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More than ten other common domains provide binding sites for attaching their protein to phosphorylated peptides in other proteinmolecules, each recognizingaphosphorylated amino acid side chain in a different protein context. As a result, protein phosphorylation and dephosphorylation very often drive the regulated assembly and disassembly of protein complexes (seeFigure 15-22).Reversible protein phosphorylation controls the activity, structure, andcellular Iocalization of both enzymes and many other types of proteins inFigure3-63 Part of the on-off switch inthe catalyticsubunitsof aspartateChangesin thetranscarbamoylase.interactionsindicatedhydrogen-bondingfor switchingthisarepartlyresponsibleenzyme'sactivesite betweenactive(yellow)and inactiveconformations.Hydrogenbonds are indicatedby thin red/ines.Theaminoacidsinvolvedin theinteractionin the T statesubunit-subunitare shown in red,while thosethat formthe activesite of the enzymein the R stateareshownin blue.Thelargedrawingsshowthe catalyticsitein the interiorofthe enzyme;the boxed sketchesshow thesamesubunitsviewedfrom the enzyme'sexternalsurface.(AdaptedfromE.R.Kantrowitzand W.N.Lipscomb,Irends1990.WithBiochem.Sci.15:53-59,permissionfrom Elsevier.)'176Chapter3: Proteinseucaryotic cells.
In fact, this regulation is so extensive that more than one-thirdof the 10,000or so proteins in a tlpical mammalian cell are thought to be phosphorylated at any given time-many with more than one phosphate. As mightbe expected, the addition and removal of phosphate groups from specific proteins often occur in responseto signals that specify some change in a cell'sstate.For example, the complicated series of events that takes place as a eucaryoticcell divides is largely timed in this way (discussedin Chapter 17), and many ofthe signals mediating cell-cell interactions are relayed from the plasma membrane to the nucleus by a cascadeofprotein phosphorylation events (discussedin Chapter 15).A EucaryoticCellContainsa LargeCollectionof ProteinKinasesand ProteinPhosphatasesProtein phosphorylation involves the enzyme- catalyzedtransfer of the terminalphosphate group of an ATP molecule to the hydroxyl group on a serine, threonine, or tyrosine side chain of the protein (Figure 3-64).
A protein kinase catalyzesthis reaction, and the reaction is essentiallyunidirectional because of thelarge amount of free energy released when the phosphate-phosphate bond inATP is broken to produce ADP (discussedin chapt er 2) . Aprotein phosphatasecatalyzesthe reversereaction of phosphate removal, or dephosphorylation. cellscontain hundreds of different protein kinases, each responsible for phosphorylating a different protein or set of proteins. There are also many different proteinphosphatases;some are highly specific and remove phosphate groups from onlyone or a few proteins, whereas others act on a broad range of proteins and aretargeted to specific substratesby regulatory subunits.
The state ofphosphorylation of a protein at any moment, and thus its activity, depends on the relativeactivities of the protein kinases and phosphatasesthat modiff it.The protein kinases that phosphorylate proteins in eucaryotic cells belongto a very large family of enzymes, which share a catal),'tic(kinase) sequence ofabout 290 amino acids. The various family members contain different aminoacid sequences on either end of the kinase sequence (for example, see Figure3-10), and often have short amino acid sequencesinserted into loops within it(red arrowheadsin Figure 3-65).
Some of these additional amino acid sequencesenable each kinase to recognize the specific set ofproteins it phosphorylates, orto bind to structures that localize it in specific regions of the cell. Other parts ofthe protein regulate the activity of each kinase, so it can be turned on and off inresponse to different specific signals,as described below.By comparing the number of amino acid sequence differences between thevarious members of a protein family, we can construct an "evolutionary tree"that is thought to reflect the pattern of gene duplication and divergence thatgave rise to the family.
Figure 3-66 shows an evolutionary tree of proteinkinases.Kinases with related functions are often located on nearby branches ofthe tree: the protein kinases involved in cell signaling that phosphorylate tyrosine side chains, for example, are all clustered in the top left corner of the tree.The other kinases shor,m phosphorylate either a serine or a threonine sidechain, and many are organized into clusters that seem to reflect their functionin transmembrane signal transduction, intracellular signal amplification, cellcycle control, and so on.Figure3-65 The three-dimensionalstructureof a proteinkinase.Superimposedon this structureareredarrowheadsto indicatesiteswhereinsertionsof 5-100aminoacidsarefound in somemembersof the proteinkinasefamily.Theseinsertionsarelocatedin loopson the surfaceof theenzymewhereother ligandsinteractwith the protein.Thus,theydistinguishdifferentkinasesand conferon them distinctiveinteractionswith other proteins.TheATp(whichdonatesa phosphategroup)and thepeptideto be phosphorylatedareheld in the activesire,whichextendsbetweenthe phosphate-bindingloop (yellow)andthe catalyticloop(orange).Seealso Figure3-10.
