Cyclin-dependent protein kinase 5 (Cdk5) and the regulation of neurofilament metabolism

M I N I R E V I E W

Cyclin-dependent protein kinase 5 (Cdk5) and the regulation ofneurofilament metabolism

Philip Grant, Pushkar Sharma and Harish C. Pant

Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA

Cyclin-dependent kinase 5 (Cdk5), a complex of Cdk5 and

its activator p35 (Cdk5/p35), phosphorylates diverse

substrates which have multifunctional roles in the nervous

system. During development, it participates in neuronal

differentiation, migration, axon outgrowth and synapto-

genesis. Cdk5, acting together with other kinases, phos-

phorylates numerous KSPXK consensus motifs in diverse

cytoskeletal protein target molecules, including neurofila-

ments, and microtubule associated proteins, tau and MAPs.

Phosphorylation regulates the dynamic interactions of

cytoskeletal proteins with one another during all aspects

of neurogenesis and axon radial growth. In this review we

shall focus on Cdk5 and its regulation as it modulates

neurofilament metabolism in axon outgrowth, cytoskeletal

stabilization and radial growth. We suggest that Cdk5/p35

forms compartmentalized macromolecular complexes of cyto-

skeletal substrates, other neuronal kinases, phosphatases and

activators (`phosphorylation machines') which facilitate the

dynamic molecular interactions that underlie these processes.

Keywords: neurofilament, Cdk5, phosphorylation,

cytoskeleton, neuron

H I S T O R I C A L I N T R O D U C T I O N

Cdk5 is a unique member of the family of cyclin-dependentkinases that regulate the progress of proliferating cellsthrough the cell cycle. Though Cdk5 is found at low levelsin proliferating cells, bound to cyclin D, it is inactive inthe cell cycle. Rather, it is most active in post mitotic cellssuch as neurons [1]. Originally, Cdk5 was identified andcloned from Hela cells as a PSSALRE kinase with a 57%sequence homology to cdc2 [2]. It has been cloned from awide variety of species including bovine brain [3,4], ratbrain [5] porcine brain [6], Xenopus and zebrafish [7], B. S.Li et al. unpublished results), all showing a remarkable99% sequence identity, suggesting a high degree ofconservation. Cdk5 is distributed in most tissues andvarious cell lines [1], active primarily in neuronal tissues,because of its association with neuron-specific activatorssuch as p35 [8] or its truncated forms, p29, p26, and p25

[6,9,10]. Other regulatory proteins, p67 and p39, are alsoexpressed primarily in the nervous system during develop-ment and in the adult [11±13]. As for cyclins, it is generallyassumed that these regulatory proteins serve to target Cdk5to specific substrates and sites within the cell [14]. Theubiquitous distribution of Cdk5, however, particularly intissues that do not express any of the above regulatoryproteins, suggests that other unidentified regulatory proteinsare to be found.

Cdk5, by virtue of phosphorylating diverse substrates,has a multifunctional role in the nervous system. In additionto its role in muscle and neuronal differentiation [15,16], italso contributes to the assembly, organization and stabilityof the axonal cytoskeleton in vertebrate neurons [17],promotes neurite outgrowth and axon guidance in devel-oping neurons (see the other two Minireviews in thisseries), participates in the regulation of synaptic transmis-sion [18] and provides an additional pathway to cellularapoptosis [19]. For some functions, the relevant targetsubstrates have been identified, particularly neurofilaments,tau and MAPs, principal elements of the axonal cyto-skeleton. Table 1 summarizes most of the identified Cdk5target substrates in the nervous system and their putativefunctions.

