IntroductionKinesins constitute a family of molecular motors that containa signature ~340 residue motor domain that transduces ATPhydrolysis into a directed walk along a microtubule (Vale andMilligan, 2000). Outside the conserved motor domain theprimary structures of kinesins diverge, and it is this part – thetail – that binds cargo and regulates motor activity. Slowly butsurely, progress is being made toward meeting the field’sprincipal challenge: identifying the cargoes and defining howthey are linked to, and released by, particular kinesins at theright time and place.

For many years it was presumed there were ‘kinesinreceptors’, that is, molecules whose sole purpose is to linkkinesins to cargo. This presumption is not supported by therecent work, which instead indicates that kinesin-cargo linkers(Bowman et al., 2000; Kamal et al., 2000; Lee et al., 2002;Nakagawa et al., 2000; Setou et al., 2000; Setou et al., 2002;Verhey et al., 2001) are ‘familiar faces’ that have otherfunctions. Most are adaptors or scaffolds. Through theirmultiple protein binding sites, they organize molecularassemblies that constitute the cargo and are themselves part ofthe cargo (Fig. 1).

One of the first proteins to illustrate this new concept wasthe adaptor protein AP-1. Through its interactions with clathrinand specific receptor-ligand complexes (Pearse, 1988), AP-1coordinates the formation of specific vesicle populations at theGolgi apparatus and the plasma membrane (reviewed inKirchhausen, 2002). The new idea contributed by kinesinresearch (Nakagawa et al., 2000) is that a site on AP-1 alsointeracts with the tail of at least one kinesin – KIF13A. Thisinteraction explains how a subset of Golgi-derived vesicles(those containing the mannose-6-phosphate receptor and itsligands) is transported along microtubules to the pre-lysosomalcompartment (Nakagawa et al., 2000). This is an appealingidea: the same protein – AP1 – that sorts membrane proteinsinto specific cargo vesicles also interacts with the motor thattransports these vesicles to their final destination.

The idea that kinesins are linked to adaptors that possessbinding sites for multiple proteins suggests we may haveunderestimated the role of microtubule-based transport in cellbiology. Given the large number of scaffold proteins andkinesins encoded by the human genome, the range of proteinsthat in principle could be part of one cargo or another is vast.As the kinesin-cargo linker molecules and the proteins theyscaffold come to light, our notion of what constitutes ‘cargo’for intracellular transport along microtubules will inevitablybroaden. For example, recent studies indicate that pre-assembled signal transduction cascades, or ‘transducisomes’(Tsunoda et al., 1997), are cargo for kinesins. Below, I discussthis novel role for kinesins at the interface of signaling andtransport.

Trafficking of scaffolds for signaling pathwaysMost signaling pathways employ scaffold proteins of one kindor another, which helps explain a fundamental tenet ofintercellular signaling: from a common collection of enzymes,individual cells assemble parallel signaling pathways thatrespond to different stimuli and produce distinct physiologicalresponses (Smith and Scott, 2002). A classic example is thescaffolding of MAP kinase (MAPK) pathways (van Drogenand Peter, 2002), which first came to light in studies of thepheromone mating response in Saccharomyces cerevisiae(Choi et al., 1994). The MAP kinase cascade (i.e. MAPKkinase kinase, MAPK kinase, and MAPK) controlling thisresponse requires a scaffold protein, Ste5, which holds thethree kinases in juxtaposition (Elion, 2001). This complexensures that these kinases phosphorylate only their specifictargets and not other potential targets in the cell. MAP kinasecascades that regulate other physiological processes aresimilarly organized on their own scaffolds. In this way,multiple MAP kinase signaling pathways, each coupled to adifferent receptor, can be isolated from each other, whileemploying some of the same components.

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The human genome has more than 40 kinesin genes whoseprotein products organize intracellular traffic alongmicrotubules. Research during the past two years hasbegun to elucidate the cargoes carried by kinesins and thenature of the kinesin-cargo linkage. Modular protein-protein interactions connect kinesins to diverse cellularmolecules, which, apart from their other functions, serve askinesin-cargo linkers. Many of these newly identifiedlinkers are scaffolds for signaling pathways, and mounting

evidence now indicates that kinesins transport pre-assembled signaling modules as vesicular cargo. Thesefindings bring together two fields, signal transduction andmolecular motors, and lead to a deeper understanding ofthe interplay between trafficking, localization andintercellular communication.

Key words: Kinesin, Scaffold, Signal transduction, Motors,Cytoskeleton, Protein trafficking

Summary

Trafficking of signaling modules by kinesin motorsBruce J. Schnapp Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland, OR 97201-3098, USA(e-mail: schnappb@ohsu.edu)

Journal of Cell Science 116, 2125-2135 © 2003 The Company of Biologists Ltddoi:10.1242/jcs.00488

Commentary

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Scaffold molecules are particularly important for theorganization of PDZ-based signaling cascades. Many of thesereside at specific subcellular sites in the nervous system, wherethe need for a high degree of localization, for example, atsynaptic junctions, is obvious. Equally important is the need tolocalize signaling cascades in epithelia. Many intercellularsignaling pathways that govern cell fate specification duringembryonic development occur in the context of epithelia(Fig. 2). Like neurons, epithelial cells are asymmetric: thebasolateral and apical domains encounter distinct extracellularenvironments. Consequently, communication from an inducingcell to a neighboring cell will depend on localization of therelevant signaling molecules.

