Understanding Signaling Pathways Controlling Gene Expression

1. Chap. 16 Signaling Pathways That Control Gene Expression • Topics • Receptor Tyrosine Kinases (RTKs) • The Ras/MAP Kinase Pathway • Phosphoinositide Signaling • TGFß Receptors and Smad Signaling • Goals • Learn the properties of RTKs. • Learn about RTK signaling via the Ras/MAP kinase signaling pathway. • Learn the general features of the PI-3 kinase signaling pathway. • Learn about the TGFß/Smad signaling pathway. EGF receptor bound to EGF

2. Major Classes of Cell-surface Receptors About one dozen classes of cell-surface receptors occur in human cells. An overview of signaling by the two receptor systems that are covered in this chapter (receptor tyrosine kinases, RTKs, and TGF-ß receptors) is shown in (Fig. 16.1a). The signal transduction pathways used by RTKs are summarized in (Fig. 16.2).

3. Activation of RTKs via Ligand Binding Receptor tyrosine kinases (RTKs) regulate cell differentiation and proliferation. Ligands of RTKs include nerve growth factor (NGF), fibroblast growth factor (FGF), and insulin. In some cases (e.g., the epidermal growth factor (EGF) RTK), ligand binding causes receptor dimerization (Fig. 16.3). In other cases (e.g., the insulin RTK), binding occurs to pre-existing dimers. RTKs exhibit intrinsic tyrosine kinase activity located within their cytosolic domains. The binding of ligand activates the kinase domains which cross-phosphorylate the two monomers of the dimeric receptor. Phosphorylation first occurs at a regulatory site known as the activation lip. Phosphorylation of the lip causes conformational changes that allow the kinase domain to phosphorylate other tyrosine residues in the receptor and in signal transduction proteins.

4. Recruitment of Signal Transduction Proteins to Activated Receptors Signal transduction system proteins interact with phosphorylated RTKs via phosphotyrosine binding domains. Two main binding domains--PTB and SH2 (Src homology domain-2)--within signal transduction proteins such as the multi-docking protein known as the insulin receptor substrate-1 (IRS-1) perform this function (Fig. 16.12). The binding of signaling proteins either directly to the receptor or to IRS-1 allows them to be phosphorylated by the receptor. Some of these signaling proteins are involved in activation of the Ras GTPase (next slides). Some such as phosphatidylinositol-3 kinase (PI-3 kinase) participate in lipid-mediated signaling pathways. RTK signaling typically is down-regulated by endocytosis of receptors from the cytoplasmic membrane.

5. RTKs and Ras/MAP Kinase Signaling Nearly all RTKs signal via Ras/MAP kinase pathways. They also may signal via other pathways. For example, the insulin receptor uses the Ras/MAP kinase pathway to regulate gene expression and the PI-3 kinase pathway to regulate enzyme activity (e.g., glycogen synthase). RTK-Ras/MAP kinase signaling controls cell division, differentiation, and metabolism. Ras is a monomeric (small) GTPase switch protein that unlike trimeric G proteins does not directly bind to receptors. Ras typically relies on guanine nucleotide-exchange factors (GEFs) for binding GTP, and on GTPase-activating proteins (GAPs) for stimulation of GTP hydrolysis. Once activated, Ras propagates signaling further inside the cell via a kinase cascade that culminates in the activation of members of the MAP kinase family. MAP kinases phosphorylate TFs that regulate genes involved in the cell cycle and in differentiation. Mutant RTKs or Ras/MAP kinase signaling proteins are associated with nearly all cancers. Dominant Ras mutations that block GAP binding and lock Ras in the "on" state promote cancer.

6. RTK Activation of Ras The mechanism by which EGF activates Ras is illustrated in Fig. 16.17. In Step 1, EGF binding causes receptor dimerization and autophosphorylation on cytosolic tyrosines. In Step 2, the adaptor protein GRB2 binds receptor phosphotyrosine residues via its SH2 domain. GRB2 contains SH3 domains that allow the GEF protein known as Sos to bind to the membrane complex. Sos then recruits Ras to the complex. In the last step of Ras activation (Step 3), Sos promotes GTP exchange for GDP on Ras. The activated Ras-GTP complex then dissociates from Sos, but remains tethered to the inner leaflet of the cytoplasmic membrane via a lipid anchor sequence. The active form of Ras then activates the MAP kinase portion of the signaling pathway (next two slides).

