Single protein kinases such as PKA, PKC, and Ca2+ calmodulin-dependent kinases (CaMKs), which result in the phosphorylation of serine and threonine residues in target proteins, play a very important role in hormone action. The discovery that the epidermal growth factor (EGF) receptor contains an intrinsic tyrosine kinase activity that is activated by the binding of the ligand EGF was an important break through. The insulin and insulin-like growth factor 1 (IGF 1) receptors also contain intrinsic ligand-activated tyrosine kinase activity. Several receptors—generally those involved in binding ligands that control cellular growth, differentiation, and the inflammatory response—either have intrinsic tyrosine kinase activity or are tightly associated with proteins that are tyrosine kinases. Another distinguishing feature of this class of hormone action is that these kinases preferentially phosphorylate tyrosine residues, and tyrosine phosphorylation is infrequent (< 0.03% of total amino acid phosphorylation) in mammalian cells. A third distinguishing feature is that the ligand-receptor interaction that results in a tyrosine phosphorylation event initiates a cascade that may involve several protein kinases, phosphatases, and other regulatory proteins.
Insulin Transmits Signals by Several Kinase Cascades
The insulin, EGF, and IGF-1 receptors have intrinsic protein tyrosine kinase activities located in their cytoplasmic domains. These activities are stimulated when their ligands bind to the cognate receptor. The receptors are then autophosphorylated on tyrosine residues, and this phosphorylation initiates a complex series of events (summarized in simplified fashion in Figure1). The phosphorylated insulin receptor next phosphorylates insulin receptor substrates (there are at least four of these molecules, called IRS 1-4) on tyrosine residues. Phosphorylated IRS binds to the Src homology 2 (SH2) domains of a variety of proteins that are directly involved in mediating different effects of insulin. One of these proteins, PI-3 kinase, links insulin receptor activation to insulin action through activation of a number of molecules, including the kinase phosphoinositide-dependent kinase 1 (PDK1). This enzyme propagates the signal through several other kinases, including PKB (also known as AKT), SKG, and aPKC (see legend to Figure 42–8 for definitions and expanded abbreviations). An alternative pathway downstream from PDK1 involves p70S6K and perhaps other as yet unidentified kinases. A second major pathway involves mTOR. This enzyme is directly regulated by amino acid levels and insulin and is essential for p70S6K activity. The mTOR-signaling system provides a dis tinction between the PKB and p70S6K branches downstream from PKD1. These pathways are involved in protein trans location, enzyme activation, and the regulation, by insulin, of genes involved in metabolism (see Figure 1). Another SH2 domain–containing protein is GRB2, which binds to IRS-1 and links tyrosine phosphorylation to several proteins, the result of which is activation of a cascade of threonine and serine kinases. A pathway showing how this insulin-receptor interaction activates the mitogen-activated protein kinase (MAPK) pathway and the anabolic effects of insulin is illustrated in Figure 1. The exact roles of many of these docking proteins, kinases, and phosphatases are actively being studied.
Fig1. Insulin signaling pathways.The insulin signaling pathways provide an excellent example of the “recognition -> hormone release -> signal generation -> effects” paradigm outlined in Figure 42–1. Insulin is released into the bloodstream from pancreatic β cells in response to hyperglycemia. Binding of insulin to a target cell-specific plasma membrane heterotetrameric insulin receptor (IR) results in a cas cade of intracellular events. First, the intrinsic tyrosine kinase activity of the insulin receptor is activated, and marks the initial event. Receptor activation results in increased tyrosine phosphorylation (conversion of specific Y residues to Y-P) within the receptor. One or more of the insulin receptor substrate (IRS) molecules (IRS 1-4) then bind to the tyrosine-phosphorylated receptor and themselves are specifically tyrosine phosphorylated. IRS proteins interact with the activated IR via N-terminal PH (pleckstrin homology) and phosphotyrosine binding (PTB) domains. IR-docked IRS proteins are tyrosine phosphorylated and the resulting P-Y residues form the docking sites for several additional signaling proteins (ie, PI-3 kinase, GRB2, and mTOR). GRB2 and PI-3K bind to IRS P-Y residues via their SH (Src homology) domains; binding to IRS-Y-P residues leads to activation of the activity of many intracellular signaling molecules such as GTPases, protein kinases, and lipid kinases, all of which play key roles in certain metabolic actions of insulin. The two best-described pathways are shown. In detail, phosphorylation of an IRS molecule results in docking and activation of the lipid kinase, PI-3 kinase; PI-3K generates novel inositol lipids that act as “second messenger”molecules. These, in turn, activate PDK1 and then a variety of downstream signaling molecules, including protein kinase B (PKB/AKT), SGK, and aPKC. An alternative path way involves the activation of p70S6K and perhaps other as yet unidentified kinases. Next, additional phosphorylation of IRS results in docking of GRB2/mSOS and activation of the small GTPase, p21Ras, which initiates a protein kinase cascade that activates Raf-1, MEK, and the p42/p44 MAP kinase isoforms. These protein kinases are important in the regulation of proliferation and differentiation of many cell types. The mTOR pathway provides an alternative way of activating p70S6K and is involved in nutrient signaling as well as insulin action. Each of the indicated signaling cascades may influence different biologic processes, as shown (Protein Translocation, Protein/Enzyme Activity, Gene Transcription/Translation, Cell Growth). All of the phosphorylation events are reversible through the action of specific phosphatases. As an example, the lipid phosphatase PTEN dephosphorylates the product of the PI-3 kinase reaction, thereby antagonizing the pathway and terminating the signal. Representative effects of major actions of insulin are shown in each of the boxes (bottom). The asterisk after phosphodiesterase indicates that insulin indirectly affects the activity of many enzymes by activating phosphodiesterases and reducing intracellular cAMP levels. (aPKC, atypical protein kinase C; GRB2, growth factor receptor binding protein 2; IGFBP, insulin-like growth factor binding protein; IRS 1-4, insulin receptor substrate isoforms 1-4; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase and ERK kinase; mSOS, mammalian son of sevenless; mTOR, mammalian target of rapamycin; p70S6K, p70 ribosomal protein S6 kinase; PDK1, phosphoinositide-dependent kinase; PI-3 kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; PTEN, phosphatase and tensin homolog deleted on chromosome 10; SGK, serum and glucocorticoid-regulated kinase.)
