module 6 spatial and temporal aspects of signalling · 2013. 5. 7. · cell signalling biology...

Cell Signalling Biology Michael J. Berridge � Module 6 � Spatial and Temporal Aspects of Signalling 6 �1

Module 6

Spatial and TemporalAspects of Signalling

Synopsis

The function and efficiency of cell signalling pathways are very dependent on their organization both inspace and time. With regard to spatial organization, signalling components are highly organized withrespect to their cellular location and how they transmit information from one region of the cell to another.This spatial organization of signalling pathways depends on the molecular interactions that occurbetween signalling components that use signal transduction domains to construct signalling pathways.Very often, the components responsible for information transfer mechanisms are held in placeby being attached to scaffolding proteins to form macromolecular signalling complexes. Sometimesthese macromolecular complexes can be organized further by being localized to specific regionsof the cell, as found in lipid rafts and caveolae or in the T-tubule regions of skeletal and cardiac cells.

Another feature of the spatial aspects concerns the localand global aspects of signalling. The spatial organization ofsignalling molecules mentioned above can lead to highlylocalized signalling events, but when the signalling mo-lecules are more evenly distributed, signals can spreadmore globally throughout the cell. In addition, signalscan spread from one cell to the next, and such intercel-lular communication can co-ordinate the activity of cellcommunities This spatial organization of signalling is wellillustrated by the elementary and global aspects of Ca2 +

signalling.The temporal aspects of signalling concern the way in-

formation is organized in the time domain. Many biolo-gical processes are rhythmical. Of particular importanceare the cellular oscillators that set up oscillating intracel-lular signals that can operate over an enormous range offrequencies to drive a wide range of cellular processes.Membrane oscillators (millisecond to second range) setup rapid membrane potential oscillations that can driveneural processing of information and pacemaker activityin contractile systems such as the heart and smooth muscle.Cytosolic oscillators (second to minute range) set up os-cillations in intracellular Ca2 + to control a large numberof cellular processes, such as fertilization, contraction ofsmooth muscle cells, ciliary beat frequency and glycogenmetabolism in liver cells. The circadian clock, which isresponsible for driving the 24 h diurnal rhythm, is a tran-scriptional oscillator.

Another important temporal aspect is timing and signalintegration, which relates to the way in which functional

Green text indicates links to content within this module; blue textindicates links to content in other modules.

Please cite as Berridge, M.J. (2012) Cell Signalling Biology;doi:10.1042/csb0001006

interactions between signalling pathways are determinedby both the order and the timing of their presentations.

The organization of signalling systems in both time andspace greatly enhances both their efficiency and versatility.

Spatial organization of signallingpathwaysMost signalling pathways function by transmitting in-formation from one component to the next (panel A inModule 6: Figure signalling hierarchies). The efficiencyand speed of this vectorial flow of information is greatlyfacilitated by the spatial organization of the signalling com-ponents that are often linked together through signal trans-duction domains (panel B in Module 6: Figure signallinghierarchies). If all of the signalling components are in placeand correctly aligned, information can flow quickly downthe signalling cascade by avoiding the delays that wouldoccur if the interacting partners had to find each other bydiffusion during the course of each signal transmission se-quence. Another important spatial feature is the locationof signalling pathways within the cell. There are a vari-ety of scaffolding/targeting proteins that function as an-chors and adaptors to hold signalling components in placeto form macromolecular signalling complexes (panel C inModule 6: Figure signalling hierarchies). These scaffoldingsystems can also function to direct macromolecular com-plexes to specific locations within the cell, such as the lipidrafts and caveolae (panel D in Module 6: Figure signallinghierarchies).

In this hypothetical system, the signalling compon-ents have a fixed location both with regard to each otherand to their location within the cell. However, thereare numerous examples of signalling components beingmuch more mobile and undergoing marked translocations

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Module 6: Figure signalling hierarchies

Stimulus

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The spatial organization of signalling pathways.The way in which information is transferred in cells is often highly organized, as illustrated in this highly schematic depiction of how componentsof signalling pathway are organized. A. The basic components of a typical signalling pathway, consisting of a receptor (R) and three signallingcomponents (X, Y and Z). B. In those cases where the signalling components are proteins, information is transmitted through protein–proteininteractions using signal transduction domains. For example, a motif on protein X recognizes a specific binding site on protein Y and so on. C. Avariety of scaffolds function to hold together the individual components of signalling pathways to create macromolecular signalling complexes. D.These macromolecular signalling complexes can be aggregated in specific locations within the cell, as occurs in lipid rafts and caveolae.

during the operation of a signalling cascade. This mobilityis particularly evident for proteins that have signal trans-duction domains that interact with various signalling lipidsin cell membranes.

Signal transduction domainsA characteristic feature of many signalling proteins is thatthey contain signal transduction domains that enable themto interact with other signalling components to set up sig-nalling pathways (Module 6: Figure signalling hierarchies).These domains participate either in protein–protein inter-actions or in protein–lipid interactions.

Protein–protein interactionsProtein–protein interactions depend upon modular pro-tein domains (e.g. SH2, SH3, PTB, 14-3-3, PDZ, WW,

SAM, FERM, ITAM, CH and LIM) that bind to spe-cific sequences on their target proteins, as summarized inModule 6: Figure modular protein domains.

Src homology 2 (SH2) domainThe Src homology 2 (SH2) domain binds to a phosphotyr-osine group located within a specific sequence on the tar-get protein (panel A in Module 6: Figure modular proteindomains). The following are some examples of signallingmolecules that use SH2 domains:

• Phospholipase Cγ (PLCγ) has two SH2 domains thatare used during translocation of the enzyme from thecytoplasm to tyrosine kinase-linked receptors at the cellsurface (Module 2: Figure PLC structure and function).

C©2012 Portland Press Limited www.cellsignallingbiology.org

http://www.cellsignallingbiology.org/csb/002/csb002.pdf#Phospholipase_C_ghttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_PLC_structure_and_function


• The signal transducers and activators of transcription(STATs) transcription factors have an SH2 domain(Module 2: Figure JAK and STAT structure) that enablesthem to attach to the Janus kinases (JAKs) (Module 2:Figure JAK/STAT function).

• The regulatory subunits of the Class IA PtdIns 3-kinasehave SH2 domains (Module 2: Figure PI 3-K family)that function to attach the catalytic subunits to varioustyrosine kinase-linked receptors at the cell surface suchas the platelet-derived growth factor receptor (PDGFR)(Module 1: Figure PDGFR activation) and the insulinreceptor (Module 2: Figure insulin receptor).

Phosphotyrosine-binding (PTB) domainA phosphotyrosine-binding (PTB) domain interacts witha unique sequence containing a phosphotyrosine group(panel B in Module 6: Figure modular protein domains).The PTB domain is an important feature of many sig-nalling molecules, such as the insulin receptor substrate(IRS) (Module 6: Figure IRS domain structure).

14-3-3 domainThe 14-3-3 proteins belong to a family of adaptor proteinsthat recognize a phosphoserine residue embedded in a spe-cific sequence within target proteins (panel C in Module 6:Figure modular protein domains). For example, the phos-phorylated transcription factor TAZ is exported from thenucleus during activation of the hippo signaling pathway(Module 2: Figure hippo signalling pathway).

Src homology 3 (SH3) domainThe Src homology 3 (SH3) domain interacts with apolyproline motif on its target proteins (panel D inModule 6: Figure modular protein domains). The ad-aptor protein growth factor receptor bound protein 2(Grb2) is a classical example of an SH3-containing pro-tein that binds to the guanine nucleotide exchange factor(GEF) Son-of-sevenless (SoS) during the activation of Rassignalling (Module 1: Figure stimuli for enzyme-linkedreceptors).

PDZ domainThe PDZ (named after postsynaptic density 95, Discs largeand zonula occludens 1) domain binds to its target via ashort peptide sequence that has a C-terminal hydrophobicresidue (panel E in Module 6: Figure modular protein do-mains). There are a large number of PDZ-containing pro-teins with a wide range of functions (Module 6: FigurePDZ-containing proteins). For example, many of the scaf-folding proteins that contain PDZ domains function toassembly large macromolecular signalling complexes.

WW domainThe WW domain binds a sequence rich in proline residues(panel F in Module 6: Figure modular protein domains).

Sterile alpha motif (SAM) domainThe sterile alpha motif (SAM) domain is a protein–proteininteraction region that has approximately 70 amino acids.It is 5 helices that are organized into a compact bundlewith a conserved hydrophobic core. This SAM domain isfound on many different proteins where it can function

in both homo- and heterotypic interactions. The follow-ing are examples of proteins that interact through SAMdomains:

• The C-terminal region of the Eph receptor has a SAMdomain that participates in a homotypic interaction dur-ing receptor dimerization (Module 1: Figure Eph re-ceptor signalling).

• The stromal interaction molecule (STIM), which is loc-ated in the endoplasmic reticulum (ER) where it func-tions to control Ca2 + entry, has a SAM domain in theN-terminal region (Module 3: Figure SOC signallingcomponents).

Calponin homology (CH) domainThe calponin homology (CH) domain is particularly evid-ent in proteins that function as part of the cytoskeleton.Tandem CH domains, such as those found in parvin, areparticularly effective in binding actin as occurs in the focaladhesion complex (Module 6: Figure integrin signalling).

FERM domainThe FERM (named after four-point-one, ezrin, radixin andmoesin) domain contains three compact modules (A–C),which has basic residues capable of binding PtdIns4,5P2.FERM domains are particularly evident on some of theproteins located on adhesion complexes such as talin andfocal adhesion kinase (FAK) (Module 6: Figure focal ad-hesion components).

