B. Alberts, A. Johnson, J. Lewis и др. - Molecular Biology of The Cell (6th edition) (1120996), страница 60
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As described in the text, the requirement for multipleupstream events to trigger these processes allows the kinase to serve as a signal integrator (Movie 3.11). (Adapted from S.C. Harrison et al., Cell112:737–740, 2003. With permission from Elsevier.)PROTEIN FUNCTION157Figure 3–65 How a Src-type protein kinase acts as a signal-integratingdevice. A disruption of the inhibitory interaction illustrated for the SH3domain (green) occurs when its binding to the indicated orange linker regionis replaced with its higher-affinity binding to an activating ligand.change that inactivates the protein.
The three-dimensional structure of a prototypical member of this family, the monomeric GTPase called Ras, is shown in Figure 3–67.The Ras protein has an important role in cell signaling (discussed in Chapter15). In its GTP-bound form, it is active and stimulates a cascade of protein phosphorylations in the cell. Most of the time, however, the protein is in its inactive,GDP-bound form. It becomes active when it exchanges its GDP for a GTP molecule in response to extracellular signals, such as growth factors, that bind to receptors in the plasma membrane (see Figure 15–47).Regulatory Proteins GAP and GEF Control the Activity of GTPBinding Proteins by Determining Whether GTP or GDP Is BoundGTP-binding proteins are controlled by regulatory proteins that determinewhether GTP or GDP is bound, just as phosphorylated proteins are turned onand off by protein kinases and protein phosphatases.
Thus, Ras is inactivated by aGTPase-activating protein (GAP), which binds to the Ras protein and induces Rasto hydrolyze its bound GTP molecule to GDP—which remains tightly bound—and inorganic phosphate (Pi), which is rapidly released. The Ras protein stays inits inactive, GDP-bound conformation until it encounters a guanine nucleotideexchange factor (GEF), which binds to GDP-Ras and causes Ras to release its GDP.Because the empty nucleotide-binding site is immediately filled by a GTP molecule (GTP is present in large excess over GDP in cells), the GEF activates Rasby indirectly adding back the phosphate removed by GTP hydrolysis. Thus, in asense, the roles of GAP and GEF are analogous to those of a protein phosphataseand a protein kinase, respectively (Figure 3–68).INPUTShas thishas thishas thisbinding been phosphatephosphatebeen removed? disrupted? been added?PPSrc-type protein kinase activity turns onfully only if the answers to all of theabove questions are yesOUTPUTMBoC6 m3.70/3.60Proteins Can Be Regulated by the Covalent Addition of OtherProteinsCells contain a special family of small proteins whose members are covalentlyattached to many other proteins to determine the activity or fate of the secondprotein.
In each case, the carboxyl end of the small protein becomes linked tothe amino group of a lysine side chain of a “target” protein through an isopeptidebond. The first such protein discovered, and the most abundantly used, is ubiquitin (Figure 3–69A). Ubiquitin can be covalently attached to target proteins in avariety of ways, each of which has a different meaning for cells.
The major form ofubiquitin addition produces polyubiquitin chains in which—once the first ubiquitin molecule is attached to the target—each subsequent ubiquitin moleculelinks to Lys48 of the previous ubiquitin, creating a chain of Lys48-linked ubiquitins that are attached to a single lysine side chain of the target protein. This formof polyubiquitin directs the target protein to the interior of a proteasome, where itis digested to small peptides (see Figure 6–84). In other circumstances, only singlemolecules of ubiquitin are added to proteins. In addition, some target proteins areGTP-binding proteinPiGTPGTPHYDROLYSISGDPGDPGTPSLOWFASTGTPONOFFOFFONACTIVEINACTIVEINACTIVEACTIVEFigure 3–66 GTP-binding proteinsas molecular switches. The activityof a GTP-binding protein (also called aGTPase) generally requires the presenceof a tightly bound GTP molecule (switch“on”).
Hydrolysis of this GTP moleculeby the GTP-binding protein producesGDP and inorganic phosphate (Pi), and itcauses the protein to convert to a different,usually inactive, conformation (switch “off”).Resetting the switch requires that the tightlybound GDP dissociates. This is a slowstep that is greatly accelerated by specificsignals; once the GDP has dissociated, amolecule of GTP is quickly rebound.158Chapter 3: Proteinsmodified with a different type of polyubiquitin chain.
These modifications havedifferent functional consequences for the protein that is targeted (Figure 3–69B).Related structures are created when a different member of the ubiquitin family, such as SUMO (small ubiquitin-related modifier), is covalently attached to alysine side chain of target proteins. Not surprisingly, all such modifications arereversible.
Cells contain sets of ubiquitylating and deubiquitylating (and sumoylating and desumoylating) enzymes that manipulate these covalent adducts,thereby playing roles analogous to the protein kinases and phosphatases that addand remove phosphates from protein side chains.An Elaborate Ubiquitin-Conjugating System Is Used to MarkProteinsSIGNAL INPROTEINKINASEGDPGEFATPGDPOFFPiADPONGTPOFFPiONGAPPROTEINPHOSPHATASEPGTPSIGNAL OUTSIGNAL OUTSIGNALING BY PHOSPHORYLATED PROTEINSIGNALING BY GTP-BINDING PROTEINFigure 3–68 A comparison of two major intracellular signaling mechanisms in eukaryoticcells.