The JAK/STAT Pathway Is Used by Hormones and Cytokines
Tyrosine kinase activation can also initiate a phosphorylation and dephosphorylation cascade that involves the action of several other protein kinases and the counterbalancing actions of phosphatases. Two mechanisms are employed to initiate this cascade. Some hormones, such as growth hormone, prolactin, erythropoietin, and the cytokines, initiate their action by activating a tyrosine kinase, but this activity is not an integral part of the hormone receptor. The hormone-receptor interaction promotes binding and activation of cytoplasmic protein tyrosine kinases, such as the Janus kinases 1 and 2, JAK1, or JAK2, or tyrosine kinase 2, TYK2.
These kinases phosphorylate one or more cytoplasmic proteins, which then associate with other docking proteins through binding to SH2 domains. One such interaction results in the activation of a family of cytosolic proteins called STATs, or signal transducers and activators of transcription. The phosphorylated STAT protein dimerizes and translocates into the nucleus, binds to a specific DNA element such as the interferon response element (IRE), and activates transcription. This is illustrated in Figure 2. Other SH2 docking events may result in the activation of PI-3 kinase, the MAP kinase pathway (through SHC or GRB2), or G-protein–mediated activation of phospholipase C (PLCγ) with the attendant production of diacylglycerol and activation of protein kinase C. It is apparent that there is a potential for “cross-talk” when different hormones activate these various signal transduction pathways.
Fig2. Initiation of signal transduction by receptors linked to Jak kinases. The receptors (R) that bind prolactin, growth hor mone, interferons, and cytokines lack endogenous tyrosine kinase. On ligand binding, these receptors dimerize and an associated, though inactive protein kinase (JAK1, JAK2, or TYK) is phosphorylated. Phospho-JAK is now activated, and proceeds to phosphorylate the receptor on tyrosine residues. The STAT proteins associate with the phosphorylated receptor and then are themselves phosphorylated by JAK-P. The phosphorylated STAT protein, STATⓅ dimerizes, translocates to the nucleus, binds to specific DNA elements, and regulates transcription. The phosphotyrosine residues of the receptor also bind to several SH2 domain-containing proteins (X-SH2), which result in activation of the MAP kinase pathway (through SHC or GRB2), PLCγ, or PI-3 kinase.
The NF-κB Pathway Is Regulated by Glucocorticoids
The DNA-binding transcription factor NF-κB is a heterodimeric complex typically composed of two subunits termed p50 and p65 (Figure 3). Normally NF-κβ is sequestered in the cytoplasm in a transcriptionally inactive form by interactions with members of the IκB (inhibitor of NF-κβ) family of proteins. Extracellular stimuli such as proinflammatory cytokines, reactive oxygen species, and mitogens lead to activation of the IKK (IκB kinase) complex, which is a het erohexameric structure consisting of α, β, and γ subunits. Activated IKK phosphorylates IκB on two serine residues. This phosphorylation targets IκB for polyubiquitylation and subsequent degradation by the proteasome. Following IκB degradation, free NF-κB translocates to the nucleus, where it binds to a number of gene enhancers and activates transcription, particularly of genes involved in the inflammatory response. Transcriptional regulation by NF-κB is mediated by a variety of coactivators such as CREB-binding protein (CBP), as described later.
Fig3.. Regulation of the NF-κB pathway.NF-κB consists of two subunits, p50 and p65, which when present in the nucleus regulates transcription of the multitude of genes important for the inflammatory response. NF-κB is restricted from entering the nucleus by IκB, an inhibitor of NF-κB. IκB binds to—and masks—the nuclear localization signal of NF-κB. This cytoplasmic protein is phosphorylated by an IKK complex which is activated by cytokines, reactive oxygen species, and mitogens. Phosphorylated IκB can be ubiquitylated and degraded, thus releasing its hold on NF-κB, and allowing for nuclear translocation. Glucocorticoids, potent anti-inflammatory agents, are thought to affect at least three steps in this process (1, 2, 3), as described in the text.
Glucocorticoid hormones are therapeutically useful agents for the treatment of a variety of inflammatory and immune diseases. Their anti-inflammatory and immunomodulatory actions are explained in part by the inhibition of NF-κB and its subsequent actions. Evidence for three mechanisms for the inhibition of NF-κB by glucocorticoids has been described: (1) glucocorticoids increase IκB mRNA, which leads to an increase of IκB protein and more efficient sequestration of NF-κB in the cytoplasm. (2) The GR competes with NF-κB for binding to coactivators. (3) The GR directly binds to the p65 subunit of NF-κB and inhibits its activation (see Figure3).
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