Immunoreceptor tyrosine-based activation motifs(ITAMs)The immunoreceptor tyrosine-based activation motifs (IT-AMs) are docking sites located on the cytoplasmic domainsof various receptors. These ITAMs function to assemblethe following signal transduction complexes:

• The Fc receptor γ (FcRγ) chains in blood platelets haveITAMs that are phosphorylated by Fyn to provide bind-ing sites for phospholipase Cγ2 (PLCγ2) (see step 2 inModule 11: Figure platelet activation).

• The CD3 subunits (γ, δ and ε) and the ζ subunits ofthe T cell receptor (TCR) have long cytoplasmic chainsthat contain ITAMs (red bars in Module 9: Figure TCRsignalling), which provide the docking sites to assemblethe receptor scaffolds responsible for activating varioussignalling pathways.

• ITAMs on the FcεRI subunits of mast cells recruit vari-ous transducing elements, such as the non-receptor tyr-osine kinases Fyn, Lyn and Syk (Module 11: FigureFcεRI mast cell signalling).

• Igα and Igβ signalling proteins recruit signalling com-ponents during the B-cell antigen receptor (BCR) activ-ation process (Module 9: Figure B cell activation).

LIM domainThe LIM domain was first identified in the three tran-scription factors LIN 11, ISL 1 and MEC3 and the firstletter of each one was used to produce the abbreviationLIM. This LIM domain consists of a tandem cysteine-richZn2 + -finger motif that is used for protein–protein inter-actions. Such LIM domains have been identified in various


http://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_Jak_Stat_structurehttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_Jak_Stat_functionhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_PI3K_familyhttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Fig1_PDGFR_activationhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_insulin_receptorhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Transcriptional_coact_with_PDZ_motif_TAZhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Hippo_signalling_pathwayhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_hippo_signalling_pathwayhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#son_of_sevenlesshttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Fig1_stimuli_for_enzyme_linked_receptorshttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Fig1_Eph_receptor_signallinghttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#Stromal_interaction_molecule_STIMhttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#Fig3_SOC_signalling_componentshttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Fynhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Phospholipase_C_ghttp://www.cellsignallingbiology.org/csb/011/csb011.pdf#Fig11_platelet_activationhttp://www.cellsignallingbiology.org/csb/009/csb009.pdf#Fig9_TCR_signallinghttp://www.cellsignallingbiology.org/csb/011/csb011.pdf#Mast_cellhttp://www.cellsignallingbiology.org/csb/011/csb011.pdf#Fig11_FcRI_mast_cell_signallinghttp://www.cellsignallingbiology.org/csb/009/csb009.pdf#B_cell_antigen_receptor_BCR_activationhttp://www.cellsignallingbiology.org/csb/009/csb009.pdf#Fig9_Bcell_activation


Module 6: Figure modular protein domains

-Y-X-X-hy

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SH3 PDZ

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Target protein Target proteinTarget protein

Target proteinTarget proteinTarget protein

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Summary of some of the major protein modules used to assemble cell signalling pathways.The fidelity of information transfer between signalling components depends upon highly precise interactions between a variety of signal transductiondomains and corresponding specific signal sequences on the target protein (see the text for further details).

Module 6: Figure PDZ-containing proteins

PDZ-containing proteins.A large number of proteins contain either single or multiple PDZ domains. Reproduced by permission from Macmillan Publishers Ltd: Nat. Rev.Neurosci., Kim, E. and Sheng, M. (2004) PDZ domain proteins of synapses. 5:771–781. Copyright (2004); http://www.nature.com/nrn; see Kim andSheng (2004).


http://www.nature.com/nrn


proteins that participate in junctional complexes such asthe particularly interesting cysteine/histidine-rich protein(PINCH) and paxillin.

Lasp-1 is an example of an adaptor protein, which con-tains an N-terminal LIM domain, that binds to actin andmay contribute to the reorganization of the cytoskeletonduring the control of parietal cell secretion of acid (Module7: Figure HCl secretion).

Protein–lipid interactionsA number of signalling molecules function by interact-ing with specific lipid messengers located in membranes.These protein–lipid interactions depend upon a numberof modular protein domains (e.g. C2, FYVE, PH, PX andENTH) that bind to specific membrane lipids such as di-acylglycerol (DAG) and various phosphoinositides suchas PtdIns4,5P2, PtdIns3P, PtdIns3,4P2 and PtdIns3,4,5P3(Module 6: Figure modular lipid-binding domains):

BAR domainThe C-terminal Bin, Amphiphysin, Rvs (BAR) domain islocated on proteins that can bind to cell membranes. BARdomains dimerize with each other to form a concave struc-ture that can bind to membranes with a positive curvatureas found on vesicles and tubules. They are found on somemembers of the sorting nexin (SNX) family that func-tion in the sorting of proteins such as the early endosometo plasma membrane trafficking of the transferrin receptor(TFR) (Module 4: Figure early endosome budding) or dur-ing the early endosome to trans-Golgi network (TGN)trafficking (Module 4: Figure endosome budding TGN).

C2 domainC2 domains are found on many proteins, where they func-tion to bind Ca2 + to induce a conformational change toform a lipid-binding domain that enables proteins to in-teract with membrane lipids. The C2 domains vary withregard to their lipid preference: some bind neutral lipids,whereas others prefer negatively charged phospholipids.Not all C2 domains bind Ca2 + . The tumour suppressorphosphatase and tensin homologue deleted on chromo-some 10 (PTEN), which hydrolyses the lipid second mes-senger PtdIns3,4,5P3, has such a Ca2 + -insensitive C2 do-main, which still functions to attach the enzyme to themembrane so that it can reach its substrate. The more clas-sical Ca2 + -sensitive C2 domains were originally describedin protein kinase C (PKC), where they are found on boththe conventional and novel PKCs (Module 2: Figure PKCstructure and action). C2 domains are also found on thesynaptotagmins that function in Ca2 + -dependent exo-cytosis (Module 4: Figure Ca2 + -induced membrane fu-sion) and on the otoferin that triggers hair cell transmitterrelease.

ENTH domainENTH is a lipid-binding domain that recognizesPtdIns4,5P2 (Module 6: Figure modular lipid-binding do-mains). This motif contains about 140 residues and is loc-ated on proteins that function in endocytosis and cyto-skeletal organization. With regard to the latter, ENTH

may play a role in mediating the PtdIns4,5P2 regulation ofactin remodelling.

Pleckstrin homology (PH) domainThe pleckstrin homology (PH) domain is capable of bind-ing to a number of lipid messengers (Module 6: Figuremodular lipid-binding domains). There are multiple PHdomains that have 100–120 residues that have little se-quence homology, but there is considerable similarity intheir tertiary structure. These different PH domains arepresent on many signalling molecules:

• Many of the phospholipase Cs (PLCs) have PH do-mains, which help the enzyme to associate with themembrane (Module 2: Figure PLC structure and func-tion).

• Protein kinase B (PKB).• Phospholipase D (PLD) has a PH domain that binds to

PtdIns4,5P2 (Module 2: Figure PLD isoforms).• Bruton’s tyrosine kinase (Btk)• The insulin receptor substrate (IRS) has a PH domain

that is used to bind IRS to the insulin receptor (Module2: Figure insulin receptor).

Phox homology (PX) domainPhox homology (PX) domains are found in many differentproteins, and have been divided into three classes:

• Class I contain small proteins where the PX domainrepresents most of the protein, and many of these belongto the sorting nexin (SNX) family (e.g. SNX3, SNX9,SNX10, SNX12, SNX22, SNX23, SNX24 and SNX26).

• Class II resemble the above, but have larger flanking re-gions. Many of these also are found within the SNX fam-ily (e.g. SNX1, SNX2, SNX4–SNX8, SNX11, SNX16,SNX20, SNX21 and SNX29).

• Class III represent proteins that contain PX domains,together with other protein domains such as the pleck-strin homology (PH) and HKD domains in phospholi-pase D 1 (PLD1) and 2 (PLD2) (Module 2: Figure PLDisoforms).

FYVE domainFYVE is a membrane-targeting motif that recognizesPtdIns3P and is often found on proteins that function inmembrane trafficking:

• The early endosome antigen 1 (EEA1) uses its FYVEdomain to associate with the phagosome (Module 4:Figure phagosome maturation).

• The Class III PtdInsP kinase, which is also known asPIKfyve, functions in the PtdIns3,5P2 signalling cas-sette (Module 2: Figure PIKfyve activation). The FYVEdomain targets PIKfyve to endomembranes.

As a result of these protein–lipid interactions, proteinstranslocate from the cytoplasm on to the cell membrane,and this is a critical part of their signalling function. Acritical event for many signalling molecules is their trans-location to the plasma membrane, as found in the followingexamples:


http://www.cellsignallingbiology.org/csb/007/csb007.pdf#Control_of_parietal_cel_secretionlhttp://www.cellsignallingbiology.org/csb/007/csb007.pdf#Fig7_HCl_secretionhttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Sorting_nexins_SNXshttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Early_endosome_to_pm_traffickinghttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Fig4_early_endosome_buddinghttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Early_endosome_to_TGN_traffickinghttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Fig4_endosome_budding_to_TGNhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#PTENhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Protein_kinase_Chttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_PKC_structure_and_activationhttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Synaptotagminshttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Ca_dependent_exocytosishttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Fig4_Ca_induced_memb_fusionhttp://www.cellsignallingbiology.org/csb/010/csb010.pdf#Hair_cell_transmitter_releasehttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#PtdIns45P2_regulation_of_actin_remodelhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Phospholipase_Chttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_PLC_structure_and_functionhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Protein_kinase_Bhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_PLD_isoformshttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Btkhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_insulin_receptorhttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Sorting_nexins_SNXshttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Membrane_invagination_and_scissionhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_PLD_isoformshttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Fig4_phagosome_maturationhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#PtdInsP_kinaseIIIhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#PtdIns35P2_signalling_cassettehttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_PIKfyve_activation


Module 6: Figure modular lipid-binding domains

PtdIns 4,5P PtdIns 3,4,5P PtdIns 3,4P PtdIns 3P

C

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Summary of the lipid signal transduction domains found on proteins that interact with specific lipids in cell membranes.Many proteins contain specific domains that enable them to interact with different signalling lipids located in cell membranes. See the text for detailsof the different proteins.