Chromatographic purification of Cdk5 relied on neuro-filament proteins as substrates because they containnumerous Lys-Ser-Pro (KSPXK) repeats (8±58 dependingon species), the specific consensus motif of cdc2 kinases[3,4,20,21]. Using dephosphorylated human NF-H assubstrate it was demonstrated that Cdk5 phosphorylatedthe tail domain, enough to shift the electrophoretic mobilityto the native phosphorylated state of phospho-NF-H [87].Moreover, the phosphorylated NF-H was particularlyreactive with antibodies that were specific for phos-phorylated KSP epitopes, found exclusively in axons ratherthan cell bodies [22], indicating that it was indeed the tail

Eur. J. Biochem. 268, 1534±1546 (2001) q FEBS 2001

Correspondence to H. C. Pant, LNC, NINDS, NIH, Bldg.36/Rm

4D-04, NIH, Bethesda, MD 20892, USA, Fax: 1 301 496 1339,

Tel.: 1 301 402 2124, E-mail: [email protected]

Abbreviations: ALS, Amyotrophic lateral sclerosis; BPAG1, Bullous

pemphigold antigen 1/dystonin; CAK, Cdk-activating kinase;

CAMKII, Ca12/calmodulin-dependent kinase II; CKI and CKII, casein

kinases I and II; DRG, dorsal root ganglion; Erk1/2, extracellular

signal-regulated kinases 1 and 2; GSK3, glycogen synthase kinase-3;

KSP, Lys-Ser-Pro; MAP, microtubule-associated protein; MAPK, MAP

kinase; MARK, MAP/microtubule affinity-regulating kinase; MT,

microtubule; NF-L, NF-M, NF-H, low, medium and high molecular

mass neurofilament proteins; PKA, cyclin-AMP-dependent protein

kinase; PKC, Ca12/phosphadidyl-dependent kinase; PP2A, protein

phosphatase 2A. SAPK, stress-associated protein kinase; BDNF,

brain-derived neurotrophic factor.

(Received 2 November 2000, accepted 12 January 2001)

domain KSP repeats that were phosphorylated. Finally, apossible functional role in the assembly of the axonalcytoskeleton was shown by the abolition of the high affinitybinding of dephosphorylated NF-H with microtubules byCdk5 phosphorylation of NF-H [17]. It is understandablethat this kinase activity, so widely conserved in thenervous system, was identified early on as a neuro-filament kinase or tau II kinase [3,23], and was suggestedto play an important role in many aspects of neuronaldevelopment and function. In this review we focusprimarily on its role in processing and regulation ofneurofilament protein assembly, phosphorylation andtransport within neurons.

N E U R O F I L A M E N T P R O T E I N S A N D T H EA X O N C Y T O S K E L E T O N

Vertebrate neurofilament proteins, NF-L (68 kDa), NF-M(115 kDa) and NF-H (190 kDa) are members of a family ofClass IV intermediate filaments, the 10 nm filaments,intermediate in size between microfilaments and micro-tubules. They are expressed primarily in neurons,assembled as heteropolymers into longitudinally arrangedfilaments, in some cases, as in large diameter axons,making up the bulk of axonal protein. Together withmicrotubules, microtubule associated proteins (tau andMAPS 1B and 2), actin and associated motor proteins, theymake up the dynamic axonal cytoskeleton. NF proteinsshare homologies with other intermediate filaments in therod coiled-coil domains, but differ in the length of theirC-terminal tail domains, with none in (NF-L) or variablenumbers of KSP repeats in NF-M/H. NF-H is probably oneof the most extensively phosphorylated proteins with over50 potential acceptor serine/threonine sites, with most inthe long C-terminal tail domain in KSP repeat motifs [24].Biochemical and mass spectrometric analyses of native

NF-H in rats [25], humans [26,27] and squid [145] revealsthat most, if not all KSP sites, are phosphorylated in vivo. Itshould be noted that neurofilaments are heterogeneous,their subunit proteins express different levels of phos-phorylation, even within the same axon. Moreover, the levelof phosphorylation also varies between different axonalbundles and nerve tracts, depending on the state of phos-phorylation of NF-M or NF-H [28]. Large diameter axonscontain the most extensively phosphorylated NF-H; smalldiameter axons such as parallel fibers in the molecular layerof the cerebellum may lack NF-H or express low levels ofphosphorylation [22].

