H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 36
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At low concentrations of cyclic AMP (cAMP), the PKAis an inactive tetramer. Binding of cAMP to the regulatory (R)subunits causes a conformational change in these subunits thatpermits release of the active, monomeric catalytic (C) subunits.(b) Cyclic AMP is a derivative of adenosine monophosphate. Thisintracellular signaling molecule, whose concentration rises inresponse to various extracellular signals, can modulate theactivity of many proteins.100-fold by the release of Ca2 from ER stores or by its import from the extracellular environment. This rise in cytosolic Ca2 is sensed by Ca2-binding proteins, particularlythose of the EF hand family, all of which contain the helixloop-helix motif discussed earlier (see Figure 3-6a).The prototype EF hand protein, calmodulin, is found inall eukaryotic cells and may exist as an individualmonomeric protein or as a subunit of a multimeric protein.
Adumbbell-shaped molecule, calmodulin contains four Ca2binding sites with a KD of ≈106 M. The binding of Ca2 tocalmodulin causes a conformational change that permitsCa2/calmodulin to bind various target proteins, therebyswitching their activity on or off (Figure 3-28). Calmodulinand similar EF hand proteins thus function as switch proteins, acting in concert with Ca2 to modulate the activityof other proteins.Switching Mediated by Guanine Nucleotide–BindingProteins Another group of intracellular switch proteins constitutes the GTPase superfamily. These proteins includemonomeric Ras protein (see Figure 3-5) and the G subunit ofthe trimeric G proteins.
Both Ras and G are bound to theplasma membrane, function in cell signaling, and play a keyrole in cell proliferation and differentiation. Other membersEF1EF3EF2protein-folding machine comprises two back-to-back multisubunit rings, which can exist in a “tight” peptide-bindingstate and a “relaxed” peptide-releasing state (see Figure3-11). The binding of ATP and the co-chaperonin GroES toone of the rings in the tight state causes a twofold expansionof the GroEL cavity, shifting the equilibrium toward the relaxed peptide-folding state.EF4TargetpeptideCa2+Calcium and GTP Are Widely Used to ModulateProtein ActivityIn the preceding examples, oxygen, cAMP, and ATP cause allosteric changes in the activity of their target proteins (hemoglobin, protein kinase A, and GroEL, respectively).
Twoadditional allosteric ligands, Ca2 and GTP, act through twotypes of ubiquitous proteins to regulate many cellularprocesses.Calmodulin-Mediated Switching The concentration ofCa2 free in the cytosol is kept very low (≈107 M) by membrane transport proteins that continually pump Ca2 out ofthe cell or into the endoplasmic reticulum. As we learn inChapter 7, the cytosolic Ca2 level can increase from 10- to▲ FIGURE 3-28 Switching mediated by Ca2/calmodulin.Calmodulin is a widely distributed cytosolic protein that containsfour Ca2-binding sites, one in each of its EF hands.
Each EFhand has a helix-loop-helix motif. At cytosolic Ca2+ concentrationsabove about 5 107 M, binding of Ca2 to calmodulin changesthe protein’s conformation. The resulting Ca2/calmodulin wrapsaround exposed helices of various target proteins, therebyaltering their activity.3.5 • Common Mechanisms for Regulating Protein FunctionActiveActive ("on")RGTPaseGDPGEFsGTP+++−GAPsRGSsGDIsGTPaseGDPOHPiATPProteinphosphataseProteinkinaseInactive ("off ")GTP85OH2OROPADPO−O−Inactive▲ FIGURE 3-29 Cycling of GTPase switch proteins betweenthe active and inactive forms.
Conversion of the active into theinactive form by hydrolysis of the bound GTP is accelerated byGAPs (GTPase-accelerating proteins) and RGSs (regulators of Gprotein–signaling) and inhibited by GDIs (guanine nucleotidedissociation inhibitors). Reactivation is promoted by GEFs(guanine nucleotide–exchange factors).▲ FIGURE 3-30 Regulation of protein activity bykinase/phosphatase switch.
The cyclic phosphorylation anddephosphorylation of a protein is a common cellular mechanismfor regulating protein activity. In this example, the target proteinR is inactive (light orange) when phosphorylated and active (darkorange) when dephosphorylated; some proteins have theopposite pattern.of the GTPase superfamily function in protein synthesis, thetransport of proteins between the nucleus and the cytoplasm,the formation of coated vesicles and their fusion with targetmembranes, and rearrangements of the actin cytoskeleton.All the GTPase switch proteins exist in two forms (Figure3-29): (1) an active (“on”) form with bound GTP (guanosinetriphosphate) that modulates the activity of specific targetproteins and (2) an inactive (“off”) form with bound GDP(guanosine diphosphate).
The GTPase activity of theseswitch proteins hydrolyzes bound GTP to GDP slowly, yielding the inactive form. The subsequent exchange of GDP withGTP to regenerate the active form occurs even more slowly.Activation is temporary and is enhanced or depressed byother proteins acting as allosteric regulators of the switchprotein. We examine the role of various GTPase switch proteins in regulating intracellular signaling and other processesin several later chapters.Nearly 3 percent of all yeast proteins are protein kinases orphosphatases, indicating the importance of phosphorylationand dephosphorylation reactions even in simple cells.
