Principles of cell signaling - UT Southwestern

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39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 620 Single photon responses of GAP-deficient mice Current (pA) 0.50 0.25 0.00 knockout heterozygous wild-type 0 2 Time (s) 4 Light flash FIGURE 14.27 G protein GAPs can accelerate signal termination upon removal of agonist, and often do not act as inhibitors during the response to receptor. The figure shows the electrical response of a mouse photoreceptor (rod) cell to a single photon of light. In mice that lack RGS9, the GAP for the photoreceptor G protein G t , the signal is prolonged for many seconds because hydrolysis of GTP bound to G t is slow. In wild-type or heterozygous mice, hydrolysis takes place in about 15 milliseconds, and the decay of the signal is much faster. Note that the maximal output is similar in wild-type and mutant mice, indicating that the GAP does not act as an inhibitor in rod cells. In humans, genetic loss of RGS9 leads to severe loss of vision that is particularly marked in bright light. Reproduced from Chen et al. Nature. 2000. 403:557–560. Permission also granted by Ching-Kang Jason Chen, Virginia Commonwealth University. While the GTPase cycle described in Figure 14.26 is general, it is highly simplified. Interactions among receptor, Gα, Gβγ, GAP, and effector are frequently simultaneous and often demonstrate complex cooperative interactions. For example, Gβγ inhibits the release of GDP (to minimize spontaneous activation), promotes the exchange catalyst activity of the receptor, inhibits GAP activity, and helps initiate receptor phosphorylation that leads to desensitization. The other components can be nearly this multifunctional. In addition, inputs from other proteins can alter the dynamics of the GTPase cycle at several points. The core G protein module is, thus, functionally versatile as a signal processor in addition to being versatile in the scope of its targets. 14.23 Small, monomeric GTPbinding proteins are multiuse switches Key concepts • Small GTP-binding proteins are active when bound to GTP and inactive when bound to GDP. • GDP/GTP exchange catalysts known as GEFs (guanine nucleotide exchange factors) promote activation. • GAPs accelerate hydrolysis and deactivation. • GDP dissociation inhibitors (GDIs) slow spontaneous nucleotide exchange. Monomeric GTP-binding proteins, which are encoded by about 150 genes in animals, modulate a wide variety of cellular processes including signal transduction, organellar trafficking, intra-organellar transport, cytoskeletal assembly, and morphogenesis. The small GTP-binding proteins that most clearly function in signal transduction are the Ras and Ras-related proteins (Ral, Rap) and the Rho/Rac/Cdc42 proteins, about 10-15 in all. They are usually about 20-25 kDa in size and are homologous to the GTP-binding domains of Gα subunits. The regulatory activities of the small GTPbinding proteins are controlled by a GTP binding and hydrolysis cycle like that of the heterotrimeric G proteins, with similar regulatory inputs. They are activated by GTP, and hydrolysis of bound GTP to GDP terminates activation. GDP/GTP exchange catalysts, known as GEFs (guanine nucleotide exchange factors, functionally analogous to GPCRs) promote activation, and GAPs accelerate hydrolysis and consequent deactivation. In addition, GDP dissociation inhibitors (GDIs) slow spontaneous nucleotide exchange and activation to dampen basal activity, an activity shared by Gβγ subunits for the heterotrimeric G proteins. While the underlying biochemical regulatory events are essentially identical for monomeric and heterotrimeric G proteins, monomeric G proteins use the basic GTPase cycle in additional ways. Signal output by heterotrimeric G proteins and many monomeric G proteins is usually thought to reflect a balance of their active (GTP-bound) and inactive (GDP-bound) states in a rapidly turning-over GTPase cycle. GEFs favor formation of more active G protein, and GAPs favor the inactive state. In contrast, probably an equal number of the monomeric G proteins behave as acute on-off switches. Upon binding GTP, they initiate a process (regulation, recruitment of other pro- 620 CHAPTER 14 Principles of cell signaling