Although NF proteins are synthesized within cell bodies,post-translational phosphorylation of NF-M and NF-H taildomains is topographically regulated [29]. A transientphosphorylation of NF-L head domain sites (Ser55 in NF-Land Ser44 in NF-M) within cell bodies may preventassembly into neurofilaments [30,31]. Although NF-Lalone can assemble into filaments in vitro NF-M and, to alesser extent NF-H, are essential for filament formationin vivo; the NFs are obligate heteropolymers [32,33].Moreover, it is the tail domains of NF-M and NF-Hsubunits that form the cross-linking sidearms seen in vivo[34]. NF oligomers migrate into the axon hillock whereassembly into neurofilaments begins. As they are trans-ported within the axon, phosphorylation of tail domain KSPrepeat motifs occurs [35] and the C-terminal domains aretransformed into projecting sidearms which interact withone another and with other cytoskeletal proteins to producea stable axonal lattice [36].

In spite of decades of study of slow axonal transport ofneurofilaments, the issue is still quite controversial [37,38].For example, it is still uncertain whether monomers,oligomers or polymers (or perhaps all, at different times)are transported. Nor is it clear that transport is indeed acontinuous slow process instead of rapid, intermittent,

Table 1. Cdk5 substrates with XS/ TPXK consensus sequences and their putative role in nerve cell function. Note for the most part the precise

sequence motif phosphrylated by Cdk-5 in each substrate has not been determined.

Substrate Abbreviation Putative function Reference

Amphiphysin Synaptic transmission [18�]

Adenomatous polyposis coli

(tumor suppressor protein)

APC Microtubule binding protein

Fast axon transport

[137�]

b-Amyloid protein precursor bAPP ? [138]

c-Src tyrosine kinase c-Src ? in neuronal cells [139]

Dopamine and cyclicAMP-regulated DARPP-32 Dopamine signaling [140]

phosphoprotein

Histone 1 H1 Generic substrate

JUN Kinase JNK3 Cell survival Unpublished resultsa

Microtubule-associated protein MAP 1B Neurite outgrowth [130]

Map kinase kinase1 MEK1 Apoptosis Unpublished resultsb

Neurofilaments NF-M & NF-H Axonal structure and radial growth [84�]

PAK I kinase PAK Actin-cytoskeleton dynamics [141]

Cdk5 activator P35 Autoregulation [105]

Munc18 P67 Synaptic transmission [142]

Synapsin Synaptic transmission [143]

Tau Microtubule bundling [144]

a B. S. Li, L. Zhang, S. Takahashi, W. Ma, Q. Su, Veerama, A. B. Kulkarni & H. C. Pant. b P. Sharma, Veerama, M. Sharma, N. D. Amin, R. K.

Siheg, P. Grant, N. Ann, A. B. Kulkarni & H. C. Pant.

q FEBS 2001 Cdk5 and neurofilament metabolism (Eur. J. Biochem. 268) 1535

asynchronous spurts and pauses [39,40]. More than onecomponent may be involved, a stable stationary component(the MT-NF cytoskeletal network) which exchanges withmore mobile NF oligomer/polymer precursors, driven ascargo by microtubule associated motors such a kinesin ordynein [41]. On the other hand, NFs may ride `piggy back'on a migrating MT `train'. What is evident is that the fate ofNFs is closely bound to the microtubules, an associationthat is regulated, in part, by Cdk5 mediated phos-phorylation, as we have already seen [17], although otherkinases (GSK3, Erk1/2, SAPK) are not precluded. Trans-port is most rapid during neurogenesis and slows into theadult as axons undergo radial growth and myelination,when NF-H phosphorylation is at its peak. Phosphorylationof KSP repeats in NF-H tail domains seems to regulate therate of transport as axons contact targets and synaptogeneisbegins [35,42]. The rate of transport is indirectly correlatedwith the level of NF phosphorylation [44]. Studies oftransport of different 32-P labeled isoforms of NF-M andNF-H in optic nerves, showed that the least phosphorylatedisoforms undergo the most rapid transport, presumablythose most likely bound to MTs. This is consistent with amodel of NFs being transported as a hypophosphorylatedcargo by MT-associated motors [43]. In the complexequilibria between dephosphorylation and phosphorylationthat regulate transport of cytoskeletal molecules andorganelles, it becomes essential to identify the kinases,phosphatases and their specific targets within the axonalcytoskeleton.