• Translocation of conventional protein kinase C (cPKC)to the plasma membrane (Module 2: Figure PKC struc-ture and activation).

• Translocation of phospholipase Cγ (PLCγ) to theplasma membrane during phosphoinositide signalling(Module 2: Figure PLC structure and function).

• Phosphoinositide-dependent kinase 1 (PDK1) andprotein kinase B (PKB) translocate to the plasma mem-brane in response to the formation of the lipid messengerPtdIns3,4,5P3.

There are other proteins that move from the cytoplasmto various intracellular membranes such as the endosomes.For example, translocation of the early endosome antigen(EEA1) on to the PtdIns3P on the phagosome membrane(Module 4: Figure phagosome maturation).

Scaffolding/targeting proteinsThere are many examples of scaffolding/targeting pro-teins that function as adaptors to assemble macromolecu-lar complexes and to target signalling complexes to specificlocations in the cell:

• Postsynaptic density (PSD) scaffolding and adaptorcomponents

• Abelson-interactor (Abi)• Arrestins• Axin• Cbl• Crk

• Dishevelled (Dsh)• A subunit of protein phosphatase 2A• Fe65• Insulin receptor substrate (IRS)• Glycogen scaffold• Growth factor receptor-bound protein 2 (Grb2)• Shc• Shank• A-kinase-anchoring proteins (AKAPs)• Membrane-associated guanylate kinase (MAGUK)• Caveolin is a scaffolding protein that organizes the

signalling function of caveolae.

Postsynaptic density (PSD) scaffolding and adaptorcomponentsMany of the scaffolding proteins contain PDZ domains(Module 6: Figure PDZ-containing proteins). A particu-larly impressive example is provided by the postsynapticdensity (PSD) scaffolding and adaptor components thathave a number of PDZ-containing proteins co-operate toform the PSD (Module 10: Figure postsynaptic density).

AxinAxin is a scaffolding protein that functions in the Wntsignalling pathway. It acts as a scaffold for a multiproteincomplex that functions to phosphorylate β-catenin to tar-get it for destruction by the proteasome (Module 2: FigureWnt canonical pathway).


http://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_PKC_structure_and_activationhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_PLC_structure_and_functionhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Phosphoinositide_dependent_kinase1http://www.cellsignallingbiology.org/csb/002/csb002.pdf#Protein_kinase_Bhttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Phagosome_maturationhttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Fig4_phagosome_maturationhttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Arrestinshttp://www.cellsignallingbiology.org/csb/010/csb010.pdf#PSD_scaffolding_and_adaptor_componentshttp://www.cellsignallingbiology.org/csb/010/csb010.pdf#Fig10postsynaptic_densityhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Wnt_signalling_pathwayshttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_Wnt_canonical_pathway


CblThe casitas B-lineage lymphoma (Cbl) family in mam-mals consists of three members: c-Cbl, Cbl-b and Cbl-3(Module 6: Figure Cbl structure). Cbl has two very differ-ent functions. Firstly, it contains various protein–proteininteraction domains that enable it to act as an adaptorprotein that contributes to the assembly of signallingcomplexes. Secondly, it contains a ubiquitin ligase (E3)region responsible for terminating the activity of manysignalling components by targeting them for degradation.Cbl structure and regulation reveals the presence of manydomains that contribute to Cbl adaptor functions and Cbldown-regulation of signalling components. Some myeloidneoplasms are caused by mutations in Cbl.

Cbl structure and regulationTwo of the Cbls are highly homologous (c-Cbl and Cbl-b),whereas Cbl-3 is much smaller with a large part of the C-terminal region missing. Cbl contains numerous structuraldomains related to its adaptor and protein degradationfunctions (Module 6: Figure Cbl structure). It has a highlyconserved N-terminal tyrosine kinase-binding (TKB) do-main, which is made up of three elements: a four-helixbundle (4H), a Ca2 + -binding EF-hand and a modified Srchomology (SH2) domain. In addition to binding proteintyrosine kinase-linked receptors (PTKRs) [e.g. epidermalgrowth factor receptor (EGFR), platelet-derived growthfactor receptor (PDGFR), colony-stimulating factor-1receptor (CSF-1R)] and non-receptor protein tyrosinekinases (e.g. Src and Syk), TKB can also interact with otherproteins such as adaptor protein-containing pleckstrin ho-mology (PH) and Src homology 3 (SH3) domains (APS),Src-like adaptor protein (SLAP), Sprouty 2 (Spry2) andtubulin. The TKB is attached through a short linker (L) tothe RING finger domain, which has the E3 ubiquitin ligaseactivity. An important aspect of the ubiquitination activityof Cbl is the ability of the RING domain to associate withan E2 ubiquitin-conjugating enzyme that functions in theCbl down-regulation of signalling components (Module1: Figure receptor down-regulation).

Both c-Cbl and Cbl-b have an extensive region ofproline-rich motifs that can bind to proteins that have Srchomology 3 (SH3) domains such as non-receptor proteintyrosine kinases (e.g. Src and Fyn), Cbl-associated protein(Cap), growth factor receptor-bound protein 2 (Grb2) andT cell ubiquitin ligand (TULA). A proline-rich sequencelocated close to the C-terminus binds to Cbl-interactingprotein of 85 kDa (CIN85), and functions to target re-ceptor complexes to the clathrin-coated vesicles by bindingto the endophilins (Module 1: Figure receptor down-reg-ulation).

Following the proline-rich region, there are a numberof tyrosine residues that are phosphorylated and contrib-ute to the regulation of Cbl activity (see below). The C-terminal region has a ubiquitin-associated (UBA) domain,which can link to either ubiquitin (primarily for Cbl-b)or to the ubiquitin-like domains found on other E3 lig-ases such as neuronal-expressed developmentally down-regulated gene 8 (Nedd8).

The activity of Cbl is regulated in a number of ways.Phosphorylation of Cbl by other signalling elements, such

as the non-receptor protein tyrosine kinases (e.g. Src),plays a critical regulatory role. For example, phosphoryla-tion of tyrosine residues in the N-terminal region providebinding sites for various proteins that have Src homology2 (SH2) domains such as the Crk-like (CrkL), PtdIns3-kinase (PtdIns 3-K) and Vav (Module 6: Figure Cblstructure). In addition, phosphorylation of two tyrosineresidues (Tyr-368 and Tyr-371) in the L region plays a crit-ical role in switching on the ubiquitination activity of Cbl.

Cbl is also regulated by interacting with other pro-teins. TULA, which is also known as suppressor of T cellsignalling-2 (Sts-2), inhibits Cbl by binding constitutivelyto the proline-rich region. One action of TULA is to in-duce the ubiquitination and degradation of c-Cbl. Anotherregulator is Spry2, an inducible inhibitor of Cbl whichbinds to the RING finger domain. By binding to RING, itprevents the binding of the E2 enzyme. Upon activation oftyrosine kinase-linked receptors, Spry2 is phosphorylated,and this causes its displacement from the RING domainto the TKB domain. The RING domain is now free tobind E2 enzymes, which are then able to begin the ubi-quitination of the receptor and Spry2. The subsequentproteasomal degradation of Spry2 is compensated for byan EGFR-dependent up-regulation of Spry2 expression.These ubiquitination reactions that occur at receptors con-tribute to the Cbl down-regulation of signalling compon-ents (Module 1: Figure receptor down-regulation).

Cbl adaptor functionsCbl functions as an adaptor in a number of processes in-cluding cell adhesion, spreading and motility. The mul-tidomain Cbl structure enables Cbl to interact with a largenumber of signalling and structural proteins. Cbl proteinsare primarily cytosolic, but they are able to translocate todifferent cellular sites, such as the plasma membrane andcytoskeleton, following activation of various signallingpathways. One of the important adaptor functions of Cblis to contribute to the skeletal and signalling events that oc-cur during cell motility, as illustrated by events that occurduring focal adhesion integrin signalling (Module 6: Figureintegrin signalling) and formation of osteoclast podosomes(Module 7: Figure osteoclast podosome).

Another adaptor role for Cbl occurs at protein-tyrosinekinase linked receptors (PTKRs). For example, dur-ing osteoclastogenesis, the phosphorylation of thecolony-stimulating factor-1 receptor (CSF-1R) providesa binding site for c-Cbl, which then functions as an ad-aptor to bind the p85-subunit of PtdIns 3-kinase (PtdIns3-K) (Module 8: Figure osteoclast differentiation). A sim-ilar sequence occurs at Tyr-1003 on the Met receptor. Thisassociation between Cbl and protein tyrosine-linked re-ceptors is also relevant to the Cbl down-regulation of sig-nalling components (Module 1: Figure receptor down-reg-ulation).