The importance of trafficking and localizing signaling

modules seems obvious in these contexts and is supported bya great deal of circumstantial evidence. However, only a fewstudies have actually provided direct evidence that propertrafficking of signaling molecules to a highly localizedsubcellular destination is essential for intercellular signalingand global expression of signaling molecules is inadequate.Among the clearest cases is the seminal study that identified acomplex of scaffold proteins, known as the LIN complex, inthe context of vulva development in Caenorhabditis elegans(Kaech et al., 1998). In C. elegans, a canonical Ras MAPkinase signaling pathway specifies an epithelial precursor cellto become a vulva cell. This pathway is activated when an EGFreceptor tyrosine kinase, LET27, in the precursor cell bindsEGF released by an ‘anchor cell’. Because the anchor cell

Journal of Cell Science 116 (11)

Kinesin

Cargovesicle

Transmembrane receptor

L inker

MotorTransmembranereceptor

Traff icking pathwayCargo L inker

K IF13A Mannose-6P receptorTrans Golgi toendosome

Mannose-6P receptor AP-1

Kinesin I ApoER2AxonsJNK signaling cascade JIP

Kinesin I APPAxonsAPP

AMPA GluR complex Dendr ites Kinesin I GRIP AMPA GluR

Binding sites for other proteins

___

(Nakagawa et al., 2000)

K IF1A GRIP-AMPA GluRDendr itesAMPA GluR complex L ipr in-α(Lee et al., 2002)

K IF1A LARAxonsPresynaptic components L ipr in-α(Lee et al., 2002)

K IF17 NMDA GluRDendr itesNMDA GluR complex L IN complex(Setou et al., 2000)

(Verhey et al., 2001)

(Kamal et al., 2000)

(Setou et al., 2002)

K IF13b ?Dlg scaffold complex Dlg(Asaba et al., 2003)

?

Fig. 1.Recently identified cargoes for kinesins are built according to a common design plan. With the exception of the transmembrane proteinAPP, kinesins are linked to cytosolic scaffold proteins that have multiple binding sites for other proteins, including a transmembrane proteinthat defines the cargo. Kinesin tails and transmembrane receptors are connected to the linkers by modular protein-protein interactions. A largefraction of the linkers discovered thus far are scaffold proteins for signaling pathways.

2127Kinesins traffic signaling modules

resides in the stroma (Fig. 2), the EGF it releases cannotpenetrate the intercellular junctions connecting the precursorcells. The products of the LIN2, LIN7 and LIN10genes preventdifferentiation of the precursors by interfering with their abilityto establish a localized cluster of LET27 at the basolateralaspect of the junctional complex. These three proteins form anevolutionarily conserved PDZ-based scaffold complex thatinteracts with LET27, as well as a number of differentreceptors in various organisms and cell types (Bredt, 1998). Inworms, null mutations in the LIN2, LIN7 or LIN10 genes leadto the same phenotype: LET27 is still present in the precursorcells, and even exists in the basolateral compartment, but it isdiffusely distributed and not concentrated at the cell junction.Second allele complementation experiments elegantly provethat specific interactions between the LIN scaffold complexand LET27 are required for proper localization of the receptorLET27 and operation of the pathway (Kaech et al., 1998).These studies provide direct experimental evidence thatexpression and global distribution of the relevant signalingmolecules in a cell are not sufficient for signaling to occurnormally. These molecules need to be properly trafficked andlocalized.

In summary, it is clear that signaling cascades must betrafficked and localized, and that these activities involvescaffold proteins. But how is the localization of signalingmodules achieved? And exactly where in the cell, and by whatmechanisms, do signaling molecules load onto scaffolds? Untilrecently, it was widely presumed that scaffold proteins andtheir signaling molecule partners arrive at their destinations,

where they assemble locally, by diffusion. Recent research onkinesin-cargo interactions instead suggests that signalingmolecules are loaded onto their scaffolds away from their finaldestinations and that active transport along microtubulesdelivers these pre-assembled signaling modules to particulardestinations. I will review how these new ideas developed fromefforts in the kinesin field to define the nature of the motor-cargo linkage and the identity of the cargoes carried bykinesins, discussing this recent work chronologically becauseof the compelling manner in which the experiments and ideasprogressed.

KIF 17 traffics glutamate receptors in the brainThe first definitive evidence that signaling molecules are cargofor kinesins developed from a yeast two-hybrid screen forpartners of the mouse neuronal kinesin KIF17 (Setou et al.,2000). The KIF17 C-terminus has a PDZ-binding motif thatbinds the first PDZ domain of mouse brain LIN-10, that is, thehomologue of C. elegansLIN-10 mentioned above. Prior to thediscovery that mouse brain LIN-10 interacts with KIF17 (Setouet al., 2000), several laboratories had established that thecomplex of LIN-10/Mint1, LIN-2/CASK and LIN-7/Velis isevolutionarily conserved both in its structure and in itsassociation with transmembrane receptors that are localized todiscrete subcellular sites (Bredt, 1998); for example, in C.elegans, the LIN complex is required for targeting EGFreceptors to cell junctions in vulval precursor cells (Kaech etal., 1998) and for localizing glutamate receptors to the

