Signalling Through Receptor Serine/Threonine Kinases

Cytoplasmic retention of receptor-regulated Smad proteins

In their basal steady state, the receptor-regulated Smads are mainly present in the cytoplasm.44 They are prevented from moving to the nucleus by a number of proteins that function as anchors. Examples are Disabled-2, Axin, cPML, SARA (all of which bind Smads 2 and 3), and ELF (Smads 3 and 4). TRAP-1

and TLP bind to Smad4, but only after treatment with TGF , and it remains uncertain whether they have a role in cytosolic retention in ‘resting’ cells.3 The best characterized of these is SARA.45

SARA operates in two ways. First, it binds the MH2 domain in the hydrophobic corridor which also binds the nuclear transport proteins46 (see Figure 20.8). Phosphorylation of the SxS motif disrupts the binding with SARA and this may constitute the nuclear translocation signal. Secondly, SARA has a FYVE domain that can bind to PI(3)P, abundant on early endosomal vesicles.47,48 Indeed,

it has been suggested that effective phosphorylation of Smads 2 and 3 only occurs when the receptors are present in the early endosomal membrane. Despite all this, there is no genetic evidence to support the idea that SARA is directly involved in signalling through Smads 2 or 3,3 and importantly, there is no interaction between Smad1 and SARA, so it is not clear if a similar protein is involved in signalling from BMP receptors.

Common mediator Smad4

Smad4 does not interact with the receptors and it lacks the C-terminal SxS phosphorylation motif, though it does form hetero-oligomeric complexes with the receptor-regulated Smads (Figure 20.10). In mammalian cells it binds to phosphorylated Smads 1, 2, 3, 5, and 8, forming complexes that transfer to the nucleus, there to act as transcription factors. It plays an important role in the interaction with cotranscription factors such as CPB or P300.

It is not certain whether Smad4 links with other Smads in the cytoplasm or the nucleus. For sure it does not interact with SARA, and no cytosolic retention proteins have been detected in resting cells, but it does carry a nuclear export signal (NES) which interacts with CRM1, a nuclear export receptor. In this

way Smad4 is retained in the cytosol unless it teams up with phosphorylated receptor-regulated Smads or with other DNA-binding transcription factors that mask the nuclear export signal.49,50

Hetero-oligomeric complex formation

Smad–Smad complexes

The C-terminal SxS phosphorylation motif of the receptor-regulated Smads resembles the phosphorylation motif of the GS domain in the T RI receptors. Following phosphorylation, the basic pocket in the MH2 domain turns its attention to fellow Smads to form hetero-oligomeric complexes (Figure 20.10).40,52 The stoichiometry of the hetero-oligomers remains a matter of some debate, but

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