Principles of cell signaling - UT Southwestern

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39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 608 (C) Catalytic subunits PKA C R R C Activation of PKA by cAMP (R) R egulatory subunits 4 cAMP R R - cAMP - cAMP - cAMP - cAMP Activated PKA C C teins throughout the cell ranging from ion channels to transcription factors, and its conserved substrate preference frequently permits prediction of substrates by sequence analysis. The cAMP response element binding protein CREB is phosphorylated by PKA on Ser 133 and is largely responsible for the impact of cAMP on transcription of numerous genes. R 2 C 2 + 4 cAMP Kinase activity as a function of [cAMP] (%) 100 80 60 40 20 10% R 2 . cAMP 4 + 2C 90% 14.15 Ca2+ signaling serves diverse purposes in all eukaryotic cells Key concepts • Ca2+ serves as a second messenger and regulatory molecule in essentially all cells. • Ca2+ acts directly on many target proteins and also regulates the activity of a regulatory protein calmodulin. • The cytosolic concentration of Ca2+ is controlled by organellar sequestration and release. 2 x 10 -9 2 x 10 -8 2 x 10 -7 [cAMP] FIGURE 14.15 PKA is a heterotetramer composed of two catalytic (C) and two regulatory (R) subunits. Binding of four molecules of cAMP to the regulatory subunits induces dissociation of two molecules of C, the active form of PKA, from the cAMP-bound regulatory subunit dimer. In the bottom panel, the cooperative binding of four molecules of cAMP generates a steep activation profile. Activity increases from approximately 10% to 90% as the cAMP concentration increases only 10-fold. An apparent threshold is introduced because there is little change in activity at low concentrations of cAMP. by phosphorylation of its activation loop. Phosphorylation occurs cotranslationally, and the activation loop phosphorylation is required for assembly of the R 2 C 2 tetramer. The PKAs are mostly cytosolic and are also targeted to specific locations by binding organelle-associated scaffolds (A-kinase anchoring proteins, or AKAPs). These AKAPs facilitate phosphorylation of membrane proteins including GPCRs, transporters, and ion channels. AKAPs can also target PKA to other cellular locations including mitochondria, the cytoskeleton, and the centrosome. AKAPs often harbor binding sites for other regulatory molecules such as phosphoprotein phosphatases and additional protein kinases, which allows for coordination of multiple signaling pathways and integration of their outputs. PKA generally phosphorylates substrates with a primary consensus motif of Arg-Arg- Xaa-Ser-Hydrophobic, placing it in a large group of kinases that recognize basic residues preceding the phosphorylation site. PKA regulates pro- Ca2+ is used as a second messenger in all cells, and is, thus, an even more widespread second messenger than cAMP. Many proteins bind Ca2+ with consequent allosteric changes in their enzymatic activities, subcellular localization, or interaction with other proteins or with lipids. Direct targets of Ca2+ regulation include almost all classes of signaling proteins described in this chapter, numerous metabolic enzymes, ion channels and pumps, and contractile proteins. Most noteworthy may be muscle actomyosin fibers, which are triggered to contract in response to cytosolic Ca2+ (see 8.21 Myosin-II functions in muscle contraction). Although free Ca2+ is found at concentrations near 1 mM in most extracellular fluids, intracellular Ca2+ concentrations are maintained near 100 nanomolar levels by the combined action of pumps and transporters that either extrude free Ca2+ or sequester it in the endoplasmic reticulum or mitochondria. Ca2+ signaling is initiated when Ca2+-selective channels in the endoplasmic reticulum or plasma membrane are opened to allow Ca2+ to enter the cytoplasm. The most important entrance channels include electrically gated channels in animal plasma membranes; a Ca2+ channel in the endoplasmic reticulum that is opened by another second messenger, inositol 1,4,5-trisphosphate (see below); and an electrically gated channel in the endoplasmic (sarcoplasmic) reticulum of muscle that opens in response to depolarization of nearby plasma membrane, a process known as excitation-contraction coupling (see 2.9 Plasma mem- 608 CHAPTER 14 Principles of cell signaling
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 609 Calcium binding causes a conformational change in calmodulin Calcium-free calmodulin calmodulin free + 4 Ca 2+ Calcium-bound calmodulin bound to target peptide of CaMK Ca 2+ target (Ca 2+ ) 4 . calmodulin . active target FIGURE 14.16 Ribbon diagrams representing the crystal structures of calmodulin free of Ca2+ and bound to four Ca2+ ions reveal the huge conformational change that calmodulin undergoes upon Ca2+ binding. Ca2+-calmodulin causes activity changes in target proteins. The bottom panel shows the activation of a target by calmodulin as a function of the intracellular free Ca2+ concentration. The requirement for binding four Ca2+ ions to induce the conformational transition results in cooperative activation of targets. Activity increases from 10% to 90% as the Ca2+ concentration increases only 10-fold. Structures generated from Protein Data Bank files 1CFD and 1MXE. Activation of target by calmodulin (%) 100 90% 80 60 40 20 10% 3 x 10 -8 3 x 10 -7 3 x 10 -6 [Ca 2+ ] brane Ca2+ channels activate intracellular functions). In addition to the proteins that are regulated by binding Ca2+ directly, many other proteins respond to Ca2+ by binding a widespread Ca2+ sensor, the small, ~17 kDa protein calmodulin. Calmodulin requires the binding of four molecules of Ca2+ to become fully active, and binding is highly cooperative, generating a sigmoid activation profile illustrated in FIGURE 14.16. Calmodulin generally binds its targets in a Ca2+dependent manner, but Ca2+-free calmodulin may remain bound but inactive in some cases. For example, calmodulin is a constitutive subunit of phosphorylase kinase that is activated upon Ca2+ binding. Higher plants again make major modifications to this paradigm. Calmodulin is not expressed as a distinct protein but, instead, is found as a domain in Ca2+-regulated proteins. In yet another variation, the adenylyl cyclase secreted by the pathogenic bacterium Bordetella pertussis is inactive outside cells but is activated by Ca2+-free calmodulin in animal cells, where its rapid production of cAMP is highly toxic. 14.16 Lipids and lipid-derived compounds are signaling molecules Key concepts • Multiple lipid-derived second messengers are produced in membranes. • Phospholipase Cs release soluble and lipid second messengers in response to diverse inputs. • Channels and transporters are modulated by different lipids in addition to inputs from other sources. • PI 3-kinase synthesizes PIP 3 to modulate cell shape and motility. • PLD and PLA 2 create other lipid second messengers. Signals that originate at the plasma membrane may have soluble regulatory targets in the cytoplasm or intracellular organelles, but integral plasma membrane proteins are also subject to acute controls. For these targets, lipid second messengers may be primary inputs. Lipids derived from membrane phospholipids or other 14.16 Lipids and lipid-derived compounds are signaling molecules 609
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Principles of cell signaling - UT Southwestern

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