BasolateralEGF release

Apical

Vulval precursor

Ras/MAPKpathway

Anchor cell

(Let-23)Receptor tyr kinase

Tight junctionBOSS

Cell R7 Cell R8

(sevenless)Receptor tyr kinase

Ras/MAPKpathway

C. elegans vulva Drosophila photoreceptor

Fig. 2.Two examples where subcellular localization of signaling modules is essential. Cell fate specification in the worm sex organ and the flyeye occur in epithelia. In each case, an inducing cell (anchor cell or cell R8) provides a ligand (secreted EGF or transmembrane protein BOSS)that interacts with a receptor tyrosine kinase (Let-27 or sevenless) to activate a canonical Ras/MAPK pathway. The MAPK pathway directs aprecursor (vulval precursor or Cell R7) toward a particular cell fate. In the case of vulva cell fate specification in C. elegans, the anchor cell is inthe stroma, and therefore Let-27, and presumably its downstream targets, must reside in the basolateral compartment to receive the signal. Inthe fly eye, both the receptor, sevenless and the ligand, BOSS, are located in the microvilli of the apical compartment.

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postsynaptic density in neurons (Rongo et al., 1998). Althoughother studies also raised the possibility that the LIN complexlocalizes glutamate receptors to postsynaptic densities in themammalian brain (Jo et al., 1999), the mechanism(s)underlying this and other LIN-associated localization eventshave not been resolved.

In light of these previous studies, the discovery that LIN-10 interacts with KIF17 immediately raised the possibilitythat postsynaptic clusters of glutamate receptors areestablished, at least in part, through KIF17-driven transport(Setou et al., 2000) (Fig. 3). In this context, the LIN complexserves as a kinesin-cargo linker: it connects KIF17 to thetransmembrane protein NR2B (a subunit of the NMDA-sensitive glutamate receptor), which itself provides theconnection to a vesicle (Figs 1 and 3). In vitro vesicle motilityexperiments support this model (Setou et al., 2000): dominantinhibitory KIF17 constructs inhibit nucleotide-dependentmicrotubule binding of NR2B-containing vesicles isolatedfrom the brain; and wild-type KIF17, but not mutants lackingthe C-terminal residues that bind LIN-10, promotes themotility of isolated NR2B-containing vesicles alongmicrotubules.

It is unclear whether the LIN complex also scaffoldssignaling molecules downstream of the receptors it carries, but

other multi-PDZ domain proteins do function in this manner(Harris and Lim, 2001; Sheng and Sala, 2001). For example,InaD has five PDZ domains that each hold a component of thephototransduction cascade in Drosophila photoreceptors(Tsunoda et al., 1997). Generation of a normal light responsedepends on this architecture. Mutations within single PDZdomains produce specific defects in the light response, eachassociated with mislocalization, to the cytoplasm or plasmamembrane, of the PDZ partner. InaD thus localizes, and isresponsible for the targeting of, a highly organizedtransducisome (Tsunoda et al., 1997).

Conventional kinesin (kinesin I)The idea that the LIN scaffold complex is also a KIF17-cargolinker (Setou et al., 2000) provided the first indication of arelationship between kinesins and the localization/traffickingof signal transduction scaffolds. Subsequent work on kinesin Igeneralized and extended this concept (Fig. 3). Kinesin I wasoriginally discovered in the context of vesicle transport inaxons (Schnapp et al., 1985; Vale et al., 1985), but its in vivofunction and cargo were uncertain for many years. It nowappears that this kinesin carries multiple signaling modules asvesicular cargo (Fig. 1).

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7

2 GUKSH3

10PTB

mLINs2, 7 & 10

PDZ

JIPMLK3/DLK

JNK

MKK 7

K inesin I K IF17

KLCKHC

...EDSTKALAESVGRAFQQFYKQFVEYTCPTEDIYLEJIP C-terminus

...ALPPTQPRPFRLESLDIPFSKAKRKK SKNSFGGEPL

K IF17 C-terminusKLC TPRs

LIN 10 PDZ domain

Reelin receptor

Apo

ER

2

NR

2B

Glutamatereceptor

Fig. 3.KIF17 and kinesin I cargo interactions compared and contrasted. The signaling scaffold JIP links kinesin I to the transmembranereceptor, ApoER2; the LIN signaling scaffold links KIF17 to the transmembrane protein, NR2B, a subunit of the NMDA-sensitive glutamatereceptor. The kinesin-linker interactions are strikingly similar, suggesting a general model for how modular protein-protein interactions underliekinesin-cargo specificity. In both cases, the kinesin-linker interactions involve a protein-binding module (TPRs or PDZ) designed to recognizespecific motifs at the C-termini of partner proteins. In the case of kinesin I, the protein-binding module (TPRs) is in the kinesin tail, and themotif is in the linker; whereas for KIF17, the module (PDZ) is in the linker and the motif in the kinesin.

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Kinesin I structure, motility and regulationNative kinesin I is a tetramer consisting of a kinesin heavychain (KHC) dimer and two kinesin light chains (KLCs) (Fig.4). The KLCs are non-motor polypeptides that associate withthe KHC dimer exclusively, that is, they are not found atappreciable levels, apart from the KHC dimer, and, as a generalrule, the KHC dimer is always associated with KLCs (Hackneyet al., 1991). For many years the functions of the KLCs wereenigmatic, but we now know they participate in two activitiesthat presumably are coordinated: linking kinesin I to cargo andregulating kinesin I motor activity.