N E U R O F I L A M E N T P H O S P H O R Y L A T I O NA N D R A D I A L G R O W T H O F A X O N S

Radial growth of axons intensifies after synaptogenesis andmyelination are initiated and it is during this period thatrobust NF-H phosphorylation occurs. A number of studiessuggest that radial axonal growth depends on NF-H andNF-M tail domain phosphorylation of NF-H and NF-M taildomains in the KSP repeat regions. This, in turn, leads toincreased net negative charge, C-terminal sidearm forma-tion, enhanced NF spacing and crossbridging to othercytoskeletal components [36,44,45].

Recently, the production of various neurofilament`knockout' and transgenic mice suggests that sidearmphosphorylation of NF-H and NF-M contributes only inpart to the radial growth of large axons [46]. Radial growthof axons may correlate with neurofilament number whichrelies more on NF-L and NF-M expression, as both aremore important to filament assembly than NF-H. Forexample, a mutation of NF-L in quail results in a `quiver'phenotype where axons have few or no neurofilaments andreduced axon caliber [47]. The targeted deletion of theNF-L gene also results in a loss of neurofilaments and areduction in axon caliber [48]. Deletion of the NF-M genealso induces a dramatic 50% reduction in axon caliber butthis too may be an effect of reduced filament number asNF-L was also drastically reduced [49]. Other dataindicate that the levels of each NF subunit interactstoichiometrically, thereby defining axonal cytoskeletalstructure.

But what role, if any, does phosphorylation play,particularly phosphorylation of NF-H sidearms? Surpris-ingly, various NF-H null mice that have been produced

yield conflicting results as the phenotypes differ signifi-cantly [50±52]. Although all studies demonstrate no effecton transcription of NF-L or NF-M, two show that acompensatory increase in NF-M protein and tubulin leavesaxonal caliber unchanged, suggesting that NF-H, and itsphosphorylation is unessential for radial growth of axonsand can be compensated by NF-M. It may, however, effectthe survival of large motor and sensory axons compared tosmaller axons [51,52]. On the other hand, a significanteffect on axon caliber of some axon tracts was seen in thethird study, implicating NF-H [50]. As this latter study wascarried out on older mice (one month), it is possible thatlarge, normally NF-H enriched axons degenerate later,when most NF-H fails to be expressed in these mice.