Dishevelled (Dsh)Dishevelled (Dsh) is a scaffolding protein that containsvarious transduction domains (DIX, PDZ and DEP),which functions in the Wnt signalling pathway by inter-acting with the frizzled (Fz) receptor (Module 2: FigureWnt canonical pathway).


http://www.cellsignallingbiology.org/csb/001/csb001.pdf#Ubiquitin_ligaseshttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Cbl_downregulation_of_signalling_componehttp://www.cellsignallingbiology.org/csb/012/csb012.pdf#Myeloid_neoplasmshttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#E_F_handhttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Protein_tyrosine_kinase_linked_receptorshttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Non_receptor_protein_tyrosine_kinaseshttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Cbl_downregulation_of_signalling_componehttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Cbl_downregulation_of_signalling_componehttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Fig1_receptor_downregulationhttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Fig1_receptor_downregulationhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#PtdIns_3_kinasehttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Cbl_downregulation_of_signalling_componehttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Fig1_receptor_downregulationhttp://www.cellsignallingbiology.org/csb/007/csb007.pdf#Osteoclast_podosomehttp://www.cellsignallingbiology.org/csb/007/csb007.pdf#Fig7_osteoclast_podosomehttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Protein_tyrosine_kinase_linked_receptorshttp://www.cellsignallingbiology.org/csb/008/csb008.pdf#Osteoclastogenesishttp://www.cellsignallingbiology.org/csb/008/csb008.pdf#CSF_1_receptor_CSF_1Rhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#PtdIns_3_kinasehttp://www.cellsignallingbiology.org/csb/008/csb008.pdf#Fig8_osteoclast_differentiationhttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Cbl_downregulation_of_signalling_componehttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Fig1_receptor_downregulationhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Wnt_signalling_pathwayshttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_Wnt_canonical_pathway


Module 6: Figure Cbl structure

4H

4H

4H

EF L

L

APSPTKRsSrc, SykSLAPSpry2Tubulin

EF

EF

SH2

Y

YY

YY

Y Y

PI 3-KNedd8CIN85

SH2

SH2

RING

VavCrkL

700

368 370

665

731

709

774

CapFyn, Src

Grb2TULA

E2Spry2

TKB domain Proline-rich

RING

RING

c-Cbl

Cbl-b

Cbl-3

P

P P

P

P

P

P

Structure of the Cbl family of adaptor proteins.The c-Cbl and Cbl-b isoform are closely homologous. The Cbl-3 isoform resembles the other two in its N-terminal region, but is missing the C-terminalregions. The double-headed arrows illustrate the ability of c-Cbl to interact with a large number of signalling components. Many of these interactionsare also evident for Cbl-b. See the text for a description of the abbreviations.

A subunit of protein phosphatase 2AThe A subunit of protein phosphatase 2A (PP2A) as-sembles the functional holoenzyme by binding both thecatalytic subunit as well as the regulatory B subunit thattargets the enzyme to specific cellular locations (Module5: Figure PP2A holoenzyme).

Fe65The Fe65 family has three members: Fe65, Fe65L1 andFe65L2. Fe65 is an adaptor protein that seems to have aprimary function in neurons where it regulates the traffick-ing of integral membrane proteins such as the β-amyloidprecursor protein (APP) (Module 12: Figure APP pro-cessing). Fe65 has three protein–protein interaction do-mains: an N-terminal WW domain and two PTB domains.It is the C-terminal PTB2 domain that binds to APP,whereas PTB1 interacts with the CP2/LSF/LBP1 tran-scription factor.

Insulin receptor substrate (IRS)The insulin receptor substrate (IRS) was one of the firstscaffolding proteins to be identified. There are three IRSproteins in humans (IRS1, IRS2 and IRS4). The first twoare expressed widely, whereas IRS4 is restricted to thebrain, kidney, thymus and β-cells. Like other scaffoldingproteins, IRS contains a number of interaction domains

that enable it to interact with various signalling compon-ents (Module 6: Figure IRS domain structure). One ofthe most important is the phosphotyrosine-binding (PTB)domain that interacts with a unique sequence contain-ing a phosphotyrosine group (Module 6: Figure modu-lar protein domains). Such an interaction occurs at thejuxtamembrane phosphotyrosine residue of the insulin re-ceptor, which is responsible for recruiting IRS into thereceptor complex (Module 2: Figure insulin receptor).

Glycogen scaffoldGlycogen functions as a scaffold to bring together many ofthe proteins that function in glycogen metabolism, such asAMP-activated protein kinase (AMPK), glycogen phos-phorylase, glycogen synthase and protein phosphatase 1(PP1) (Module 6: Figure glycogen scaffold). For some ofthese proteins, their attachment to glycogen is facilitatedby various adaptors, such as the protein targeting to gly-cogen (PTG), GM and GL in the case of PP1 (Module 5:Figure PP1 targeting to glycogen).

Growth factor receptor-bound protein 2 (Grb2)Growth factor receptor-bound protein 2 (Grb2) isan adaptor protein that functions to link tyrosinekinase-linked receptors to the mitogen-activatedprotein kinase (MAPK) signalling system


http://www.cellsignallingbiology.org/csb/005/csb005.pdf#Protein_phosphatase_2Ahttp://www.cellsignallingbiology.org/csb/005/csb005.pdf#Fig5_PP2A_holoenzymehttp://www.cellsignallingbiology.org/csb/012/csb012.pdf#b_amyloid_precursor_protein_APPhttp://www.cellsignallingbiology.org/csb/012/csb012.pdf#Fig12_APP_processinghttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#LBP_family_of_transcrpition_factorshttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_insulin_receptorhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#AMP_activated_protein_kinasehttp://www.cellsignallingbiology.org/csb/005/csb005.pdf#Protein_phosphatase1_PP1http://www.cellsignallingbiology.org/csb/005/csb005.pdf#Fig5_PP1_targeting_to_glycogenhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#MAP_kinase_signalling


Module 6: Figure IRS domain structure

YGSS

YGSS

YGDV

PH

PH

IRS1

IRS2

PTB

PTB

14-3-3 JNK-binding

JNK-binding

14-3-3

YICM

YRRV

YILS

YSLT

YTEM

YTLM

YPEE

YPED

YMPM

YGDI

YMPM

YMPM YMPMYKAS

YMRM

YMMM

YLNVYKAP

YGKL

YVLM

YMNM

YPLP

YGPE

YINI

YSLP

YPPL

YAAT

YTEM

YVNIYMKM

YMNL

YMTM

YMTM

YVDTYADM

YIDL

YIAI

YASI

YASI

Domain structure of the insulin receptor substrate (IRS).There are three insulin receptor substrate (IRS) isoforms. The domain structures of the main IRS1 and IRS2 isoforms illustrate the position ofthe phosphotyrosine-binding (PTB) domain and the pleckstrin homology (PH) domain. The latter is unusual in that it has a low affinity for lipids,but resembles the structure of PTB. The sequence motifs are potential tyrosine phosphorylation sites that enable IRS to recruit various signallingmolecules. A typical example is PtdIns 3-kinase, which associates with IRS through its Src homology 2 (SH2) domains (Module 2: Figure insulinreceptor).

Module 6: Figure glycogen scaffold

The scaffolding function of glycogen.Glycogen acts to bring together a number of signalling components, such as AMP-activated protein kinase (AMPK), glycogen phosphorylase, glycogensynthase, glycogenin, protein targeting to glycogen (PTG) and protein phosphatase 1 (PP1). Reproduced from Curr. Biol., volume 13, Polekhina, G.,Gupta, A., Michell, B.J., van Denderen, B., Murthy, S., Feil, S.S.C., Jennings, I.G., Campbell, D.J., Witters, L.A., Parker, M.W., Kemp, B.E. and Stapleton,D., AMPKβ subunit targets metabolic stress sensing to glycogen, pp. 867–871. Copyright (2003), with permission from Elsevier; see Polekhina et al.(2003).


http://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_insulin_receptor


(Module 1: Figure stimuli for enzyme-linked re-ceptors). It is particularly important for theextracellular-signal-regulated kinase (ERK) pathway(Module 2: Figure ERK signalling). The central Srchomology 2 (SH2) domain binds to the pTyr-Xaa-Asnmotifs found on activated receptors or on cytoplasmicscaffolding proteins. The N-terminal Src homology 3(SH3) domain binds the Pro-Xaa-Xaa-Pro motif on theRas guanine nucleotide exchange factor Son-of-sevenless(SoS). Typical examples of this adaptor function occuron the platelet-derived growth factor (PDGF) receptor(Module 1: Figure PDGFR activation) and on the vascularendothelial growth factor (VEGF) receptor (Module 9:Figure VEGF-induced proliferation).

Grb2 contributes to the Cbl down-regulation of sig-nalling components by helping to attach Cbl to activatedreceptors (Steps 2 and 3 in Module 1: Figure receptordown-regulation).

ShcThe Src homology 2 (SH2)-domain-containing protein(Shc) is a highly versatile adaptor protein that can as-sociate with a number of signalling components. Thereare three Shc genes (ShcA, ShcB and ShcC). ShcB andShcC are mainly confined to the nervous system. Thereis an N-terminal phosphotyrosine-binding (PTB) domainand the C-terminal region has an adaptin-binding domain(ABD) followed by an Src homology (SH2) domain. Loc-ated between the PTB and ABD domains there are tyr-osine residues that are phosphorylated to provide addi-tional binding sites for adaptors such as growth factorreceptor-bound protein 2 (Grb2) (Module 2: Figure ERKsignalling).

ShankThe Shank family (Shank1–3) are scaffolding proteins thatare major binding partners for Homer in the postsynapticdensity (Module 10: Figure postsynaptic density). The N-terminus has a domain of ankyrin repeats, an SH3 domain,a PDZ domain, a long proline-rich sequence that has bind-ing sites for Homer and cortactin. These multiple domainsenable Shank to interact with other scaffolding moleculesand with many signalling components.

Disruption of Shank3 has been identified as a candidategene for Phelan–McDermid syndrome, which is one of theautisim spectrum disorders.

A-kinase-anchoring proteins (AKAPs)The A-kinase-anchoring proteins (AKAPs) are a diversefamily of scaffolding proteins that function to locateprotein kinase A (PKA) (primarily PKA II) and othersignalling components to specific cellular targets. PKAis attached to a specific binding region on the AKAP,which also has targeting sequences that enable it to as-sociate with different cellular structures [Module 2: Fig-ure protein kinase A (PKA)]. In addition to bindingPKA, many of the AKAPs [e.g. AKAP350, AKAP220 andWiskott–Aldrich syndrome protein (WASP) verprolin ho-mologous 1 (WAVE1)] also bind to a range of other com-ponents related to both cyclic AMP and other signalling

systems. By establishing large macromolecular signallingcomplexes, the AKAPs provide a platform where the par-allel processing of information and the cross-talk betweendifferent signalling pathways can occur.