As the vesicle-microtubule interface can only accommodateone or two motor molecules, kinesin I (Block et al., 1990;Howard et al., 1989) and other kinesins that carry small cargo(Tomishige et al., 2002) must be processive, that is, they mustwalk along the microtubule without letting go. If this were notthe case, continuous transport of cargoes over long distanceswould not be possible because the kinesin-cargo complexwould diffuse away from the microtubule. Althoughprocessivity would seem to imply that the bulk of kinesin Ishould be bound tightly to microtubules, most kinesin I isactually soluble, unbound to either microtubules or cargo.Moreover, the microtubule-stimulated ATPase rate of isolatedkinesin I is too low (Hackney, 1995) to account for the ~600nm sec–1 rate of kinesin-driven movement, given that one ATPhydrolytic event powers each 8 nm step along the microtubule(Schnitzer and Block, 1997; Svoboda et al., 1993). Whatreconciles these apparent inconsistencies is that kinesin I motoractivity is turned off in the absence of cargo. If it were not, themotor population would walk to the end of the tracks andaccumulate at the cell periphery. By negatively regulatingmotor activity, the cell maintains the concentration of aprocessive motor such as kinesin I at a uniform levelthroughout the cell and in large excess of cargo. An appealinghypothesis is that a cargo controls its own destiny by bindingto and activating motors on demand (Verhey et al., 1998).

Kinesin I appears to be turned off in the absence of cargo bya self-inhibition mechanism that depends on its tail andinvolves folding of the KHC dimer (reviewed in Verhey andRapoport, 2001; Woehlke and Schliwa, 2000) (Figs 4 and 5).The KHC dimer contains coiled coils interrupted by shortunstructured regions that act as hinges (Fig. 4). Isolated nativekinesin I is primarily in a folded conformation (Fig. 5) thatprevents the motor domains from engaging the microtubule,and thereby inhibits motility and ATP hydrolysis. The principalevidence supporting this model comes from mutationalanalyses, which indicate that hinge 2 (Friedman and Vale,1999; Seiler et al., 2000) and C-terminal residues of KHC(Hackney and Stock, 2000; Seiler et al., 2000; Stock et al.,1999; Verhey et al., 1998) are essential for inhibition. The C-terminal residues, which carry excess positive charge, mightinteract with a region of excess negative charge in the neckcoiled coil (Stock et al., 1999) to stablilize the foldedconformation.

Although such self-inhibition by folding is an integralproperty of the KHC dimer, the KLCs are needed for fullrepression of KHC in vivo (Verhey et al., 1998): in the absenceof KLCs, epitope-tagged KHCs colocalize with microtubulesand accumulate at the ends of cell processes, that is, at theminus ends of microtubules. In vitro experiments also supportthe idea that the KLCs contribute to inhibition (Friedman and

Vale, 1999). Although the exact mechanism by which KLCsinhibit kinesin I motor activity is unclear, the KLC structure-function relationships provide some clues (Verhey et al., 1998).KLCs consist of at least two structurally distinct regions (Fig.4): an N-terminal region of heptad repeats and a C-terminalregion that has six tetratrico peptide repeats (TPRs) – protein-binding modules present in diverse proteins (Blatch and Lassle,1999). The heptad repeats alone are sufficient for binding KHC(Fig. 4) and inhibiting the ability of KHC to engagemicrotubules. By contrast, the KLC TPRs are required forneither of these activities. In view of the idea that at some pointKHC motor activity must be activated in relation to cargobinding, it was appealing to consider that the partner(s) of theKLC TPRs might be involved in cargo binding or motoractivation (Verhey et al., 1998). Thus, identifying the partnersof the KLC TPRs became a priority.

Kinesin I carries a MAP kinase cascadeWe now know that the KLC TPRs are kinesin I cargo-bindingdomains. Initially this came to light following the discoverythat the TPRs interact with a family of MAP kinase scaffoldproteins – the JIPs (JNK-interacting proteins, known also asJSAPs) (Bowman et al., 2000; Verhey et al., 2001). The threeJIP isoforms in mammals are scaffolds for the MAP kinasecascade that activates Jun N-terminal kinase (JNK) (Davis,2000). In common with InaD and Ste5, JIPs juxtapose acascade of kinases, ultimately enabling the efficient andspecific phosphorylation of JNK at two sites by a MAPKK(Davis, 2000). Activated JNK phosphorylates downstreamtargets, including the transcription factor Jun, and regulatesvarious physiological activities (Weston and Davis, 2002),including apoptosis in the brain (Morishima et al., 2001).Among the many different transmembrane receptorsimplicated in JNK signaling is ApoER2, the Reelin receptor(Stockinger et al., 2000). The Reelin signaling pathway has avery important role in neurogenesis (Rice and Curran, 2001),which is consistent with the enrichment of kinesin I and JIPsin the brain.