Allclasses of proteins—including structural proteins, enzymes,membrane channels, and signaling molecules—are regulatedby kinase/phosphatase switches. Different protein kinases andphosphatases are specific for different target proteins and canthus regulate a variety of cellular pathways, as discussed inlater chapters. Some of these enzymes act on one or a few target proteins, whereas others have multiple targets. The latterare useful in integrating the activities of proteins that are coordinately controlled by a single kinase/phosphatase switch.Frequently, another kinase or phosphatase is a target, thus creating a web of interdependent controls.Cyclic Protein Phosphorylationand Dephosphorylation RegulateMany Cellular FunctionsAs noted earlier, one of the most common mechanisms forregulating protein activity is phosphorylation, the additionand removal of phosphate groups from serine, threonine, ortyrosine residues.
Protein kinases catalyze phosphorylation,and phosphatases catalyze dephosphorylation. Althoughboth reactions are essentially irreversible, the counteractingactivities of kinases and phosphatases provide cells with a“switch” that can turn on or turn off the function of various proteins (Figure 3-30). Phosphorylation changes a protein’s charge and generally leads to a conformational change;these effects can significantly alter ligand binding by a protein, leading to an increase or decrease in its activity.Proteolytic Cleavage Irreversibly Activatesor Inactivates Some ProteinsThe regulatory mechanisms discussed so far act as switches,reversibly turning proteins on and off.
The regulation ofsome proteins is by a distinctly different mechanism: the irreversible activation or inactivation of protein function byproteolytic cleavage. This mechanism is most common in regard to some hormones (e.g., insulin) and digestive proteases. Good examples of such enzymes are trypsin andchymotrypsin, which are synthesized in the pancreas and secreted into the small intestine as the inactive zymogenstrypsinogen and chymotrypsinogen, respectively. Enterokinase, an aminopeptidase secreted from cells lining the smallintestine, converts trypsinogen into trypsin, which in turncleaves chymotrypsinogen to form chymotrypsin. The delayin the activation of these proteases until they reach the intestine prevents them from digesting the pancreatic tissue inwhich they are made.86CHAPTER 3 • Protein Structure and FunctionHigher-Order Regulation Includes Controlof Protein Location and Concentrationactivity state into another or to the release of active subunits (see Figure 3-27).The activities of proteins are extensively regulated in orderthat the numerous proteins in a cell can work together harmoniously.
For example, all metabolic pathways are closelycontrolled at all times. Synthetic reactions take place whenthe products of these reactions are needed; degradative reactions take place when molecules must be broken down.All the regulatory mechanisms heretofore described affect aprotein locally at its site of action, turning its activity onor off.Normal functioning of a cell, however, also requires thesegregation of proteins to particular compartments such asthe mitochondria, nucleus, and lysosomes.
In regard to enzymes, compartmentation not only provides an opportunityfor controlling the delivery of substrate or the exit of productbut also permits competing reactions to take place simultaneously in different parts of a cell. We describe the mechanisms that cells use to direct various proteins to differentcompartments in Chapters 16 and 17.In addition to compartmentation, cellular processes areregulated by protein synthesis and degradation.
For example,proteins are often synthesized at low rates when a cell has little or no need for their activities. When the cell faces increased demand (e.g., appearance of substrate in the case ofenzymes, stimulation of B lymphocytes by antigen), the cellresponds by synthesizing new protein molecules. Later, theprotein pool is lowered when levels of substrate decrease orthe cell becomes inactive. Extracellular signals are often instrumental in inducing changes in the rates of protein synthesis and degradation (Chapters 13–15). Such regulatedchanges play a key role in the cell cycle (Chapter 21) and incell differentiation (Chapter 22).Two classes of intracellular switch proteins regulate avariety of cellular processes: (1) calmodulin and relatedCa2-binding proteins in the EF hand family and (2) members of the GTPase superfamily (e.g., Ras and G), whichcycle between active GTP-bound and inactive GDP-boundforms (see Figure 3-29).KEY CONCEPTS OF SECTION 3.5Common Mechanisms for RegulatingProtein FunctionIn allostery, the binding of one ligand molecule (a substrate, activator, or inhibitor) induces a conformationalchange, or allosteric transition, that alters a protein’s activity or affinity for other ligands.■In multimeric proteins, such as hemoglobin, that bindmultiple ligand molecules, the binding of one ligand molecule may modulate the binding affinity for subsequent ligand molecules.
Enzymes that cooperatively bind substratesexhibit sigmoidal kinetics similar to the oxygen-bindingcurve of hemoglobin (see Figure 3-26).■■The phosphorylation and dephosphorylation of aminoacid side chains by protein kinases and phosphatases provide reversible on/off regulation of numerous proteins.■Nonallosteric mechanisms for regulating protein activity include proteolytic cleavage, which irreversibly convertsinactive zymogens into active enzymes, compartmentationof proteins, and signal-induced modulation of protein synthesis and degradation.■3.6 Purifying, Detecting,and Characterizing ProteinsA protein must be purified before its structure and themechanism of its action can be studied. However, becauseproteins vary in size, charge, and water solubility, no singlemethod can be used to isolate all proteins.
To isolate oneparticular protein from the estimated 10,000 different proteins in a cell is a daunting task that requires methods bothfor separating proteins and for detecting the presence of specific proteins.Any molecule, whether protein, carbohydrate, or nucleicacid, can be separated, or resolved, from other molecules onthe basis of their differences in one or more physical orchemical characteristics. The larger and more numerous thedifferences between two proteins, the easier and more efficient their separation. The two most widely used characteristics for separating proteins are size, defined as either lengthor mass, and binding affinity for specific ligands. In this section, we briefly outline several important techniques for separating proteins; these techniques are also useful for theseparation of nucleic acids and other biomolecules. (Specialized methods for removing membrane proteins from membranes are described in the next chapter after the uniqueproperties of these proteins are discussed.) We then considergeneral methods for detecting, or assaying, specific proteins,including the use of radioactive compounds for trackingbiological activity.
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