There are a number of different AKAPs that function inspecific locations within the cell:

• Plasma membrane (AKAP79/150, AKAP18, Yotiao)• Mitochondria [Wiskott–Aldrich syndrome protein

(WASP) verprolin homologous 1 (WAVE1), D-AKAP1,Rab32)

• Centrosome (AKAP350, pericentrin)• Microtubules [microtubule-associated protein-2

(MAP2)]• Cytoskeleton [Wiskott–Aldrich syndrome pro-

tein (WASP) verprolin homologous 1 (WAVE1),AKAP-Lbc (AKAP13), gravin (AKAP12)]

Some of the AKAPs function in a context-dependentmanner in that they assemble a different set of signallingcomponents depending on where they are expressed.For example, Wiskott–Aldrich syndrome protein (WASP)verprolin homologous 1 (WAVE1) can act in neurons toregulate actin remodelling during the outgrowth of neur-ons, whereas in the liver, it associates with the mitochon-dria, where PKA acts to phosphorylate Bad to inhibit ap-optosis.

In the following list of AKAPs, their common nameshave been used and many of these refer to their molecu-lar masses. The name in parentheses is that given by theHUGO Gene Nomenclature Committee:

D-AKAP1 (AKAP1)D-AKAP1 is a scaffolding protein for protein kinase A(PKA) and protein phosphatase 1 (PP1). It is targeted to themitochondria by a conventional mitochondrial targetingsequence.

AKAP150 (AKAP5)The human isoform is AKAP79, so the protein isoften referred to as AKAP79/150. It is attachedto the plasma membrane by binding to phosphol-ipids. AKAP79, which binds protein kinase A (PKA)and protein phosphatase 2A (PP2A), is linked toα-amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA) receptors through synapse-associated protein 97(SAP97) (Module 10: Figure postsynaptic density).

mAKAP (AKAP6)Muscle AKAP (mAKAP) associates with both proteinkinase A (PKA) and the phosphodiesterase PDE4D3. Oneof the functions of mAKAP is to link PKA to the type 2ryanodine receptor (RYR2) (Module 3: Figure ryanodinereceptor structure).

AKAP18 (AKAP7)Dual palmitoyl groups target AKAP18 to the plasmamembrane, where it brings protein kinase A (PKA) intoclose association with various voltage-operated channels(VOCs) such as the L-type Ca2 + channels in skeletalmuscle (Module 3: Figure CaV1.1 L-type channel) and


http://www.cellsignallingbiology.org/csb/001/csb001.pdf#Fig1_stimuli_for_enzyme_linked_receptorshttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#ERK_pathwayhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_ERK_signallinghttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Son_of_sevenlesshttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Platelet_der_growth_fact_receptor_PDGFRhttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Fig1_PDGFR_activationhttp://www.cellsignallingbiology.org/csb/009/csb009.pdf#Vascular_endothelial_growth_factor_VEGFhttp://www.cellsignallingbiology.org/csb/009/csb009.pdf#Fig9_VEGF_induced_proliferationhttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Cbl_downregulation_of_signalling_componehttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Fig1_receptor_downregulationhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_ERK_signallinghttp://www.cellsignallingbiology.org/csb/010/csb010.pdf#Postsynaptic_densityhttp://www.cellsignallingbiology.org/csb/010/csb010.pdf#Fig10postsynaptic_densityhttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Cortactinhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Protein_kinase_A_http://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_protein_kinase_Ahttp://www.cellsignallingbiology.org/csb/011/csb011.pdf#Badhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Protein_kinase_A_http://www.cellsignallingbiology.org/csb/005/csb005.pdf#Protein_phosphatase1_PP1http://www.cellsignallingbiology.org/csb/002/csb002.pdf#Protein_kinase_A_http://www.cellsignallingbiology.org/csb/005/csb005.pdf#Protein_phosphatase_2Ahttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#AMPA_receptorshttp://www.cellsignallingbiology.org/csb/010/csb010.pdf#Fig10postsynaptic_densityhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Protein_kinase_A_http://www.cellsignallingbiology.org/csb/005/csb005.pdf#PDE4Dhttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#Ryanodine_receptors_2_RYR2http://www.cellsignallingbiology.org/csb/003/csb003.pdf#Fig3_ryanodine_receptor_structurehttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#Fig3_CaV1_1_Ltype_channel


in cardiac muscle (Module 3: Figure CaV1.2 L-typechannel). AKAP 18 has an important role in themodulation of the CaV1.1 L-type channel.

AKAP350 (AKAP9)AKAP350 exists in different splice variants with differentnames and different functions. For example, AKAP350 isalso known as centrosome- and Golgi-localized proteinkinase N (PKN)-associated protein (CG-NAP), whichis targeted to the centrosome (microtubule-organizingcentres), where it functions as a scaffold to assemble acomplex containing protein kinase A (PKA), protein phos-phatase 2A (PP2A), protein kinase Cε (PKCε), proteinphosphatase 1 (PP1), phosphodiesterase (PDE) and caseinkinase I (CKI). On the other hand, one of the splice vari-ants, called Yotiao, is found in synaptic regions, whereit binds to the cytoplasmic region of the NR1 subunitof N-methyl-d-aspartate (NMDA) receptors (Module 10:Figure postsynaptic density).

D-AKAP2 (AKAP10)Associates with protein kinase A (PKA).

AKAP220 (AKAP11)This scaffolding protein brings together three of the en-zymes [protein kinase A (PKA), protein phosphatase1 (PP1) and glycogen synthase kinase-3β (GSK-3β)]that function in glycogen metabolism (Module 7: Figureskeletal muscle E-C coupling).

Gravin (AKAP12)Gravin is thought to associate with the plasma membranethrough both phospholipid binding and an N-terminalmyristoyl group. It functions to target protein kinases A(PKA) and C (PKC) to the neuromuscular junction, andcan also associate with the β-adrenergic receptor.

AKAP-Lbc (AKAP13)This AKAP functions in the assembly of stress fibres,where it acts to bring together protein kinases A (PKA),C (PKC) and D (PKD) and Rho.

Membrane-associated guanylate kinases (MAGUKs)In humans there are 22 membrane-associated guanylatekinases (MAGUKs), which are a heterogeneous group ofmodular scaffolding proteins with multiple cellular func-tions. They often participate in the assembly of multipro-tein complexes on the inner surface of the plasma mem-brane where they contribute to junctional complexes andthe regulation of receptors and ion channels. There are anumber of MAGUK family groups:

• Membrane-associated guanylate cyclase kinase, WWand PDZ domain-containing (MAGI)

• Calcium/calmodulin-dependent serine protein kinase(CASK)

• Membrane protein, palmitoylated (MPP)• Zona occludens (ZO)• Disc, large homology (DLG)• CARMA1• Cytoplasmic Ca2 + channel β subunits (CACNBs)

Membrane-associated guanylate cyclase kinase, WW andPDZ domain containing (MAGI)There are three human membrane-associated guanylate cy-clase kinase, WW and PDZ domain containing (MAGI)proteins (MAGI1–3) (Module 6: Figure MAGUKs).

Calcium/calmodulin-dependent serine protein kinase(CASK)Calcium/calmodulin-dependent serine protein kinase(CASK), which is the paralogue of the LIN-2 proteinin Caenorhabditis elegans, belongs to the membrane-associated guanylate kinase (MAGUK) family of scaffold-ing molecules (Module 6: Figure MAGUKs). CASK seemsto have two functions: it is a membrane-associated scaffoldprotein associated with intercellular junctions and it canalso function as a transcriptional co-regulator. CASK con-sists of an N-terminal PDZ domain, a central SH3 domainand a C-terminal guanylate-kinase homology domain. Itis unusual in that it has an N-terminal Ca2 + /calmodulin-dependent protein kinase II (CaMKII) domain. The C-terminal guanylate-kinase domain of CASK is a pseudok-inase that is involved in targeting the protein to the nuc-leus in neuronal cells where it interacts with the T-brain(TBR1) transcription factor and the CASK-interactingnucleosome-assembly protein (CINAP), which regulatesthe expression of neuronal genes.

Membrane protein, palmitoylated (MPP)There are seven human membrane protein, palmitoylated(MPP) proteins (MPP1–7), which belong to themembrane-associated guanylate kinase (MAGUK) fam-ily (Module 6: Figure MAGUKs). MPP1 may function inneutrophil chemotaxis by regulating the phosphorylationof PKB.

Disc, large homology (DLG)There are five human Disc, large homology(DLG) proteins (DLG1–5), which belong to themembrane-associated guanylate kinase (MAGUK) family(Module 6: Figure MAGUKs). The DLGs have multiplescaffolding functions especially for orchestrating thepositioning of signalling components such as receptorsand ion channels in discrete cellular domains. For ex-ample, DLG1, which is also known as synapse-associatedprotein 97 (SAP97), is one of the postsynaptic density(PSD) signalling elements (see 1 in Module 10: Figurepostsynaptic density). DLG1/SAP97 plays a role inAMPA receptor trafficking during synaptic plasticity.The expression of Kv4 and Kv1.5 channels can also beregulated by DLG1.