The KLC TPRs recognize a motif found at the extreme C-termini of JIP 1 and JIP2 (Fig. 3) (Verhey et al., 2001). TPRdomains in other proteins likewise recognize the C-termini oftheir partners (Gatto et al., 2000; Scheufler et al., 2000;Terlecky et al., 1995). This establishes a striking parallelbetween the kinesin-I–JIP linkage and the KIF17-LIN scaffoldlinkage (Fig. 3) with respect to how kinesin-cargo specificityis governed. Both interactions involve binding modules, PDZdomains or TPRs, that recognize motifs in the C-termini oftheir partners. In the case of kinesin I, the module is in thekinesin and the motif in the linker, whereas in the case ofKIF17, the module is in the linker and the motif in the kinesin.But the interactions are comparable fundamentally.

The significance of the JIP-KLC TPR interaction came tolight in three complementary studies (Bowman et al., 2000;Byrd et al., 2001; Verhey et al., 2001). First, Bowman and co-workers identified Sunday Driver (SYD) (Bowman et al., 2000),the Drosophila homologue of mammalian JIP3, in a mutantscreen for genes that produce in larvae a behavioral phenotypecalled tail flipping, which is characteristic of kinesin I KHC andKLC null nutants (Hurd and Saxton, 1996; Saxton et al., 1991).This phenotype is associated with axonal jams – accumulations

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Folding (892-918)

Inactivation (892-940)

Inactivation

COOH

COOH

Motor domain(1-368)

Hinge 1(369-413)

Stalk coi l 1(414-566)

Hinge 2(567-593)

Stalk coi l 2(594-810)

Stalk -tai l linker(811-818)

Tail coi l(820-918)

C-term(919-963)

QIAKPIRPG 919-927

Motor catalytic core(1-325)

Neck coi l(337-368)

Neck linker(326-334)

Heptadrepeats

Tetratri co peptid e repeats

APP, JIP CBD

Folding (349-357)

Neurospora CBD (824-875)

GRIP CBD (807-934)

A. Kinesin I

Motor catalytic core(1-354)

Neck linker(355-363)

Neck coi l(364-397)

Motor domain(1-397)

(398-428)Hinge

Stalk coi l 1(429-462)

(515-572)FHA

Stalk coi l 2(625-679)

(1581-1680)PH domain

COOH

1695

COOH

MBS (638-881)

LBD (650-1105)

B. KIF1A

Fig. 4.Domain organization of kinesin I and KIF1A. For kinesin I, its heavy and light polypeptide chains are shown in red and green, respectively.Amino-acid residues in parentheses are referenced to the mouse ubiquitous isoform. Cargo-binding domains (CBD) are indicated by black on whitelabels; domains involved in regulation of motor activity are indicated by black on white labels. KIF1A, like other KIF1 kinesins, have much shortercoiled-coil regions than kinesin I, and these promote dimerization of KIF1 polypeptides that are brought into close proximity on the cargo surface(Klopfenstein et al., 2002; Tomishige et al., 2002). Thus, KIF1 polypeptides are monomers in the cytosol (Nangaku et al., 1994; Okada et al., 1995),and through dimerization, presumably on the cargo surface, acquire the ability to walk along microtubules processively, like native kinesin I does(Tomishige et al., 2002). It is appealing to consider that dimerization regulates motor activity of KIF1 kinesins like folding regulates kinesin I activity(see text). The function of the FHA domain, which marks all KIF1 family members, is unknown, but may be involved in regulating dimerization.Liprin and MAGUK cargo-binding domains (LBD and MBS) overlap. The pleckstrin homology domain (PH) is probably a separate cargo-bindingdomain (Klopfenstein et al., 2002). The amino-acid residues in parentheses are referenced to the mouse isoform of KIF1A.

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of membrane organelles in peripheral nerves (Hurd and Saxton,1996) – and arises as the level of maternal kinesin I declineduring development, which leads to death. Although the exactrelationship between axonal jams and a specific kinesin Itransport function is unclear, the similar phenotypes of KLC,KHC and SYD mutants indicate that the products of the threegenes are involved in the same process. The additional findingthat SYD co-precipitates with kinesin I in vivo and interactsdirectly with KLC TPRs suggested SYD functions byinteracting directly with kinesin I. But these studies left openthe question of whether SYD/JIP3 and its associated signalingmolecules are kinesin I regulators, cargoes or both.

Simultaneously, studies by Verhey et al. (Verhey et al., 2001)provided evidence that JIPs are cargoes of kinesin I inmammals. Earlier work had indicated that JIPs are highlyenriched at the tips of neuronal processes (Kelkar et al., 2000;Meyer et al., 1999), that is, at the minus ends of microtubules,suggesting that they are transported there by kinesin I. The factthat dominant inhibitory KLC constructs (heptad repeatswithout TPRs or TPRs without heptad repeats) block neuritetip localization of JIPs supports the hypothesis that JIPs arekinesin I cargo (Verhey et al., 2001). In addition, the effects ofmutations in JIP1 or JIP2 C-terminal residues on KLC TPRbinding and on neurite tip localization correlate (Verhey et al.,2001). Thus, the steady-state distribution of JIP1 and JIP2 inneuronal cell lines depends on the interaction between theconserved C-terminal residues of JIP1 and JIP2 and the KLCTPRs of kinesin I.

