^AGAC| | icacgtgim^ 3-bp spacer


^AGAC| | icacgtgim^ 3-bp spacer

Transcription ceptors separates the domains, permitting binding of importin p to the NLS. Simultaneously a complex containing two molecules of Smad3 (or Smad2) and one molecule of a co-Smad (Smad4) forms in the cytosol. This complex is stabilized by binding of two phosphorylated serines in each Smad3 to phosphoserine-binding sites in both the Smad3 and the Smad4 MH2 domains. The bound importin p then mediates translocation of the heteromeric R-Smad/co-Smad complexes into the nucleus. After importin p dissociates inside the nucleus, the Smad2/Smad4 or Smad3/Smad4 complexes cooperate with other transcription factors to activate transcription of specific target genes.

Within the nucleus R-Smads are continuously being de-phosphorylated, which results in the dissociation of the R-Smad/co-Smad complex and export of these Smads from the nucleus. Because of this continuous nucleocytoplasmic shuttling of the Smads, the concentration of active Smads within the nucleus closely reflects the levels of activated TGFp receptors on the cell surface.

Virtually all mammalian cells secrete at least one TGFp isoform, and most have TGFp receptors on their surface. However, because different types of cells contain different sets of transcription factors with which the activated Smads can bind, the cellular responses induced by TGFp vary among cell types. in epithelial cells and fibroblasts, for example, TGFp induces expression not only of extracellular-

M FIGURE 14-2 TGFp-Smad signaling pathway. Step OS: In some cells, TGFp binds to the type III TGFp receptor (RIII), which presents it to the type II receptor (RII). Step 1b : In other cells, TGFp binds directly to RII, a constitutively phosphorylated and active kinase. Step 2 : Ligand-bound RII recruits and phosphorylates the juxtamembrane segment of the type I receptor (RI), which does not directly bind TGFp. This releases the inhibition of RI kinase activity that otherwise is imposed by the segment of RI between the membrane and kinase domain. Step 3: Activated RI then phosphorylates Smad3 (shown here) or another R-Smad, causing a conformational change that unmasks its nuclear-localization signal (NLS). Step 4 : Two phosphorylated molecules of Smad3 interact with a co-Smad (Smad4), which is not phosphorylated, and with importin p (Imp-p), forming a large cytosolic complex. Steps 5 and 6 : After the entire complex translocates into the nucleus, Ran-GTP causes dissociation of Imp-p as discussed in Chapter 12. Step 7 : A nuclear transcription factor (e.g., TFE3) then associates with the Smad3/Smad4 complex, forming an activation complex that cooperatively binds in a precise geometry to regulatory sequences of a target gene. Shown at the bottom is the activation complex for the gene encoding plasminogen activator inhibitor (PAI-1). See the text for additional details. [See Z. Xiao et al., 2000, J. Biol. Chem. 275:23425; J. Massague and D. Wotton, 2000, EMBO J. 19:1745; X. Hua et al., 1999, Proc. Nat'l. Acad. Sci. USA 96:13130; and A. Moustakas and C.-H. Heldin, 2002, Genes Devel. 16:1867.]

matrix proteins (e.g., collagens) but also of proteins that inhibit serum proteases, which otherwise would degrade the matrix. The latter category includes plasminogen activator inhibitor 1 (PAI-1). Transcription of the PAI-1 gene requires formation of a complex of the transcription factor TFE3 with the Smad3/Smad4 complex and binding of all these proteins to specific sequences within the regulatory region of the PAI-1 gene (see Figure 14-2, bottom). By partnering with other transcription factors, Smad2/Smad4 and Smad3/ Smad4 complexes induce expression of proteins such as p15, which arrests the cell cycle at the G1 stage and thus blocks cell proliferation (Chapter 21). These Smad complexes also repress transcription of the myc gene, thereby reducing expression of many growth-promoting genes whose transcription normally is activated by Myc.

The various growth factors in the TGFp superfamily bind to their own receptors and activate different sets of Smad proteins, resulting in different cellular responses. The specificity exhibited by these related receptors is a common phenomenon in intercellular signaling, and the TGFp signaling pathway provides an excellent example of one strategy for achieving such response specificity. As just discussed, for instance, binding of any one TGFp isoform to its specific receptors leads to phosphorylation of Smad2 or Smad3, formation of Smad2/Smad4 or Smad3/Smad4 complexes, and eventually transcriptional activation of specific target genes (e.g., the PAI-1 gene). On the other hand, BMP proteins, which also belong to the TGFp superfamily, bind to and activate a different set of receptors, leading to phospho-rylation of Smad1, its dimerization with Smad4, and activation of specific transcriptional responses by Smad1/Smad4. These responses are distinct from those induced by Smad2/ Smad4 or Smad3/Smad4.

Oncoproteins and I-Smads Regulate Smad Signaling via Negative Feedback Loops

Smad signaling is regulated by additional intracellular proteins, including two cytosolic proteins called sSnoN and ,Ski (Ski stands for "Sloan-Kettering Cancer Institute"). These proteins were originally identified as oncoproteins because they cause abnormal cell proliferation when overexpressed in cultured fibroblasts. How they accomplish this was not understood until years later when SnoN and Ski were found to bind to the Smad2/Smad4 or Smad3/Smad4 complexes formed after TGFp stimulation. SnoN and Ski do not affect the ability of the Smad complexes to bind to DNA control regions. Rather, they block transcription activation by the bound Smad complexes, thereby rendering cells resistant to the growth-inhibitory actions normally induced by TGFp (Figure 14-3). Interestingly, stimulation by TGFp causes the rapid degradation of Ski and SnoN, but after a few hours, expression of both Ski and SnoN becomes strongly induced. The increased levels of these proteins are thought to dampen long-term signaling effects due to continued exposure to TGFp.


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