Zona occludens (ZO)The three zona occludens (ZO1–3) proteins, which belongto the membrane-associated guanylate kinase (MAGUK)family (Module 6: Figure MAGUKs), are scaffoldingproteins located within the tight junctions (TJs) thatform between epithelial cell layers, such as endothelialcells (Module 7: Figure endothelial cell), and in the my-elin sheaths formed by oligodendrocytes and Schwanncells. Tight junctions consist of multiple proteins thatfall into three main groups: integral membrane proteins,


http://www.cellsignallingbiology.org/csb/003/csb003.pdf#Fig3_CaV1_2_Ltype_channelhttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#Modulation_CaV1_1_Ltype_channelhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Protein_kinase_A_http://www.cellsignallingbiology.org/csb/005/csb005.pdf#Protein_phosphatase_2Ahttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Protein_kinase_Chttp://www.cellsignallingbiology.org/csb/005/csb005.pdf#Protein_phosphatase1_PP1http://www.cellsignallingbiology.org/csb/005/csb005.pdf#Phosphodiesterasehttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Casein_kinase_Ihttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#NMDA_receptorshttp://www.cellsignallingbiology.org/csb/010/csb010.pdf#Fig10postsynaptic_densityhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Glycogen_syntahse_kinase_3bhttp://www.cellsignallingbiology.org/csb/007/csb007.pdf#Fig7_skeletal_muscle_EC_couplinghttp://www.cellsignallingbiology.org/csb/010/csb010.pdf#Postsynaptic_densityhttp://www.cellsignallingbiology.org/csb/010/csb010.pdf#Fig10postsynaptic_densityhttp://www.cellsignallingbiology.org/csb/007/csb007.pdf#Endothelial_cellshttp://www.cellsignallingbiology.org/csb/007/csb007.pdf#Fig7_endothelial_cell


Module 6: Figure MAGUKs

GK domain

GK domain

GK domain

GK domain

GK domain

GK domain

GK domain

GK domain

PDZPDZPDZPDZPDZPDZ

PDZ

PDZ

PDZPDZ

PDZ

PDZ

PDZ

PDZ

PDZPDZ

PDZCARD

CAMKCASK

MPP 1-7

ZO 1-3

DLG 1-4

1-4

DLG 5

Carma

CACNB 1-4( subunits of VOCs)

WW

SH3

SH3

L27

MAGI 1-3

MAGUKs

Domain organization of the MAGUK family.Members of the membrane-associated guanylate kinases (MAGUKs) family of scaffolding proteins have a number of protein–protein interactiondomains.

cytoplasmic plaque proteins and the cytoskeletal ele-ments. Claudin, occludin, tricellulin, junctional adhesionmolecule-A (JAM-A), JAM4, coxsackie adenovirus re-ceptor (CAR) and endothelial cell-selective adhesion mo-lecule (ESAM) are some of the key integral proteins. Theyfunction to hold together the two membranes to form atight seal. The ZO1–3 proteins provide a link between theintegral membrane proteins and the cytoskeleton. For ex-ample, they can bind both to claudin and to F-actin.

CARMA1The full name for CARMA1 is caspase-recruitment do-main (CARD) membrane-associated guanylate cyclase(MAGUK) protein 1. As its name implies, CARMA1belongs to the membrane-associated guanylate kinase(MAGUK) family of scaffolding proteins. It containsa caspase-recruitment domain (CARD), a coiled-coildomain, a PDZ domain, an SH3 domain and a C-terminal guanylate kinase (GK) domain (Module 6: FigureMAGUKs). CARMA1 plays a major role in the activationof the NF-κB signalling pathway in both T cells and Bcells. In T cells, for example, one of the signalling com-ponents activated by the T-cell receptor (TCR) signallingsystem is protein kinase Cθ (PKCθ), which is responsiblefor phosphorylating CARMA1, which is then recruitedinto the immunological synapse (Module 9: Figure TCRsignalling). CARMA1 then associates with a pre-existingcomplex that consists of the CARD protein Bcl10 and themucosa-associated lymphoid tissue protein-1 (MALT1,which is also known as paracaspase) to form the CARMA–

Bcl10–Malt1scaffolding complex. CARMA1 and Bcl10 in-teract through their CARD domains. MALT1 has an N-terminal death domain followed by two Ig-like domainsand a C-terminal caspase-like domain. This complex thenactivates the IκB kinase (IKK) resulting in activation of theNF-κB signalling pathway.

Alterations in the MALT and Bcl10 genes have beenlinked with MALT lymphomas.

Cytoplasmic Ca2 + channel β subunits (CACNBs)The cytoplasmic Ca2 + channel β subunits (CACNBs) area sub-family of the membrane-associated guanylate kinase(MAGUK) proteins (Module 6: Figure MAGUKs). Thereare four CACNB genes (CACNB1–4) with additionalsplice variants that code for the β1-4 subunits that con-trol voltage-operated Ca2 + channels.

The CACNBs are made up of a core Src homology do-main 3 (SH3) and a guanylate kinase (GK) domain joinedtogether by a variable linker. The GK domain interactswith high affinity to the Ca2 + channel α-subunits to regu-late channel opening and closing (Module 3: Figure CaV1.1L-type channel).

Microtubule-associated protein-2 (MAP2)The microtubule-associated protein-2 (MAP2) proteinfunctions to links together protein kinase A (PKA) andtubulin.


http://www.cellsignallingbiology.org/csb/002/csb002.pdf#NF_kB_signalling_pathwayhttp://www.cellsignallingbiology.org/csb/009/csb009.pdf#Tcell_receptor_signallinghttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Protein_kinaseC_thetahttp://www.cellsignallingbiology.org/csb/009/csb009.pdf#Immunological_synapsehttp://www.cellsignallingbiology.org/csb/009/csb009.pdf#Fig9_TCR_signallinghttp://www.cellsignallingbiology.org/csb/012/csb012.pdf#MALT_lymphomashttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#Beta_subunithttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#Voltage_operated_channelshttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#Fig3_CaV1_1_Ltype_channel


PericentrinPericentrin attaches to the centrosome through apericentrin–AKAP350 centrosomal targeting (PACT) do-main. It is included in this list of AKAPs because it is ascaffold for protein kinase A (PKA) and also binds proteinkinase C (PKC).

Wiskott–Aldrich syndrome protein (WASP) verprolinhomologous 1 (WAVE1)Wiskott--Aldrich syndrome protein (WASP) verprolin ho-mologous 1 (WAVE1) is a multifunctional scaffolding pro-tein that brings together different sets of signalling mo-lecules depending on where it is located in the cell. Whenit is associated with the mitochondria, it brings togetherprotein kinase A (PKA), protein phosphatase 1 (PP1), Badand glucokinase. However, when it is operating to controlactin polymerization, it recruits a different set of proteins,such PKA, Abl, Rac, WAVE-associated Rac Gap protein(WRP) and the actin-related protein 2/3 complex (Arp2/3complex) (Module 4: Figure actin remodelling).

Rab32This scaffolding protein binds protein kinase A (PKA) andis associated with the mitochondria.

Macromolecular signalling complexesComponents of many signalling pathways are often collec-ted together to form large molecular complexes (panel C inModule 6: Figure signalling hierarchies). The close appos-ition of signalling components that are often arranged onmolecular scaffolds greatly enhances the efficiency of in-formation transfer. There are numerous examples of suchmacromolecular signalling complexes:

• The T cell receptor (TCR) uses its receptor subunits andscaffolding elements such as the proteins LAT (linkerfor activation of T cells) and Src homology 2 (SH2)-domain containing leukocyte protein of 76 kDa (SLP-76) to assemble a large group of signalling molecules(Module 9: Figure TCR signalling).

• The platelet-derived growth factor receptor (PDGFR)provides phosphorylated residues to assemble the com-ponents of a number of signalling pathways (Module 1:Figure PDGFR activation).

• In the Wnt signalling pathway there is a large β-catenindegradation complex that functions to regulate thephosphorylation and degradation of β-catenin (Module2: Figure Wnt canonical pathway).

• The scaffolding protein KSR1 holds together compon-ents of the extracellular-signal-regulated kinase (ERK)signalling pathway (Module 2: Figure ERK signalling).

• The ryanodine receptors (RYRs) not only function asCa2 + channels, but also assemble many of the signallingcomponents responsible for modulating their activity(Module 3: Figure ryanodine receptor structure).

• The postsynaptic density (PSD) in neurons is a largecollection of different, but interacting macromolecularsignalling complexes (Module 10: Figure postsynapticdensity).

• Integrin receptors located in the focal adhesion complexassemble a large number of signalling components manyof which function to assemble the actin cytoskeleton(Module 6: Figure integrin signalling).

• Gene transcription is regulated by transcriptosomes thatconsist of transcription factors, co-regulators such as theco-activators and co-repressors that recruit chromatinremodelling enzymes such as histone acetyltransferases(HATs), histone deacetylases (HDACs) and proteinmethylases.

Lipid rafts and caveolaeLipid rafts and caveolae are specialized regions of the mem-brane that have a number of signalling functions. Theyprovide a plasma membrane-associated scaffolding systemfor organizing signalling components. The lipid compos-ition of rafts and caveolae provides a unique membranemicroenvironment rich in lipids, such as cholesterol andsphingomyelin, which create a liquid-ordered phase do-main where a variety of signalling components aggregate.Caveolae structure depends upon the coat protein caveolin,which has a caveolin-scaffolding domain capable of bind-ing to the many components responsible for the signallingfunction of caveolae. Lipid rafts closely resemble that ofthe caveolae and may perform a similar function of organ-izing signalling components, and this may be an importantfeature of the immunological synapse.

Lipid composition of rafts and caveolaeLipid rafts and caveolae are characterized by having a lipidcomposition quite different from that of the surroundingplasma membrane (Module 6: Figure caveolae organiza-tion). These special domains are particularly rich in cho-lesterol and sphingomyelin, but they also contain highlevels of glycosphingolipids, ceramide, PtdIns4,5P2 anddiacylglycerol (DAG). Many of these lipids are closely as-sociated with various signalling pathways and highlightthe importance of these zones as sites of informationtransfer across the plasma membrane. The surroundingplasma membrane, which is rich in phospholipids withkinked unsaturated fatty acid tails, forms a highly fluid‘liquid-disordered phase’, within which there is consid-erable lateral movement of membrane proteins. In con-trast, the high concentration of saturated hydrocarbonswithin the lipid rafts and caveolae form a ‘liquid-orderedphase’ because the straight fatty acid chains and the choles-terol pack tightly together to give a highly ordered struc-ture (Module 6: Figure caveolae molecular organization).In effect, the plasma membrane is separated into spatialdomains.