These studies generalize the model of kinesin-cargoarchitecture established initially in the context of KIF17 (Fig.3) and also contribute a new idea: the JIP scaffold is pre-loadedwith its kinase cascade prior to transport. This idea was broughtto light by the finding that not only JIP but the kinasesscaffolded by JIPs, and the transmembrane receptor ApoER2,all co-precipitate from brain extracts with kinesin I when thelatter is isolated either with an antibody or by nucleotide-dependent microtubule co-sedimentation (Verhey et al., 2001).Furthermore, kinesin I dominant inhibitory constructs thatinhibit neurite tip localization of JIP likewise inhibitlocalization of the MAPKK scaffolded by JIP (Verhey et al.,2001). These findings fit nicely with the notion of atransducisome (Tsunoda et al., 1997) and raise the possibilitythat transducisomes are also trafficking units. The findings alsosupport the idea that signaling scaffolds, in addition tojuxtaposing kinases in a cascade, carry information about thetrafficking and localization of the cascade. This model differsfrom the conventional view that signaling molecules assembleon scaffolds at their final destination.

Does signaling through the cascade feedback to regulate themotor? One appealing proposal (Verhey and Rapoport, 2001)is that activation of the Reelin pathway by ApoER2 causeskinesin I to dissociate from the JIP scaffold upon fusion of thevesicle with the nerve terminal membrane. Although thisspecific hypothesis has not been tested, a third line ofinvestigation (Byrd et al., 2001) establishing an interactionbetween JIP scaffolding proteins and kinesin I indicates that

Cargo

Cargo

Cargo bindin gand act ivation

Cargo Cargo

Folded, inact ivekinesin I tetramer

Fig. 5. Auto-inhibition of kinesin I motor activity by folding. In the absence of cargo, kinesin I is in a folded conformation wherein the C-terminus of KHC interacts with the motor domain to prevent binding to the microtubule. How kinesin I is activated is unknown. One hypothesisis that cargo binding (to either the KLC or the KHC cargo binding sites) induces a conformational change that frees the motor domain toengage the microtubule.

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kinases scaffolded by JIP regulate intracellular vesicle traffic,including cargo transport by kinesin I. These studies identifiedunc-16, which encodes the JIP 3/SYD homologue, in a geneticscreen for molecules that organize presynaptic terminals in theC. elegansnervous system. In C. elegans, as in mammals andflies, JIP 3 scaffolds JNK and its upstream kinases and isdependent on kinesin I for its normal distribution in neurons(Byrd et al., 2001). Partial loss-of-function alleles of kinesin I,unc-16 or JNK and its upstream kinases produce the samephenotype – mislocalization of synaptobrevin (Byrd et al.,2001) – and together enhance this phenotype; hence thesegenes must function in the same process. Signaling via kinasescarried by kinesin I thus does regulate vesicle transport, but thenature of this regulation is unclear. Whether kinesin I itself isregulated directly by the kinases it carries is yet to beaddressed.

One kinesin – multiple cargo-linkersAPPA key question is whether each member of the kinesin familyis committed to carrying one or multiple cargoes.Investigations of kinesin I support the latter idea. For example,the KLC TPRs interact not only with JIPs but with amyloidprecursor protein (APP) (Kamal et al., 2000). APP is atransmembrane protein that in vivo is subjected to proteolyticcleavages, including a final, intramembrane, presenilin-dependent cleavage (Selkoe, 1998). This cleavage produces theextracellular amyloid β-peptide of Alzheimer’s disease andreleases an intracellular tail fragment of unknown function.Despite intensive research, the normal cellular function of APPis not known, but the proteolytic processing of APP isstrikingly similar to that of Notch (Artavanis-Tsakonas et al.,1999), the transmembrane receptor for a signaling pathwayinvolved in cell fate specification. Cleavage of Notch producesa cytoplasmic fragment that enters the nucleus and regulatestranscription. Other signaling molecules, for example, E-cadherin (Marambaud et al., 2002) and the ErbB-4 receptortyrosine kinase (Ni et al., 2001), are similarly processed.Recent studies extend the parallel between Notch and APP bydemonstrating that the cytoplasmic tail of APP forms amultimeric complex with the nuclear adaptor protein Fe65 andthe histone acetyltransferase, Tip60 (Cao and Sudhof, 2001).

Definitive evidence now demonstrates that APP istransported in axons by kinesin I (Gunawardena and Goldstein,2001; Kamal et al., 2000). Earlier studies (reviewed in Selkoe,1998) demonstrated that APP is synthesized in theendoplasmic reticulum, glycosylated in the Golgi apparatus,and packaged into vesicular structures that are transporteddown axons (Yamazaki et al., 1995). Transport is blocked byanti-sense oligos against kinesin I (Amaratunga et al., 1993;Ferreira et al., 1992). The new work establishes that the 47 C-terminal residues constituting the cytoplasmic domain of APPinteract directly with the KLC TPRs (Kamal et al., 2000) andthat kinesin I is associated with a defined class of axonallytransported vesicles that contain APP and the transmembraneproteins involved in its proteolytic cleavage (Kamal et al.,2001). That the interaction between APP and KLC is requiredfor APP transport is clear from several experiments. Sciaticnerves from mice lacking one of the three known KLCisoforms (KLC1, the neuronally enriched isoform) transport

