with the beta/gamma subunits. The hormone disengages from the receptor. The increased cAMP binds to the cAMP binding sites on the regulatory subunits of protein kinase A, causing them to dissociate from the catalytic subunits which are now able to act as a kinase phosphorylating their substrates.
2. Protein kinase A, when activated, regulates three enzymes involved in glucose/glycogen metabolism. Name them and describe how they are regulated by protein kinase A. (6 pts)
Glycogen phosphorylase kinase. When phosphorylated itself it becomes activate and phosphorylates glycogen phosphorylase. When phosphorylated it becomes active and causes glycogen to release glucose 1-phosphate which can enter glycolysis.
Glycogen synthetase: When phosphorylated it becomes inactive and cannot catalyze the synthesis of glycogen from glucose monomers.
Phosphoprotein phosphatase inhibitor: When it becomes phosphorylated, it becomes active and capable of binding to phosphprotein phosphatase. This prevents the phosphatase from removing the controlling phosphates on the enzymes mentioned above.
3. The activation of phospholipase C produces two molecules that are each involved in signal transduction. What are they and what do they do? (8 pts)
IP3: It travels to the endoplasmic reticulum membrane, binds to a calcium channel there, causing it to open and to release calcium into the cytoplasm. Calcium is involved in many signaling activities. The calcium influx also causes the movement of protein kinase C to the cell membrane where it becomes active (see below) as a kinase in signal transduction also. The calcium channels also move to the cell membrane where they bind another calcium channel located there, causing it to open allowing calcium to enter the cell.
DAG: Diacylglycerol remains in the membrane and activates protein kinase C. (see above)
4. What role do the following play in signaling? (6 pts)
A. SH2 domains
Bind to phosphotyrosines on activated receptor tyrosine kinase cytoplasmic domains. Then bind to other proteins to transduce the signals initiated by the receptor.
B. PTB domains
Bind to phosphotyrosines also, as described above. They are usually part of a multidocking protein that themselves become tyrosine phosphorylated and serve as sites for the binding of other SH2 domain signaling proteins.
C. SH3 domains
Part of a signaling protein that itself contains an SH2 domain or that interacts with an SH2 domain containing protein to continue transducing the signal. It is a domain rich in prolines.
5. Name the molecule that plays the following role in the signaling pathway begun by epidermal growth factor binding to its receptor. (6 pts)
A. What is the GEF?
Sos
B. What contains an SH2 domain?
GRB2
C. What contains an SH3 domain?
GRB2
6. Following ras activation, there are four protein kinases that work to activate the c-fos gene in the EGF signaling pathway. Name them and describe where they work in the kinase cascade. (8 pts)
Raf kinase, a serine/threonine kinase, gets released from 14-3-3, moves to the membrane, and phosphorylates MEK, activating it.
MEK phosphorylates MAP kinase at both serines and tyrosines, activating it.
MAP kinase dimerizes and phosphorylates p90 in the cytoplasm and then moves into the nucleus and phosphorylates transcription factor TCF.
P90 moves into the nucleus once it is activated as above and phosphorylates SRF.
The phosphorylated SRF dimerizes and binds to the SRE upstream promoter element along with the phosphorylated TCF. Together they help activate the transcription of the c-fos gene.
7. We identified four categories of proteins that regulate the activity of cyclin/cdk complexes during progression of the cell cycle. Name the four categories. (8 pts)
Protein kinases that can attach phosphates to critical amino acids that either activate or inactivate the complex kinase activity.
Phosphatases that remove critical phosphates, reversing the effects described above.
Inhibitors that bind the complexes to prevent their ability to phosphorylate their substrates.
Ubiquitin protein ligases that target the complexes for degredation in the proteosome system.
8. Explain how a cell that is signaled to begin the cell-cycle is able to pass the restriction point in late G1. (7 pts)
In mid G1 the cyclin/cdk complex phosphorylates some of the Rb protein, releasing it from transcription factor E2F which can then activate the cyclin/cdks for late G1. In late G1 the cyclin/cdk complex further phosphorylates Rb completing releasing all of the E2F. E2F can then activate the transcription of S-phase specific proteins as well as its own transcription, moving the cell into S.
9. Describe how mdm2 and p53 are involved in the DNA damage checkpoint that causes cell cycle arrest in G1. (8 pts)
In the absence of DNA damage signals, the half life of p53 is very short because it is bound to mdm-2 which signals it for ubiquitination and degredation in the proteosome system. However, when DNA is damaged, the ATM/R kinase is activated and phosphorylates both mdm-2 and p53. This releases p53 from mdm-2 and increases its stability. The p53 then acts as a transcription factor and activates the transcription of p21 and of itself. P21 is an inhibitor of multiple cdk/cyclin complexes and its binding helps induce cell-cycle arrest.
10. There are three additional checkpoints that regulate the cell cycle, in addition to the DNA damage checkpoints. Name them. You do not need to describe how they work. (6 pts)
Intra S phase checkpoint, insuring that all DNA replication is complete before cells enter mitosis.
Spindle-assembly checkpoint, insuring that all kinetochores are attached to the mitotic spindle before anaphase is allowed to progress.
Spindle-position checkpoint, insuring that all chromosomes have properly segregated before before telophase and cytokinesis is allowed to be completed.
11. Describe what would happen to apoptosis if the bad protein was mutated in a way that prevented its phosphorylation. Give all the details. (8 pts)
When survival factors are present, they activate a cell membrane receptor that initiates a kinase cascade involving PI-3 kinase activating protein kinase B and protein kinase B phosphorylating bad. When bad is phosphorylated, it gets sequestered by 14-3-3 in the cytoplasm. But if not, it remains in the mitochondrial membrane where it binds to bcl2/x and prevents it from inhibiting the formation of the bax-bax dimer and ion channel. Therefore, the bax ion channel would initiate the events of apoptosis even in the presence of survival factors. Ions would flow in from the cytoplasm, cytochrome c would be released from the mitochondria, bind apaf-1, initiate formation of the apoptosome, and cause the activation of the caspases with subsequent destruction of cellular components and cell death.
12. Identify the following as either a proto-oncogene or as a tumor suppressor gene. (7 pts)
A.
bcl2
proto-oncogene
B. bax
tumor-suppressor gene
C. ras
proto-oncogene
D. Rb
tumor-suppressor gene
E. Myc
proto-oncogene
F. p53
tumor-suppressor gene
G. src
proto-oncogene
13. Describe one of the two reciprocal translocations we discussed in class that are involved in creating an oncogene. Include all details. (8 pts)
Refer to figures 25-20 (bcr-abl activation; 9/22) and 25-23 (c-myc activation; (8/14).
14. Name the three categories of genes that are involved in the formation of cancers and indicate whether they are genetically dominant or recessive. (6 pts)
Proto-oncogenes: dominant
Tumor-suppressor genes: recessive
Caretaker genes: recessive