There is a phase separation in the membrane, with thebulk of the membrane being in a fluid state, whilethe lipids are much more ordered in the rafts and ca-veolae. The semi-crystalline state of the latter makesthem resistant to detergents, which can dissolve awaythe liquid-disordered regions of the bulk of the mem-brane to leave behind the rafts and caveolae. Many of theearlier names for these membrane domains reflected this


http://www.cellsignallingbiology.org/csb/004/csb004.pdf#WAVEhttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Arp2_3_complexhttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Fig2_actin_remodellinghttp://www.cellsignallingbiology.org/csb/009/csb009.pdf#Fig9_TCR_signallinghttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Platelet_der_growth_fact_receptor_PDGFRhttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Fig1_PDGFR_activationhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_Wnt_canonical_pathwayhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_ERK_signallinghttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#Ryanodine_receptors_RYRshttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#Fig3_ryanodine_receptor_structurehttp://www.cellsignallingbiology.org/csb/010/csb010.pdf#Postsynaptic_densityhttp://www.cellsignallingbiology.org/csb/010/csb010.pdf#Fig10postsynaptic_densityhttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#Gene_transcriptionhttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Histone_acetyltransferase_HAThttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Histone_deacetylase_HDACshttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Protein_methylationhttp://www.cellsignallingbiology.org/csb/009/csb009.pdf#Immunological_synapse


Module 6: Figure caveolae organization

Lipidraft

Receptors Channels

PDGFEGFInsulinMuscarinic

LipidsCholesterolSphingomyelinCeramide

Transducers

G SSrcRasRaf-1PKC

SHCCAMAdenyl cyclaseeNOSMAP kinase

GlycosphingolipidsPtdIns4,5PDiacylglycerol

PMCANCX

Pumps/Exchangers

Caveolus

BradykininAdrenergicEndothelin

2

NO

NO

cAMP

cAMP

IP

IP

3

3

Erk1/2

Erk1/2

L-type VOC

Ca2+

Ca2+

Ca2+

TrpC1

K

K

K

+

+

ATP

BK channel

Plasma membrane

The organization and signalling function of lipid rafts and caveolae.The rafts and caveolae are specialized regions of the plasma membrane (green zones), which are characterized by having a lipid composition(particularly rich in cholesterol and sphingomyelin) distinct from that of the remaining plasma membrane. Apart from its distinct shape, the caveolusdiffers from the raft by having a cytoplasmic coat of caveolin molecules (yellow). These specialized regions of the plasma membrane containa number of signalling components (receptors, transducers, channels, pumps and exchangers) responsible for initiating many of the major cellsignalling pathways.

low solubility in detergents, i.e. detergent-resistant mem-branes (DRMs), detergent-insoluble, glycolipid-enrichedmembranes (DIGs), glycolipid-enriched membranes(GEMs) or the Triton X-100-insoluble floating fraction(TIFF). These structures are now usually referred to aslipid rafts and caveolae. However, there remains some de-bate as to the functional equivalence of these two struc-tures. While caveolae structure is clearly different to thatof the rafts (Module 6: Figure caveolae organization), thesetwo domains do have many similarities, especially with re-gard to their role in signalling. It is important to appreciate,however, that the rafts and caveolae may carry out somedifferent functions. Here most attention will be focusedon the caveolae.

Caveolae structureCaveolae are flask-shaped invaginations of the plasmamembrane. The organization of the caveolae is maintainedby a cytoplasmic coat of integral membrane proteins (theyellow layer in Module 6: Figure caveolae organization),of which caveolin is the major component. Caveolae havebeen observed in many cell types and are particularlyevident in endothelial cells and various muscle cells. Incardiac muscle, the numerous openings of the caveolaeare clearly evident in freeze–fracture images (Module 6:Figure cardiac caveolae). An important feature of cave-

olae, which is particularly evident in cardiac and smoothmuscle cells, is their close association with the sarcoplas-mic reticulum (SR). The full extent of these close asso-ciations is very evident in tangential sections, where thecaveolae lie in the interstices between the highly retic-ulated SR. What is not clear from these early electronmicroscopic studies is whether this peripheral SR nearthe plasma membrane is connected to the SR that liesdeeper within the cell that is responsible for excitation–contraction coupling in muscle cells. An interesting possib-ility is that this peripheral SR located close to the caveolaemight have a separate function, such as the control of store-operated Ca2 + entry (Module 3: Figure capacitative Ca2 +

entry).In the case of endothelial cells, caveolae function in a

transcellular pathway to transport large molecules such asalbumin from the plasma to the interstitial space (Module7: Figure endothelial cell).

A similar association between the caveolae and the SRhas been described in smooth muscle cells (Module 6: Fig-ure smooth muscle caveolae). As for the cardiac cell, thecaveolae can be seen lying in holes in the flat SR sheet.In addition to associating with the caveolae, portions ofthe SR also come into close contact with the plasma mem-brane to form junctional zones that could have variousfunctions. They might function like the junctional zones


http://www.cellsignallingbiology.org/csb/003/csb003.pdf#Fig3_capacitative_Ca_entryhttp://www.cellsignallingbiology.org/csb/007/csb007.pdf#Fig7_endothelial_cell


Module 6: Figure cardiac caveolae

T

T

T

T

TFreeze-fracture

Tangential section

Caveolae in rat cardiac ventricular cells.In the freeze–fracture image shown at the top, there are numerous openings of the caveolae (yellow arrows) in the membrane between the T-tubuleinvaginations. In the tangential section shown at the bottom, the caveolae have different shapes – rounded, dumb-bells (green) or trilobed (yellow) –and are surrounded by a network of the sarcoplasmic reticulum (red arrows). Reproduced from J. Ultrastruct. Res., Vol. 65, Gabella, G., Inpocketingsof the cell membrane (caveolae) in the rat myocardium, pp. 135–147. Copyright (1978), with permission from Elsevier; see Gabella (1978).

in cardiac muscle to trigger the release of internal Ca2 + .Alternatively, they might represent regions where the SRfunctions to control the opening of plasma membrane ionchannels such as the store-operated channels (SOCs), orthey might be regions where Ca2 + sparks activate Ca2 + -sensitive K+ channels such as the large-conductance (BK)channels (Module 7: Figure smooth muscle cell spark) andthe ATP-sensitive K+ (KATP) channels (Module 6: Fig-ure caveolae organization). In the case of arterial smoothmuscle cells, the Kir6.1 subunit of the KATP channel islocated in the caveolae where it appears to be associatedwith caveolin-1. There are indications that the BK chan-nels found in the caveolae of uterine smooth muscle cellsappear to be regulated by cholesterol within the caveolarmembrane. An excessive build-up of cholesterol that oc-curs during obesity may increase the risk of complicationsin pregnancy. By enhancing BK channel activity, uterinecontractility will be reduced during labour and this may

account for the increased incidence of Caesarean sectionsin obese women.

Signalling function of caveolaeThe caveolae have been implicated in a number of sig-nalling processes. Caveolae contain a high concentrationof the lipid precursors that are used for signalling. Forexample, the sphingomyelin signalling pathway is thoughtto be localized to the caveolae by virtue of the factthat most of sphingomyelin in the plasma membrane isconcentrated at these sites. Components of a number ofsignalling pathways are located at caveolae (Module 6:Figure caveolae molecular organization). Many of thesepathways are associated with the caveolins, which areintegral membrane proteins that have two importantfunctions. Firstly, they bind to the ‘liquid-ordered phase’,forming a dense mat beneath the membrane that isresponsible for maintaining the flask-like shape of the


http://www.cellsignallingbiology.org/csb/003/csb003.pdf#Store_operated_channelshttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#Large_conductance_BK_channelshttp://www.cellsignallingbiology.org/csb/007/csb007.pdf#Fig7_Smooth_muscle_cell_sparkhttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#ATP_sensitive_potassium_channelshttp://www.cellsignallingbiology.org/csb/007/csb007.pdf#Uterine_smooth_muscle_cellshttp://www.cellsignallingbiology.org/csb/012/csb012.pdf#Obesityhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Sphingomyelin_signalling_pathway


Module 6: Figure smooth muscle caveolae

SR

SR

SR

SR

Coronary smoothmuscle cells

Caveolae in coronary smooth muscle cells of the mouse.The tangential section at the top reveals the large number of concentriccaveolae (yellow arrows), many of which are surrounded by an extensiveinterconnected network of the sarcoplasmic reticulum (SR). In the lon-gitudinal section at the bottom, the caveolae are shown opening to thesurface. The SR makes close contact with both the caveolae (green ar-rows) and the plasma membrane, where it forms a typical junctional zone(red arrows). Such junctions may play a role in store-operated Ca2 + entry(Module 3: Figure capacitative Ca2 + entry). Reproduced from J. Ultra-struct. Res., Vol. 67, Forbes, M.S., Rennels, M.L. and Nelson, E., Caveolarsystems and sarcoplasmic reticulum in coronary smooth muscle cells ofthe mouse, pp. 325–339. Copyright (1979), with permission from Elsevier;see Forbes et al. 1979.

caveolae. Secondly, they have a scaffolding function,which depends on a caveolin scaffolding domain in theN-terminal region that binds a number of signallingcomponents [e.g. epidermal growth factor receptor(EGFR), platelet-derived growth factor receptor(PDGFR), GαS, Gαi1, Gαi2, adenylyl cyclase (AC),Ha-Ras, Src, Fyn, endothelial nitric oxide synthase(eNOS), protein kinase Cα (PKCα) and mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated protein kinase (ERK) kinase (MEK)], whichhave distinct caveolin-binding motifs. Some proteintyrosine phosphatases (PTPs), i.e. PTP1B, PTP1C, SHP-2[Src homology 2 (SH2) domain-containing PTP-2],phosphatase and tensin homologue deleted on chromo-some 10 (PTEN) and leucocyte common antigen-related

(LAR), have caveolin-1-binding motifs and may functionby being recruited into lipid rafts or caveolae.