It is evident that assembly of axonal cytoarchitecture is acomplex process, involving dynamic interactions betweenphosphorylated sidearms on NF-H and NF-M with micro-tubules, actin, and other NF associated proteins such asplectins [53], one of which, BPAG1, is found in neurons[54]. Plectins, long filamentous molecules (500 kDa) cross-link microtubules to intermediate filaments and to actin inmost cells [38,55]. A BPAG1 knockout mouse displaysmarked degeneration of motor neuron axons, reduction ofneurofilaments and the accumulation of phosphorylatedNFs in perikarya of the DRG and ventral motor neurons, apathology resembling the cell bodies of some neurodegen-erative disorders such as ALS [54]. The binding of plectinsto intermediate filaments (vimentin) in mitotic cells is adynamic process, regulated by phosphorylation of a specificKTPXK site in the C-terminal repeat domain of plectin by acdc2 kinase [56,57]. Moreover, phosphorylation by PKA orPKC may dissociate plectin from its attachment to anintermediate filament and promote its association withactin, a shift from a stable to a more dynamic cytoskeleton.Plectin seems to function more than a scaffolding in thecell cytoskeleton; it also regulates actin dynamics duringcell motility [58]. It is interesting to speculate that suchchanges in the structure of the axonal cytoskeleton could beregulated, in part, by Cdk5 phosphorylation which alsorecognizes the KTP motif found in plectin, or its neuralhomologue, BCAP. Regulated phosphorylation/dephos-phorylation of various components of the axonal cyto-skeleton may shift dynamic changes associated with axonalelongation and guidance to stabilization of the cytoskeletonand radial growth associated with synaptogenesis andmyelination. Although phosphorylation of NFs may con-tribute to interfilament spacing, significant radial growth oflarge axons depends on the assembly of microtubules,neurofilaments and actins into a dynamic, space fillingcytoskeletal lattice by plectin-like linker proteins [55,59].

N E U R O F I L A M E N T A S S O C I A T E DK I N A S E S

One could predict that large macromolecules such asneurofilament proteins, with multiple phosphate acceptorsites, would be phosphorylated by several different kinases.Such large molecules would be likely to undergo sequentialmultisite phosphorylation by synergistically interactingkinases [60]. In this view, phosphorylation at one site byone kinase would increase rates of phosphorylation of othersites by a different kinase. For example, tau, prephos-phorylated by a number of nonproline directed kinases such

1536 P. Grant et al. (Eur. J. Biochem. 268) q FEBS 2001

as PKA or CKI, markedly increased GSK3 phosphorylationof tau [61]. Likewise Cdk5 and GSK3 interact synergisti-cally in tau phosphorylation. Consequently, we find thatpreparations of NF proteins isolated from mammalian andsquid neural tissues (e.g. axoplasm from the giant axon), docontain active endogenous second messenger dependentkinases such as PKA, PKC, and CAMK II [62±71]. Thesekinases, preferentially phosphorylating head domainacceptor sites, may regulate neurofilament assembly [31].Presumably, for assembly to occur, the site is dephos-phorylated as the NF subunits are transported into the axon[29].

Second messenger independent kinases such as CKI andCKII are also firmly bound to NF preparations frommammalian and squid nervous tissues. In vitro, theyactively phosphorylate Ser/Thr residues in the glutamicacid rich region of the C-terminal tail domain shared by allthree subunits [62,64,68,72±74]. It has been suggested thatCKI phosphorylation at these sites, adjacent to a conservedsequence motif found in all NF subunits, from lamprey tohumans, may regulate dynamic exchanges between NFoligomers and polymers during axonal transport [75].

N E U R O F I L A M E N T- A S S O C I A T E DP R O L I N E D I R E C T E D K I N A S E S

It is now evident that two families of proline directedkinases, the cyclin-kinases including Cdk5 and the MAPkinases such as Erk1/2, SAP and p38 kinases are theprincipal kinases that phosphorylate the multiple KSP sitesin the NF-M and NF-H tail domains [76±86]. In vivophosphorylation of the NF-H proteins by Cdk5 has beenshown to occur in transfection and antisense studies. Cotransfection of COS cells with rat NF-H and Cdk5/p35, orGSK3 showed that both enzymes phosphorylated NF-H atdifferent sites [80]. Only Cdk5/p35, however, altered theelectrophoretic migration of NF-H to that of brain NF-Hsuggesting that the KSPXK repeat sites were phos-phorylated. This was confirmed by an independentcotransfection study with rat NF-H and Cdk5/p35 in thehuman SW13cl2Vim2 cell line [85]. The41 KSPXY repeats(largely of the KSPXXXK motif ) between residues 508 and763 of rat NF-H were not phosphorylated by Cdk5/p35,whereas the specific KSPXK motifs in the constructcontaining residues 769±1072 were phosphorylated with aconcomitant shift in electrophoretic mobility to that of thenative phosphorylated NF-H. The specificity of Cdk5 forthe KSPXK motif was further demonstrated in an in vitrocomparison of Cdk5 and Erk2 phosphorylation of differentKSP containing substrates [86]. Here it was shown that theKSPXXXK motif, which predominates in the tail domain ofrat NF-H, is preferentially phosphorylated by Erk1/2 whileCdk5, which requires an adjacent basic residue, has agreater preference for the KSPXK motif. This explains whythe human NF-H, with its 34 KSPXK repeats with basicresidues is a preferred substrate for Cdk5 and exhibits asignificant electrophoretic shift after phosphorylationcompared to rodent NF-H [87].

