CHEM-E8120 Cell Biology, Examination April 8th
Questions 1 to 2: Problem analyzing and solving, each question gives maximally 5 points
Questions 3 and 4: Essay questions, Q3 gives maximally 5 points, Q4 gives maximally 10 points
Problem solving:
First, analyze the problem. If you are unsure about certain details of the problem clearly indicate it in your
answers and mention your assumptions!
Question 1. (maximally 5 points)
When activated, the platelet-derived growth factor (PDGF) receptor phosphorylates itself on multiple
tyrosines. These phosphorylated tyrosines serve as assembly sites for several SH2-domain
containing proteins that include phospholipase C-y (PLCy), a Ras-specific GTPase-activating protein
(GAP), a subunit of phosphoinositide 3-kinase (PI3K), and a phosphotyrosine phosphatase (PTP)
(Figure 1A).
PDGF binding stimulates several changes in the target cell, one of which is an increase in DNA
synthesis, as measured by incorporation of radioactive thymidine or bromodeoxyuridine into DNA.
To determine which of the bound proteins is responsible for activation of DNA synthesis, you
construct several mutant genes for the PDGF receptor that retain individual or combinations of
tyrosine phosphorylation sites. When expressed in cells that do not make a PDGF receptor of their
own, each of the receptors is phosphorylated at its tyrosines upon binding of PDGF. As shown in
Figure 1B, DNA synthesis is stimulated to different extents in cells expressing the mutant receptors.
What roles do PI3K, GAP, PTP, and PLCy play in the stimulation of DNA synthesis by PDGF?
Figure 1. A) The signaling complex assembled on the PDGF receptor. Numbers refer to the positions of the
phosphorylated amino acids in the sequence of the PDGF receptor. B) Stimulation of DNA synthesis by the normal
PDGF receptor and by receptors missing some phosphorylation sites. Stimulation by the normal receptor is set
arbitrarily at 100%. The presence of a phosphorylation site (P site) is indicated by +; absence of a site by –.
Question 2. (maximally 5 points)
Purified importin (=nuclear import receptor) in the presence of Ran and GTP promotes uptake of a
labeled substrate into nuclei (Figure 2). No uptake occurs in the absence of GTP, and Ran alone is
unable to promote nuclear uptake. Importin by itself causes a GTP-independent accumulation of
substrate at the nuclear periphery, but does not promote nuclear uptake (Figure 2A). The substrate
is labeled with a fluorescent dye (fluorescin).
To define the steps in the uptake pathway, you first incubate nuclei with substrate in the presence of
importin. You then wash away free importin and substrate and incubate a second time with Ran and
GTP (Figure 2B).
A. Why do you think the substrate accumulates at the nuclear periphery, as is seen in the absence
of GTP or with importin alone in the presence of GTP?
B. To the extent these data allow, define the order of events that leads to uptake of substrate into the
nucleus.
2A
2B
Figure 2. Effects of Ran, importin, and GTP on nuclear uptake of fluorescein labeled substrate. (A)
Comparison of various combinations of Ran and importin in the presence and absence of GTP. (B) Twostage incubation of cell remnants with importin and substrate and then with Ran and GTP. Circles are the
nuclei; bound or imported substrate stains the nuclei yellow.
Essay questions
Question 3. Essay question, maximally 5 points
Explain the general role of G-proteins and how their activity is regulated. How do small GTPase and
G-protein-coupled receptors differ?
Question 4. Essay question, maximally 10 points
Cellular uptake of galactose is mediated by a specific transporter, which is an integral membrane
protein localized to the cell surface. The transporter is only expressed after stimulation of a cell
membrane localized receptor tyrosine kinase (RTK) by galactose. After activation of the RTK, the
signal is conveyed through a signaling cascade into the nucleus of the cell. When the signal reaches
the nucleus, the gene encoding the galactose transporter is activated.
A) Explain how a signaling cascade functions (max. 2 points)
B) Give a detailed account of the events taking place after the signal reaches the nucleus until
said galactose transporter reaches its target destination. (max. 8 points)