less APP than nerves from normal animals (Kamal et al., 2000)and, in Drosophila, deletion of the APP-like (appl) geneproduces the axonal jam phenotype in larvae that characterizeskhc and klc mutants (Gunawardena and Goldstein, 2001).Finally, the fraction of endogenous APP that has beenphosphorylated at Thr 668 by the neuronal kinase CDK5(Iijima et al., 2000) accumulates at the tips of neurites inmammalian neuron-like cells in culture (Ando et al., 1999), andthe localization of this modified APP is abolished byoverexpression of dominant inhibitory KLC constructs (e.g. theTPRs or heptad repeats of KLC) (Muresan et al., 2001). Thisfinding, together with the observation that phosphorylation ofAPP Thr 668 by CDK5 promotes the interaction between theAPP C-terminus and KLC TPRs in vitro (Muresan et al., 2001),supports the hypothesis that the transport of APP requires aninteraction between the cytoplasmic domain of APP and theKLC TPRs. The idea that APP is a kinesin-I-cargo linkerestablishes a precedent for direct interactions between kinesinsand transmembrane proteins and thus suggests that thegeneralized architecture in which kinesins are linked totransmembrane proteins indirectly through soluble scaffolds(see Fig. 1), although common, is not universal.

Still unclear is whether APP and the JIP scaffold are carriedon the same cargo vesicles. Structural studies of the TPR motifindicate that three TPR repeats would be sufficient to bind apartner (Gatto et al., 2000; Lapouge et al., 2000; Scheufler etal., 2000); hence it is conceivable that APP and JIPs bindsimultaneously to the six TPR repeats in a single KLC.Alternatively, a single kinesin I motor could carry JIPs andAPP simultaneously by devoting one KLC to each partner. Arelated question is whether the JNK signaling cascade carriedby kinesin I and APP are involved in the same signalingpathway.

GRIPThe KLC TPRs do not provide the only cargo-binding site onkinesin I. Earlier studies focusing on Neurosporakinesin I,which lacks KLCs, identified a candidate cargo binding regionon the KHC (Fig. 4) (Seiler et al., 2000). Seiler et al. searchedfor functional KHC domains, using KHC deletion mutantcDNAs expressed as transgenes and evaluating their ability torescue the reduced growth rate of a KHC-deficient strain ofNeurospora. They identified in the tail coiled coil a domain of51 residues (Fig. 4) that is highly conserved among KHCs fromdifferent species. In Neurospora, this region is essential forlocalizing tagged KHC constructs to small vesicles destined forsecretion at the hyphal tip (Seiler et al., 2000). Setou et al.subsequently used this region in a yeast two-hybrid screen ofmouse brain cDNAs (Setou et al., 2002) to identify theglutamate receptor interacting protein 1 (GRIP1), a knownmulti-PDZ-domain protein at synaptic junctions (Dong et al.,1997; Srivastava et al., 1998), as the kinesin I partner. A regionof GRIP1 between the sixth and seven PDZ domains binds toa region of KHC that overlaps the cargo-binding domaindefined originally in Neurospora(Fig. 4).

GRIP1, and the related protein GRIP2, scaffold severalneuronal signaling proteins (Wyszynski et al., 2002). Inparticular, the fifth GRIP1 PDZ domain interacts with the C-terminal sequence (-ESVKI) of subunits 2 and 3 (GluR2/3) ofAMPA-sensitive glutamate receptors (Wyszynski et al., 1999),

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which mediate excitatory synaptic transmission in the brain.Indeed, GRIP was initially identified through this interaction,and a large fraction of GluR2 exists as a complex with GRIP(Wyszynski et al., 1999). Although its exact function is unclear,GRIP presumably contributes to the localization, atpostsynaptic densities, of a large multi-protein complex thattransduces presynaptic activity into postsynaptic responses.

GRIP1, GluR2 and kinesin I are colocalized within thedendrites and cell bodies of cultured hippocampal neurons, andco-immunoprecipitate from vesicle fractions (Setou et al.,2002). That the interaction between kinesin I and GRIP1 isfunctionally important for trafficking of AMPA-senstitiveglutamate receptors is evident from dominant inhibitoryexperiments: overexpression of the KHC GRIP1-binding sitereduces the amount of both GRIP1 and GluR2 in dendrites.

The work described above provides further support for ageneralized model of kinesin-dependent trafficking ofsignaling modules that involves vesicles and soluble scaffolds.It also raises interesting questions regarding the logic of thistrafficking. Using binding sites on KHC, kinesin I carriesAMPA-sensitive glutamate receptor vesicles within dendrites,but using binding sites on KLCs it carries JIP or APP vesiclesin axons. How does kinesin I, which is distributed uniformlyin both axons and dendrites, transport some cargo vesicles todendrites and others to axons? One interesting proposal is thatoccupation of a particular cargo-binding site directs the motorto distinct microtubules in axons and dendrites (Setou et al.,2002).

One cargo – multiple kinesins?The interaction between GRIP and kinesin I is apparently notthe sole means of delivering AMPA-sensitive glutamatereceptors to postsynaptic densities. GRIP is also carried byKIF1A (Lee et al., 2002) indirectly through the neuronalscaffold protein Liprin-α. The sixth PDZ domain of GRIPinteracts with the Liprin-α C-terminus (Wyszynski et al.,2002), and the Liprin-α N-terminal coiled coil interacts withthe KIF1A tail between residues 650 and 1105 (Lee et al.,2002). This KIF1A region overlaps with a recently identifiedprotein-binding domain termed the MAGUK-binding stalkdomain or MBS (Asaba et al., 2003) (Fig. 4), which warrantsan explanation before we return to the transport of AMPA-sensitive glutamate receptors by KIF1A.