7. Ras Activation of MAP Kinase Ras activates MAP kinase via a phosphorylation cascade that proceeds from Ras to Raf kinase, to MEK kinase, and finally to MAP kinase (Fig. 16.20). MAP kinase then dimerizes and enters the nucleus (next slide).

8. MAP Kinase Activation of Transcription In the final steps of RTK-Ras/MAP kinase signaling, MAP kinase phosphorylates and activates the p90RSK kinase in the cytoplasm (Fig. 16.22). Both kinases enter the nucleus where they phosphorylate ternary complex factor (TCF) and serum response factor (SRF), respectively. The phosphorylated forms of these TFs bind to serum response element (SRE) enhancer sequences that control genes such as c-fos. c-fos activates the expression of genes that propel cells through the cell cycle. SREs occur in a number of genes that are regulated by growth factors present in serum.

9. Signaling via Phosphatidyl-inositol 3-phosphates RTKs also can signal via formation of phosphoinositide compounds. Like GPCRs, they signal via the IP3/DAG pathway. However, RTKs activate the PLCg isoform of phospholipase C, not PLCß as occurs with GPCRs. PLCg binds to activated RTKs via SH2 domains. In addition, RTKs can signal via PI 3,4-bisphosphate and PI 3,4,5-trisphosphate formed by the enzyme PI-3 kinase (Fig. 16.25). PI-3 kinase is recruited to the membrane by SH2 domain-mediated binding to activated RTK phosphotyrosine residues. The PI 3-phosphate compounds synthesized by PI-3 kinase activate protein kinase B (PKB) (next slide).

10. Activation of Protein Kinase B Signaling downstream of PI 3-phosphates is conducted by PKB (Fig. 16.26). PKB is recruited to the membrane via binding to PI 3-phosphates via its PH domain. There it is phosphorylated and activated by the PDK1 & PDK2 kinases. PDK1 also is recruited to the membrane via binding to PI 3-phosphates. Activated PKB then enters the cytosol, where it phosphorylates target proteins. In insulin receptor signaling, PKB phosphorylates and inactivates glycogen synthase kinase, stimulating glycogen synthesis. PKB also is a potent inhibitor of apoptosis. PI 3-phosphate signaling ultimately is terminated by cleavage of 3-phosphates from phosphoinositides by the PTEN phosphatase. PTEN is inactive in many advanced cancers.

11. Biological Roles of TGFß Growth factors are proteins that play important roles in regulating cell differentiation, division, and movement. Activating mutations in growth factor receptors or their signaling pathways commonly are associated with cancers. Transforming growth factor ß (TGFß) plays widespread roles in regulating development in both vertebrates and invertebrates. Despite their names, all three types of human TGFßs exert anti-proliferative effects on target cells. Therefore, the loss of a TGFß receptor can lead to transformation of a cell to a cancerous state. TGFß is secreted from cells as an inactive precursor. It subsequently undergoes proteolytic processing and attaches to the extracellular matrix. It is released from the matrix after the receipt of an appropriate signal, and then carries out paracrine signaling on neighboring cells.

12. TGFß/Smad Pathway The signal transduction pathway by which TGFß regulates TF activity is illustrated in Fig. 16.28. The points to know for this pathway are 1) TGFß binds to cell surface receptors causing phosphorylation of these receptors, 2) the activated receptors phosphorylate certain Smad TFs exposing their nuclear localization signals, and 3) Smad TFs enter the nucleus where they combine with other TFs and activate the transcription of target genes. Smad TF activity ultimately is shut down via the action of transcription repressors, whose activity is induced by TGFß via a feedback loop. These repressors bind to the Smad TFs, and recruit a histone deacetylase to the activated promoter, which then causes chromatin condensation and gene silencing. Because TGFß has antiproliferative effects on cells, overexpression of the repressors results in cellular transformation and cancer.