The attachment of signalling components to caveolinbrings these elements together, thus increasing the effi-ciency of information transfer. In some cases, the closeassociation can lead to inactivation of certain signallingpathways.

Caveolae and Ca2 + entryA number of Ca2 + channels are found either in or attachedto the caveolae (Module 6: Figure caveolae molecular or-ganization). Examples of the former are the CaV1 family ofL-type channels and the canonical transient receptor po-tential TRPC1 channels. There is an interaction betweencaveolin-1 and the TRPC1 entry channels. Somewhatmore problematical are the inositol 1,4,5-trisphosphatereceptors (InsP3Rs), which have been localized to thecaveolae region, but their exact topology is uncertain.There are suggestions that they might be embedded in theplasma membrane and can thus function in Ca2 + entry.Alternatively, they might be embedded in endoplasmicreticulum (ER) regions that are tightly associated withthe caveolae (Module 6: Figure smooth muscle caveolae).The InsP3R can be immunoprecipitated by either anti-caveolin-1 or anti-TRPC1 antibodies, suggesting that allthese components may interact with each other at thecaveolae to form a store-operated Ca2 + entry complex,consistent with the conformational coupling hypothesis(Module 3: Figure conformational coupling hypothesis).

Caveolin inactivation of signalling pathwaysMany signalling components are inactivated when they arebound to caveolin. This inactivation phenomenon may beparticularly important during the onset of cell transform-ation and cancer, during which there is a down-regulationof caveolin. As the expression of caveolin declines, the ca-veolae disappear and the associated signalling elements areno longer inactivated and begin to signal constitutively; acharacteristic of many cancer cells. Examples of the sig-nalling systems that are inactivated by caveolin includeendothelial nitric oxide (NO) synthase (eNOS) (Module2: Figure eNOS activation), the mitogen-activated proteinkinase (MAPK) signalling pathway, Src family kinases andPKCα. In the case of the Cav1− / − mouse, the inhibitionof eNOS was removed, resulting in a dramatic increase insystemic NO levels.

The fact that caveolin can inhibit the MAPK cascademay be particularly important with regard to the role ofmitogen-activated protein kinase (MAPK) signalling incardiac hypertrophy. Removal of the caveolin 3, whichis specifically expressed in cardiac cells, resulted in an in-crease in the MAPK signalling pathway and an increase incardiac hypertrophy in mice.

CadherinsThere is a large superfamily of cadherins, which are highlydynamic cell–cell adhesion molecules. From a signallingpoint of view, the cadherins are of interest in that theyare the target of various signalling pathways that can


http://www.cellsignallingbiology.org/csb/003/csb003.pdf#Fig3_capacitative_Ca_entryhttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Epidermal_growth_factor_receptor_EGFRhttp://www.cellsignallingbiology.org/csb/001/csb001.pdf#Platelet_der_growth_fact_receptor_PDGFRhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Adenylyl_cyclasehttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Endothelial_NOShttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Protein_kinase_Chttp://www.cellsignallingbiology.org/csb/005/csb005.pdf#Protein_tyrosine_phosphataseshttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#PTENhttp://www.cellsignallingbiology.org/csb/005/csb005.pdf#LARhttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#CaV1_family_Ltype_channelshttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#TRPC1http://www.cellsignallingbiology.org/csb/003/csb003.pdf#InsP3_receptors_InsP3Rshttp://www.cellsignallingbiology.org/csb/003/csb003.pdf#Fig3_conformational_coupling_hypothesishttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Endothelial_NOShttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#Fig2_eNOS_activationhttp://www.cellsignallingbiology.org/csb/002/csb002.pdf#MAP_kinase_signallinghttp://www.cellsignallingbiology.org/csb/012/csb012.pdf#MAPkinase_signalling_in_cardiac_hypertro


Module 6: Figure caveolae molecular organization

eNOSHa-Ras

RafMEK

EGFRPDGFR

G S

Adenylyl

cyclasePLC

NO Erk1/2InsP3

CyclicAMP

Cholesterol

Sphingomyelin

Glycerolipids

Caveolin

A schematic diagram to illustrate how caveolin is thought to organize various signalling components.The caveolin molecules that oligomerize with each other are embedded in the membrane through a hydrophobic region. Another region that is closeto this hydrophobic domain has the caveolin scaffolding domain (shown in black). A number of signalling components have caveolin-binding motifs(shown in red), which enable them to associate with this scaffolding region.

adjust cell–cell interactions by altering cadherin adhesive-ness. Conversely, there is increasing evidence that some ofthe cadherins may initiate cell signalling pathways to con-trol various cellular pathways such as the establishment ofplanar cell polarity (PCP) during development.

Cadherins have a wide range of functions both duringdevelopment and in the turnover of adult tissues. With re-gard to the former, they are responsible for many of themorphogenic processes that occur during development.They play a role in separating cells into distinct layers byforming tissue boundaries and they contribute to cell mi-gration and the formation of synapses during brain devel-opment. After tissues have formed, they continue to func-tion in adult life. In the brain, they contribute to learningand memory by strengthening new synapses. Cadherinsorchestrate the replacement of cells in tissues that turnoverrapidly, such as the gut and epidermis. In order for newcells to move into the regenerating tissues, the cadher-ins must strike a fine balance by allowing cells to movewhile maintaining tissue integrity. When such tissue integ-rity breaks down, cells dissociate and changes in cadherinprofiles are a feature of metastatic cells. In the endothe-lium, they regulate junctional permeability where there isa controlled cell–cell separation to allow the passage ofneutrophils.

These multiple and diverse functions are carried outby the cadherin superfamily that has been divided intodifferent groups:

• Classical cadherins• Desmosomal cadherins• Protocadherins• Atypical cadherins

Classical cadherinsThe classical cadherins are a large family of multifunctionalproteins. They tend to be named after the tissue where theywere first discovered as illustrated below:

• E-cadherin (epithelial cadherin) is expressed in epithelialcells where it is mainly associated with the zonula adher-ens that functions to hold cells together. The expressionof E-cadherin is regulated by zinc-finger E-box bindinghomeobox 1 (ZEB1), which is also known as transcrip-tion factor 8 (TCF8), and zinc-finger E-box bindinghomeobox 2 (ZEB2), which is also known as Smad-interacting protein 1 (SIP1). The expression of ZEB1and ZEB2 is regulated by miR-200.


http://www.cellsignallingbiology.org/csb/008/csb008.pdf#Planar_cell_polarity_PCPhttp://www.cellsignallingbiology.org/csb/004/csb004.pdf#miR_200


Module 6: Figure cadherin superfamily

FlamingoCelsr1-3

Fat

Atypical cadherins

EC repeat

EC1EC2EC3EC4EC5

Ca2+

Flamingo box

EGF domain

Laminin AG domain

DachsousDesmosomalcadherins

Classicalcadherins

Proto-cadherins

Cadherin superfamily of cell–cell adhesion molecules.Most of the cadherins are single-membrane-spanning proteins characterized by having large extracellular domains containing multiple Ca2 + -bindingextracellular cadherin (EC) domains (shown in green). The exception is the Drosophila cadherin called Flamingo and its mammalian homologuesCelsr1–Celsr3, which have seven membrane-spanning regions. The way in which some of these cadherin interact with each other during cell signallingis shown in Module 6: Figure classical cadherin signalling and in Module 8: Figure planar cell polarity signalling.

Mutations in the ZEB1 gene are associated withposterior polymorphous corneal dystrophy-3 (PPC3) andlate-onset Fichs endothelial corneal dystrophy.

• N-cadherin (neural cadherin) is expressed mainly in thenervous system where it contributes to synapse forma-tion (see step 7 in Module 10: Figure postsynaptic dens-ity). The fourth extracellular cadherin domain (EC4) ofN-cadherin interacts with thefibroblast growth factorreceptor (FGFR) thus enabling this cadherin to contrib-ute to mitogenic signalling. The N-cadherin complexalso functions in the peg-socket junctional complex thatforms between pericytes and endothelial cells (Module9: Figure angiogenesis signalling).

• M-cadherin functions in the interaction between musclecells and satellite cells (Module 8: Figure satellite cellactivation).

• R-cadherin (retinal) was original identified in the ret-ina, but also functions in other regions of the brain. Itinteracts with cadherin-6 during brain development.

• VE-cadherin (vascular endothelial cadherin).

This nomenclature can be confusing because it soonbecame clear that many of cadherins shown above werenot tissue-specific. This lettering system was switched to

a numbering system for many of the remaining cadherins,of which some are shown below:

• Cadherin-6 is mainly expressed in the kidney and brain.During brain development, differential expression ofcadherin-6 and R-cadherin may set up the compartmentboundary between the cerebral cortex and the striatum.

• Cadherin-23 appears to form the helical tip link fila-ment that connects the ends of stereocilia on hair cells(Module 10: Figure tip link). Mutation of cadherin-23 is one of the causes of the deaf/blindness Ushersyndrome.

Most attention has focused on E-cadherin, which is usedas the basis for the following general description of cad-herin function. As part of their role in cell adhesion, cad-herins provide a membrane anchor for actin and th

module 6 spatial and temporal aspects of signalling · 2013. 5. 7. · cell signalling biology...

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