In addition to their presence in non-motor proteins, MBSdomains are found in all members of the KIF1 subfamily ofkinesins (Asaba et al., 2003), next to their FHA domains (Fig.4). The MBS domain was originally identified in GAKIN(Hanada et al., 2000) – the human homologue of KIF13b –through its interaction with the protein human disks large(hDlg) (Asaba et al., 2003). Dlg is a member of the membraneassociated guanylate-kinase MAGUK family of scaffoldproteins, which are composed of one or more PDZ domains,an SH3 domain and a guanylate kinase-like (GUK) domain thatlacks enzymatic activity and instead functions as a protein-binding module (Anderson, 1996). In humans, the KIF13bMBS interacts with the GUK domain of Dlg (Asaba et al.,2003). This interaction is extremely interesting because Dlgand other MAGUK proteins localize signaling complexes atspecialized membrane sites such as tight junctions and synapticjunctions (Muller et al., 1996). For example, DrosophilaDlg

and its orthologues scaffold large protein complexes at pre- andpostsynaptic membranes and at the basolateral membrane ofepithelial cells (Lue et al., 1994). The scaffold protein LIN 2(Fig. 2) is also a MAGUK protein, and as discussed above, inC. elegansit localizes an EGF receptor at tight junctions andglutamate receptors at synaptic junctions. The finding thatMBS domains exist in all KIF1 kinesins, and the demonstrationthat the human KIF13b-MBS–Dlg interaction is needed tomaintain Dlg localization in MDCK cells (Asaba et al., 2003),establishes a relationship between KIF1 kinesins and theMAGUKs. The finding that the Liprin-α-binding site overlapsthe MBS in KIF1A suggests that the MBS also interacts withscaffold proteins that lack GUK domains. Thus, one canimagine that the MBS of KIF1A, like the KLC TPRs of kinesinI, interacts with more than one type of cargo linker.

The GRIP–Liprin-α interaction is clearly necessary forlocalization of AMPA-sensitive glutamate receptors atpostsynaptic sites on the dendrites of cultured hippocampalneurons, as overexpressed GRIP or Liprin-α constructs lackingthe interaction sites diminish the population of localizedAMPA-sensitive glutamate receptors (Wyszynski et al., 2002).The evidence that KIF1A transports glutamate receptors on theLiprin-α–GRIP scaffold is less direct. Liprin-α accumulates inthe cell bodies of hippocampal neurons that overexpress aKIF1A construct lacking the motor domain (Lee et al., 2002).One presumes localization of AMPA-sensitive glutamatereceptors would be diminished, but this has not been showndirectly. Nevertheless, these studies again provide evidence fora signaling module carried as vesicular cargo by a kinesin.Because GRIP and Liprin-α have multiple binding sites forother proteins, this scaffold complex could in principle holdcomponents of a signaling pathway downstream of theglutamate receptor or other receptors that interact with GRIP(reviewed in Wyszynski et al., 2002).

The implication that GRIP is carried by both KIF1A andkinesin I raises several questions. Do KIF1A and Kinesin Imediate parallel, functionally redundant transport pathways forthe AMPA receptor? Or is AMPA receptor delivery achievedin two consecutive transport steps – might, for example, onemotor transport the cargo from the Golgi apparatus to theprimary dendrite, but the other mediate localized traffickingwithin the dendritic tree? Assuming GRIP is carried by KIF1Awithin dendrites, how does this one, ubiquitously distributed(Lee et al., 2002), motor also deliver synaptic vesicleprecursors within axons (Hall and Hedgecock, 1991;Yonekawa et al., 1998)?

Summary and perspectiveThe recent progress on kinesin-cargo interactions provides abasis for understanding how kinesins interface with otheraspects of cell biology, particularly protein trafficking andsignal transduction. At these interfaces, a number of generalprinciples have emerged. It now appears that many cellularmolecules have adapted through evolution to serve as kinesin-cargo linkers. In several cases, modular protein-proteininteractions govern these interactions. With one exception(APP), identified kinesin-cargo linkers are soluble scaffoldproteins whose multiple binding sites organize functionallyrelated proteins into a single cargo. These propertiessignificantly broaden our expectations about the kinds of cargo

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carried along microtubules by kinesin motors, and therebydeepen our understanding of biological processes such asintercellular signaling. For example, the new work indicatesthat scaffolds for signaling pathways not only direct the flowof information in the cascade, but control trafficking andlocalization of the cascade through their interactions withkinesin tails. The findings that a single kinesin (e.g. kinesin I)has multiple cargo-binding sites and can carry diverse cargo,together with the idea that one molecule (e.g. AMPA receptor)can be cargo for different kinesins (KIF1A and kinesin I),suggest an unanticipated degree of plasticity to proteintrafficking by motor proteins. The future is now rich withquestions, whose resolution is likely to change our ideas aboutintracellular trafficking fundamentally. Chief among these isthe question of exactly how and where cargoes are assembled,and the regulatory principles by which cargoes activate, and letgo of, particular kinesins at the right time and place.

I am indebted to the members of my laboratory, particularly VirgilMuresan (now at Case Western) for many helpful discussions.Research in my laboratory is supported by grants from the NIH anda grant from the Human Frontiers Program.

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