In both cases, a signaling protein is activated by the addition of a phosphate group andinactivated by the removal of this phosphate. Note that the addition of a phosphate to a proteincan also be inhibitory. (Adapted from E.R. Kantrowitz and W.N. Lipscomb, Trends Biochem. Sci.15:53–59, 1990.)MBoC6 m3.73/3.63NH2GTPPswitchhelixHow do cells select target proteins for ubiquitin addition? As an initial step, thecarboxyl end of ubiquitin needs to be activated.
This activation is accomplishedwhen a protein called a ubiquitin-activating enzyme (E1) uses ATP hydrolysisenergy to attach ubiquitin to itself through a high-energy covalent bond (a thioester). E1 then passes this activated ubiquitin to one of a set of ubiquitin-conjugating (E2) enzymes, each of which acts in conjunction with a set of accessory(E3) proteins called ubiquitin ligases. There are roughly 30 structurally similarbut distinct E2 enzymes in mammals, and hundreds of different E3 proteins thatform complexes with specific E2 enzymes.Figure 3–70 illustrates the process used to mark proteins for proteasomaldegradation.
[Similar mechanisms are used to attach ubiquitin (and SUMO) toother types of target proteins.] Here, the ubiquitin ligase binds to specific degradation signals, called degrons, in protein substrates, thereby helping E2 to form apolyubiquitin chain linked to a lysine of the substrate protein. This polyubiquitin chain on a target protein will then be recognized by a specific receptor in theproteasome, causing the target protein to be destroyed. Distinct ubiquitin ligasesrecognize different degradation signals, thereby targeting distinct subsets ofintracellular proteins for destruction, often in response to specific signals (seeFigure 6–86).SIGNAL INCOOHPPsite of GTPhydrolysisFigure 3–67 The structure of the Rasprotein in its GTP-bound form.
Thismonomeric GTPase illustrates the structureof a GTP-binding domain, which is presentin a large family of GTP-binding proteins.The red regions change their conformationm3.72/3.62when the GTPMBoC6moleculeis hydrolyzedto GDP and inorganic phosphate by theprotein; the GDP remains bound to theprotein, while the inorganic phosphate isreleased. The special role of the “switchhelix” in proteins related to Ras isexplained in the text (see Figure 3–72and Movie 15.7).PROTEIN FUNCTIONN159MULTIUBIQUITYLATIONMONOUBIQUITYLATIONLys63POLYUBIQUITYLATIONubiquitinLys48Lys63Lys48COHNlysine side chainof target proteinisopeptidebondhistone regulationHC(A)endocytosisproteasomaldegradation(B)Protein Complexes with Interchangeable Parts Make Efficient Useof Genetic InformationThe SCF ubiquitin ligase is a protein complex that binds different “target proteins”at different times in the cell cycle, covalently adding polyubiquitin polypeptidechains to these targets.
Its C-shaped structure is formed from five protein subunits, the largest of which serves as a scaffoldonm6.93/3.64which the rest of the complexMBoC6is built. The structure underlies a remarkable mechanism (Figure 3–71). At oneend of the C is an E2 ubiquitin-conjugating enzyme. At the other end is a substrate-binding arm, a subunit known as an F-box protein. These two subunits areseparated by a gap of about 5 nm. When this protein complex is activated, theF-box protein binds to a specific site on a target protein, positioning the proteinin the gap so that some of its lysine side chains contact the ubiquitin-conjugatingenzyme.
The enzyme can then catalyze repeated additions of ubiquitin polypeptide to these lysines (see Figure 3–71C), producing polyubiquitin chains that markthe target proteins for rapid destruction in a proteasome.ubiquitinubiquitinactivatingenzymeE1ATP(A)Figure 3–69 The marking of proteinsby ubiquitin. (A) The three-dimensionalstructure of ubiquitin, a small protein of 76amino acids.
A family of special enzymescouples its carboxyl end to the aminogroup of a lysine side chain in a targetprotein molecule, forming an isopeptidebond. (B) Some modification patterns thathave specific meanings to the cell. Notethat the two types of polyubiquitylationdiffer in the way the ubiquitin moleculesare linked together. Linkage throughLys48 signifies degradation by theproteasome (see Figure 6–84), whereasthat through Lys63 has other meanings.Ubiquitin markings are “read” by proteinsthat specifically recognize each type ofmodification.E1COO–SHE1DNA repairSCObinding toubiquitinconjugatingenzymeAMP+P PE1SE2SHCOSHE2SCOubiquitin-conjugatingenzyme primed withubiquitinε-amino groupon lysineside chainNH2NH2E2E2E3degradation signalon target proteinE3target proteinbound toubiquitin ligasefirst ubiquitinchain addedto target proteintarget protein withpolyubiquitinchain(B)Figure 3–70 The marking of proteins with ubiquitin.
(A) The C-terminus of ubiquitin is initially activated by being linked via a high-energy thioesterbond to a cysteine side chain on the E1 protein. This reaction requires ATP, and it proceeds via a covalent AMP-ubiquitin intermediate. Theactivated ubiquitin on E1, also known as the ubiquitin-activating enzyme, is then transferred to the cysteine on an E2 molecule. (B) The addition ofa polyubiquitin chain to a target protein. In a mammalian cell, there are several hundred distinct E2–E3 complexes. The E2s are called ubiquitinconjugating enzymes. The E3s are referred to as ubiquitin ligases.
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