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Fig. 7. B
regulates the basal level of the type I receptor and
B
regulates its activity. (A) Outline of the
experimental procedure to isolate B- and B-containing active
PP2A holocomplexes, and to assay their ability to affect the kinase activity
of ALK5. (B) The presence of neither PP2A complex affects the kinase
activity of ALK5 in vitro. Endogenous ALK5 complexes immunopurified from
untreated or TGF-β-treated HaCaT cells were incubated with recombinant
Smad2 substrate in the absence or presence of B-subunit-specific PP2A
complexes purified as in Fig.
6. C-terminal Smad2 phosphorylation was detected by
immunoblotting. The activity of the PP2A complexes was confirmed by their
ability to dephosphorylate pS259 of Raf-1 (lower panel). (C) Knockdown
of B promotes ALK4 clustering. Animal caps from embryos expressing
either HA-ALK4 mRNA alone (top row) or in combination with morpholino against
B (MoB, middle row) were incubated for 1 hour in the presence or
absence of Activin and stained with anti-HA antibody. HA-ALK4 clusters in
response to Activin and in untreated embryos injected with MoB. Okadaic
acid (OA) treatment (bottom row) also induces HA-ALK4 clustering and thus
mimics B knockdown. (D) B knockdown strongly decreases
basal protein levels of ALK5. HaCaT cells were transfected with siRNAs and
treated with TGF-β as indicated. Extracts were immunoblotted with
antibodies against ALK5, phospho-Smad2, pan B-subunits and Smad2/3. (E)
B knockdown has no effect on TβR-II levels. HaCaT cells were
transfected with the indicated siRNAs. Extracts were immunoblotted with
antibodies against TβR-II, pan B-subunits and Smad2/3. Prior to
electrophoresis, extracts were treated with or without PNGase F to remove
N-linked sugars from TβR-II and visualise it more clearly. (F)
B knockdown or B overexpression decreases protein levels of
HA-ALK4. Xenopus embryos were injected at the one-cell stage with
HA-ALK4 and GFP mRNAs, as well as with morpholinos or B mRNA as
indicated, cultured until uninjected embryos had reached stage 9 and analysed
by immunoblotting. (G) Model of the modulation of
TGF-β/Activin/Nodal signalling by B and B. B
normally stabilises the type I receptors ALK4 and ALK5, and B knockdown
promotes their basal degradation. B normally restricts ligand-dependent
activation of ALK4 and ALK5, and B knockdown facilitates such
activation. When overexpressed, B additionally inhibits endogenous
B by replacing it in the PP2A holoenzyme owing to its higher affinity
for the catalytic subunit (not shown).
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