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 621 teins). They then maintain this activity, sometimes for many seconds or minutes, until they are acted upon by a GAP. For example, the monomeric G protein Ran regulates nucleocytoplasmic trafficking of protein and RNA in both directions, cooperating with carrier proteins known as karyopherins (see 5.15 The Ran GTPase controls the direction of nuclear transport). In the nucleus, high Ran GEF activity promotes GTP binding. Nuclear Ran-GTP then binds import karyopherins to drive dissociation of newly arrived cargo and promote return of the karyopherin to the cytoplasm. It also binds export karyopherins to permit binding of outgoing cargo. Outside the nucleus, high Ran GAP activity promotes GTP hydrolysis. Cytoplasmic Ran-GDP dissociates from both the export karyopherins to allow dissociation of outgoing cargo and from the import karyopherins to allow them to bind cargo for import. Thus, for monomeric G proteins such as Ran, each phase of the GTPase cycle determines a specific, coupled step in a parallel regulatory cycle. A second major difference between the monomeric and the heterotrimeric G proteins is the structures of the GEFs, GAPs, and GDIs. Both GEFs and GAPs for monomeric GTP-binding proteins are structurally heterogeneous (although some clearly related families are evident). In addition, mechanisms for regulating these GEFs and GAPs are equally diverse. They include phosphorylation by protein kinases; allosteric regulation by heterotrimeric and/or monomeric G proteins, by second messengers and by other regulatory proteins; subcellular sequestration or recruitment to scaffolds; and assorted other mechanisms. The Ras proteins were the first small GTPbinding proteins to be discovered. They were identified as oncogene products because they cause malignant growth if they are either overexpressed or persistently activated by mutation; they are among the most commonly mutated genes in human tumors. Several viral ras genes figure prominently as oncogenes. Mammalian cells contain three ras genes (H, N, and K). They may share inputs and outputs to varying extents, and they can compensate for each other in some genetic screens. It has been difficult to assign unique functions to the individual Ras proteins. Inputs to the Ras proteins are diverse and speak to the importance of Ras proteins as a crucial node in signaling. Ras GEFs and GAPS are regulated by both receptor and nonreceptor Tyr kinases through direct phosphorylation and by recruitment of the regulators to the plasma membrane. Other cytoplasmic serine/threonine kinases also converge on Ras activation. Rap1, another member of the Ras family, may also fit directly into this network because it is suspected of competing with Ras proteins for protein kinase targets; in vivo it can suppress the oncogenic activity of Ras. Rap1 is regulated independently, however, and acts on independent signaling pathways as well. One of its GAPs is stimulated by the G i class of G proteins, for example, and its several GEFs are stimulated by Ca2+, diacylglycerol, and cAMP. Ras proteins generally regulate cell growth, proliferation, and differentiation by modulating the activities of multiple effector proteins. The best known and best studied Ras effector is the protein kinase Raf, which initiates a MAPK cascade. FIGURE 14.28 shows well established Ras effectors. Rho, Rac, and Cdc42 are related monomeric GTP-binding proteins that are involved in generating signals that affect cell morphology. Each class of proteins regulates its own array of effectors and is controlled by separate groups of GEFs, GAPs, and GDIs. Effectors regulated by this family include phospholipases C and D, multiple protein and lipid kinases, proteins that nucleate or reorganize actin filaments, and components of the neutrophil oxygen activating system, among others (see 8.14 Small G proteins regulate actin polymerization). 14.24 Ras has three main effectors Function Effector Target Protein kinase cascade Raf MAPK Lipid kinase PI 3-kinase Akt Exchange factor RalGDS Exocyst Protein phosphorylation/ dephosphorylation is a major regulatory mechanism in the cell Key concepts • Protein kinases are a large protein family. • Protein kinases phosphorylate Ser and Thr, or Tyr, or all three. • Protein kinases may recognize the primary sequence surrounding the phosphorylation site. • Protein kinases may preferentially recognize phosphorylation sites within folded domains. FIGURE 14.28 Ras-GTP binds to many proteins. Three well established effectors include Raf, PI 3-kinase, and RalGDS. Activation of these effectors activates a MAPK pathway, increases PI 3-kinase activity, and promotes assembly of a protein complex involved in exocytosis of secretory vesicles. 14.24 Protein phosphorylation/dephosphorylation is a major regulatory mechanism in the cell 621
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Principles of cell signaling - UT Southwestern

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