(Adaptedfrom D.R.Knightonet al.,Science253:407-414,1991.With permissionfrom AAAS.)olADPO: P-OIoOHIIs e nn eCHs i d ec h a i nCH,(A)--,k in a s e*--lphosphatasek in a s e-_.:--_-/oFFphosphatase(B)Figure3-64 Proteinphosphorylation.Manythousandsof proteinsin a typicaleucaryoticcellaremodifiedby the covalentadditionof a phosphategroup.(A)Thegeneralreaction,shownhere,transfersa phosphategroupfrom ATPto anaminoacidsidechainof the targetproteinby a protein kinase.Removalof thephosphategroup is catalyzedby a secondenzymera proteinphosphatase.In thisexample,the phosphateis addedto a serinesidechain;in othercases,the phosphateisinsteadlinkedto the -OH groupof athreonineor a tyrosinein the protein.(B)Thephosphorylationof a proteinby aproteinkinasecan eitherincreaseordecreasethe protein'sactivity,dependingon the siteof phosphorylationand thestructureof the protein.177PROTEINFUNCTIONFigure3-66 An evolutionary tree ofselectedprotein kinases.Although acellcontainshundredshighereucaryoticand the humanof suchenzymes,genomecodesfor morethan 500,onlYin this bookaresomeof thosediscussedsnown.CdcTPDGFreceptorEGFtyrosinereceprorkinasesubfamilycyclic-AMPd e p e n d e n tk i n a s ecyclic-GMPd e p e n d e n tk i n a s ep r o t e i nk i n a s eCIGFBreceptorCa2*/calmodulin-m y o s i nl i g h t dependent kinasec h a i nk i n a s e sr e c e p t o rs e r i n ek i n a s es u b f a m i l yAs a result of the combined activities of protein kinases and protein phosphatases,the phosphate groups on proteins are continually turning over-beingadded and then rapidly removed.
Such phosphorylation cyclesmay seem wasteful, but they are important in allowing the phosphorylated proteins to switchrapidly from one state to another: the more rapid the cycle, the faster a population of protein molecules can change its state of phosphorylation in responsetoa sudden change in the phosphorylation rate (see Figure 15-11). The energyrequired to drive this phosphorylation cycle is derived from the free energy ofATP hydrolysis, one molecule of which is consumed for each phosphorylationevent.ShowsHowaTheRegulationof Cdkand SrcProteinKinasesProteinCanFunctionasa MicrochipThe hundreds of different protein kinases in a eucaryotic cell are organized intocomplex networks of signaling pathways that help to coordinate the cell's activities, drive the cell cycle, and relay signals into the cell from the cell's environment.
Many of the extracellular signals involved need to be both integrated andamplified by the cell. Individual protein kinases (and other signaling proteins)serve as input-output devices, or "microchips," in the integration process. Animportant part of the input to these signal processing proteins comes from thecontrol that is exerted by phosphates added and removed from them by proteinkinases and protein phosphatases,respectively.In general, specific sets of phosphate groups serve to activate the protein,while other sets can inactivate it.
A cyclin-dependent protein kinase (Cdk) provides a good example.Kinasesin this classphosphorylate serinesand threonines,and they are central components of the cell-cycle control system in eucaryoticcells,as discussedin detail in Chapter 17.In avertebrate cell, individual Cdk proteins turn on and off in succession, as a cell proceeds through the differentphases of its division cycle.r'A/hena particular kinase is on, it influences variousaspectsof cell behavior through effects on the proteins it phosphorylates.A Cdk protein becomes active as a serine/threonine protein kinase onlywhen it is bound to a second protein called a cyclin.
But, as Figure 3-67 shows,the binding of cyclin is only one of three distinct "inputs" required to activate theCdk. In addition to cyclin binding, a phosphate must be added to a specific threonine side chain, and a phosphate elsewherein the protein (covalently bound toa specific tyrosine side chain) must be removed. Cdk thus monitors a specific setINPUTSOUTPUTFigure 3-67 How a Cdk protein acts asan integrating device.<TAGA>Theof thefunctionof thesecentralregulatorsin Chapter17'cellcycleis discussed178Chapter3: Proteinsfattyacid5 0 0a m i n o a c i d sof cell components-a cyclin, a protein kinase, and a protein phosphatase-andit acts as an input-output device that turns on if, and only if, each of these components has attained its appropriate activity state.
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