Biologia Cel·lular 2 2022-2023 Name: Anna Aguilera Romero Problem 1

Please find information about the techniques used in this problem in the file

“Teaching material BC5-8” uploaded in Aula Global.

The TGF-, type II receptor (TR-II) is a transmembrane serine/threonine kinase that, upon
ligand binding, recruits and phosphorylates a second transmembrane kinase, TR-I, as a
requirement for signal transduction. TR-I is phosphorylated by TR-II in the GS domain, a 30
amino acid region preceding the kinase domain. The functional role of three serines and two
threonines in the TR-I GS domain and two threonines located immediately nearby was
investigated by mutational analysis.

Figure 1. The GS domain of TR-I. The GS domain is depicted in gray and its sequence indicated
below. The mutants used in the work are indicated underneath the sequence. Residue K232,
located within the kinase domain, is responsible for ATP binding.

For the study of TGF- receptors and signaling, Mv1Lu mutant cell lines were obtained that
lacked TR-I (R mutants) or cell lines lacking TR-II (DR mutants).
Biologia Cel·lular 2 2022-2023 Name: Anna Aguilera Romero Problem 1

Figure 2. Effects of mutations in the TTSGSGSG motif. Luciferase activity in transiently

transfected R-1B cells. Three micrograms of p3TP-Lux (a TGF--responsive luciferase reporter
construct) and 1g of each receptor construct containing the indicated mutations were co-
transfected into R-1B cells (lacking TR-I). On the next day, media were changed to low serum
media, and TGF- was added at 100 pM. 20 h later, cells were lysed and luciferase activity was
determined. Each assay was carried out in duplicate in at least two experiments.

Figure 3. Effects of Thr200 and 204 mutations. (A) Luciferase assays were carried out in R-IB
cells transfected with the indicated constructs as described in the legend to Figure 2. (B)
Inhibition of DNA synthesis in stably transfected R-IB cells. Cells were seeded sparsely into 24-
well plates and incubated in low serum media in the presence of the indicated concentrations
of TGF- for 24 h. [125I]deoxyuridine incorporation into DNA was then determined. The data are
presented as percent incorporation relative to transfectants that did not receive TGF-.
Biologia Cel·lular 2 2022-2023 Name: Anna Aguilera Romero Problem 1

Figure 4. Biological effects of the mutations to aspartic acid. (A) Luciferase assays were carried
out in R-1B cells transfected with the indicated constructs as described in the legend to Figure
2. Data are the average of duplicate determinations ± SD. (B) The indicated constructs subcloned
in a doxycycline-inducible vector were stably transfected into R-1B cells. [125I]deoxyuridine
incorporation into DNA was determined in the presence of the indicated TGF, concentrations,
with and without doxycycline (dox) induction. Data are the average of triplicate determinations.
(C) The TR-II-defective cell line DR-26 was transiently co-transfected with the p3TP-Lux reporter
construct and TR-II or increasing concentrations of TR-I (WT) or TR-I (T204D) vector. Some
TR-II transfectants were incubated with TGF before lysis as described in Figure 2. Luciferase
activity was then determined. Data are the average of duplicate determinations ±SD.

Figure 5. (A) Basal phosphorylation of TR-I (T204D) in intact cells. R-1B cells transiently
transfected with the indicated HA-tagged constructs were labeled in parallel dishes with
[35S]methionine or [32P]orthophosphate. Receptors were immunoprecipitated and analyzed by
SDS-PAGE and autoradiography. (B) In vitro kinase assay. The indicated HA-tagged receptor
constructs were immunoprecipitated from transiently transfected COS-1 cells and incubated
with [y-32P]ATP. Phosphorylated immunoprecipitates were analyzed by SDS-PAGE and
autoradiography (top). To control for the amount of receptor protein present in the assays,
separate aliquots of the cell lysates were resolved by SDS-PAGE and immunoblotted with anti-
HA antibody (bottom).
Biologia Cel·lular 2 2022-2023 Name: Anna Aguilera Romero Problem 1

Questions (very short answers).

1. What is the consequence of mutating all the serine and threonine residues of the GS
domain of TR-I for TGF-dependent gene transcription?

If the serine and threonine residues of the GS domain of TβR-I are mutated, it loses its binding
domain with TR-II. Consequently, it can’t be phosphorylated and has no activity.

2. What is the consequence of mutating the TβR-I ATP binding residue lysine K232 to
arginine for TGFβ-dependent gene transcription?

As we can see in the figure 3A, it loses all the activity.

3. Which are the consequences of mutating threonines 200 and 204 of TβR-I to valine for
TGFβ-dependent gene transcription and cell proliferation?

If we change threonine 200 it loses all the activity, otherwise, if we change the 204, it loses its
activity partially.

4. Which are the consequences of mutating threonines 200 and 204 of TβR-I to aspartic
acid for TGFβ-dependent gene transcription and cell proliferation?

If threonine 200 is changed it loses all the activity, otherwise, the change in the 204 involve
constitutive and multiplicity activity.

5. Why TβR-I WT transfection in Figure 4C does not lead to increases in luciferase

activity? Which would be the result if the transfected cells would have been treated
with TGFβ?

In this experiment, the cells are modified (DR-26), they are defective of TβR-II, that’s why TβR-I
it cannot be activated, and it would be no activity. Even though they treat the cells with TGF-β
it wouldn’t be any activity because the problem is the lack of TβR-II.
Biologia Cel·lular 2 2022-2023 Name: Anna Aguilera Romero Problem 1

6. What type of mutant is TβR-I T204D accordingly to the evidences presented in Figures
4 and 5?

This type of mutant is an oncological mutation due to the protein shows constitutive and
multiplicated activity.

7. Why the result of the immunoprecipitation and kinase assay of TβR-I WT in Figure 4B
is positive for 32P incorporation?

It is positive because they put all the kinase complex, and it has plenty of activity.

8. Why the result of the immunoprecipitation and kinase assay of TβR-I K232R mutant in
Figure 4B is negative for 32P incorporation?

It is negative because of even though they put all the kinase complex, the mutation in this
aminoacid blocks the binding domain to TβR-II, and it loses its activation.