▲ FIGURE 14-27 Recruitment and activation of protein kinase B (PKB) in PI-3 kinase pathways. In unstimulated cells, PKB Is In the cytosol with Its PH domain bound to the catalytic domain, Inhibiting Its activity. Hormone stimulation leads to activation of PI-3 kinase and subsequent formation of phosphatidylinositol

(PI) 3-phosphates (see Figure 14-26). The 3-phosphate groups serve as docking sites on the plasma membrane for the PH domain of PKB and another kinase, PDK1. Full activation of PKB requires phosphorylation both in the activation lip and at the C-terminus by PDK1. [Adapted from A. Toker and A. Newton, 2000, Cell 103:185, and M. Scheid et al., 2002, Mol. Cell Biol. 22:6247.]

The Insulin Receptor Acts Through the PI-3 Kinase Pathway to Lower Blood Glucose

The insulin receptor is a dimeric receptor tyrosine kinase that can initiate the Ras-MAP kinase pathway, leading to changes in gene expression. Insulin stimulation also can initiate the PI-3 kinase pathway just described, leading to activation of protein kinase B. In insulin-stimulated liver, muscle, and fat cells, activated protein kinase B acts in several ways to lower blood glucose and promote glycogen synthesis.

The principal mechanism by which insulin causes a reduction of the blood glucose level is by increasing import of glucose by fat and muscle cells. This effect is mediated by protein kinase B, which through mechanisms that are not fully understood causes movement of the GLUT4 glucose transporter from intracellular membranes to the cell surface (Chapter 15). The resulting increased influx of glucose into these cells lowers blood glucose levels.

In both liver and muscle, insulin stimulation also leads to activation of glycogen synthase (GS), which synthesizes glycogen from UDP-glucose (see Figure 13-16). This represents another mechanism for reducing glucose concentration in the circulation. In resting cells (i.e., in the absence of insulin), glycogen synthase kinase 3 (GSK3) is active and phos-phorylates glycogen synthase, thereby blocking its activity. Activated protein kinase B phosphorylates and thereby inactivates GSK3. As a result, GSK3-mediated inhibition of glycogen synthase is relieved, promoting glycogen synthesis.

Activated Protein Kinase B Promotes Cell Survival by Several Pathways

In many cells activated protein kinase B directly phosphory-lates pro-apoptotic proteins such as Bad, thereby preventing activation of an apoptotic pathway leading to cell death (Chapter 22). Activated protein kinase B also promotes survival of many cultured cells by phosphorylating the transcription factor Forkhead-1 on as many as three serine or threonine residues. In the absence of growth factors, Forkhead-1 is unphosphorylated and localizes to the nucleus, where it activates transcription of several genes encoding pro-apoptotic proteins. When growth factors are added to the cells, protein kinase B becomes active and phosphorylates Forkhead-1. This allows the cytosolic phosphoserine-binding protein 14-3-3 to bind Forkhead-1 and thus sequester it in the cytosol. (14-3-3 is the same protein that retains phos-phorylated Raf protein in the cytosol; see Figure 14-21.) Withdrawal of growth factor leads to inactivation of protein kinase B and dephosphorylation of Forkhead-1, thus favoring apoptosis. A Forkhead-1 mutant in which the three serine target residues for protein kinase B are mutated is "constitutively active" and initiates apoptosis even in the presence of activated protein kinase B. This finding demonstrates the importance of Forkhead-1 in controlling apoptosis of cultured cells.

PTEN Phosphatase Terminates Signaling via the PI-3 Kinase Pathway

Like virtually all intracellular signaling events, phosphorylation by PI-3 kinase is reversible. The relevant phosphatase, termed PTEN phosphatase, has an unusually broad specificity. Although PTEN can remove phosphate groups attached to serine, threonine, and tyrosine residues in proteins, its ability to remove the 3-phosphate from PI 3,4,5-trisphosphate is thought to be its major function in cells. Overexpression of PTEN in cultured mammalian cells promotes apoptosis by reducing the level of PI 3,4,5-trisphosphate and hence the activation and anti-apoptotic effect of protein kinase B.

The gene encoding PTEN is deleted in multiple types of advanced human cancers, and its loss is thought to lead to uncontrolled growth. Indeed, cells lacking PTEN have elevated levels of PI 3,4,5-trisphosphate and PKB activity. Since protein kinase B exerts an anti-apoptotic effect, loss of PTEN indirectly reduces the programmed cell death that is the normal fate of abnormally controlled cells. In certain cells, such as neuronal stem cells, absence of PTEN not only prevents apoptosis but also leads to stimulation of cell-cycle progression and an enhanced rate of proliferation. Thus knockout mice that cannot express PTEN have big brains with excess numbers of neurons, attesting to PTEN's importance in control of normal development. I

The Receptor for a Particular Growth Factor Often Is Linked to Multiple Signaling Pathways

Interaction of different signaling pathways permits the fine-tuning of cellular activities required to carry out complex developmental and physiological processes. As we have noted previously, both RTKs and cytokine receptors can initiate signaling via the Ras-MAP kinase pathway, DAG/IP3 pathway, and PI-3 kinase pathway (see Table 14-1). In addition, cytokine receptors can act through their associated JAK kinases to directly activate STAT transcription factors.

Activation of multiple signal-transduction pathways by many receptors allows different sets of genes to be independently controlled by the same or different receptors. Occasionally these pathways can induce opposite effects. For example, genetic manipulation of the Ras-MAP kinase and PI-3 kinase pathways during muscle differentiation indicates that these pathways have opposite phenotypic effects: activation of the Ras-MAP kinase pathway inhibits myocyte differentiation into myotubes, whereas activation of the PI-3 kinase pathway promotes it.

The initiation of tissue-specific signaling pathways by stimulation of the same receptor in different cells is exemplified by the EGF receptor. Genetic studies analogous to those described earlier for development of R7 cells in Drosophila demonstrated the central importance of EGF-stimulated signaling via the Ras-MAP pathway in development of the vulva in C. elegans. Other genetic studies, however, showed that stimulation of the EGF receptor triggers a Ras-independent pathway in some tissues. For example, one of the many functions of EGF in C. elegans is to control contractility of smooth muscle, which in turn regulates the extrusion of oocytes from one compartment of the hermaphrodite gonad to another, where they are fertilized. Coupling of the EGF receptor to Ras is not required for the EGF-induced contractions of the gonad. Analysis of several different types of mutations led researchers to conclude that in C. elegans smooth muscle, the EGF receptor is linked to the IP3/DAG pathway. Ligand binding to the receptor leads to activation of PLCy activity, an increase in IP3, and release of intracellular Ca2+ stores. The increased cytoso-lic Ca2+ level then promotes muscle contraction.

In Chapter 15 we will encounter several other examples of how stimulation of the same receptor in different cell types activates different signaling pathways that produce very diverse effects on the metabolism and fate of the cell.

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