R E G U L A T I O N O F C D K 5 A C T I V I T Y

An excellent extensive review of cyclin-dependent kinasesregulation in the cell cycle at the level of molecular

structure of catalytic and regulatory subunits, and activa-tion/inhibition by phosphorylation has been published [14].Though information about Cdk5 regulation in postmitoticcells is much less understood, some regulatory models ofcell cycle Cdks may apply.

Cdk5 is a unique member of the family of cyclin-dependent kinases; though ubiquitously distributed in mosttissues, including proliferating cells, it is functionalprimarily in post mitotic neuronal tissues because theyspecifically express the noncyclin activator proteins, p35,p39 and such proteolytic fragments of p35 as p25, and p21.Moreover, unlike most cdc2 kinases, Cdk5 does not have tobe activated by phosphorylation at a specific site (S159,equivalent to T160 in cdc2 kinase), by a CAK kinase forfull kinase activity, nor phosphorylated or inhibited by aTyr15 specific Wee1 kinase, nor is kinase activity inhibitedby known cdc2 inhibitors, p21cip/WAF1 or p27kip1. This wasconfirmed in reconstitution studies using fusion proteins oftruncated p21, 23,25 as well as p35 with the catalytic Cdk5subunit; activity is restored in the absence of any otherkinase activity [88]. Analyses of reconstituion assays usinga group of systematically fragmented mutant forms of p35have shown that the active domain of the p35 activator forCdk5 is similar in structure to the cyclin A activationdomain, spanning residues 150±291 in the total sequence of300 amino acids [89]. The region of the cyclin fold thatbinds to Cdk5 may lie between residues 135±227.Independently, another laboratory dissecting the moleculeeven further, demonstrated that two sites were essential forfull activation of Cdk5, a high affinity binding site betweenresidues 150±200, and a short sequence between 279 and291 at the C-terminal end, required for full activation [90].A `claw model' of activation binding of p35 to Cdk5,involving a binding domain and activation loops at the C-and N-terminal ends, has been proposed, based on a seriesof mutant studies in our own laboratory (N. D. Amin, R. W.Albers & H. C. Pant, unpublished results). In all studies, itwas clear that p25 and p21 were more active regulatorsin vitro than the intact p35.

Although these recombination studies support the claimthat Cdk5 activation is completely independent of anyadditional phosphorylations at such sites as Ser/159-Thr160, either by other kinases or autophosphorylation, itappears that activation can be significantly enhanced bothin vivo and in vitro by site-specific phosphorylation ofCdk5 by kinases in PC12 extracts [91]. PC12 cells,incubated with inorganic 32P, disclosed that Cdk5 wasindeed phosphorylated. A bacterially expressed Cdk5/p25complex, phosphorylated by a PC12 extract in vitro, alsoexhibited a five-fold increase in H1 phosphorylationactivity. The data also showed that phosphorylation ofCdk5 by the crude PC12 lysate markedly increased its rateof activation by p25. A telling point is that mutation of theSer159 in the T-loop domain of Cdk5 inhibited theactivation by the lysate. As seems to be true for cdc2kinases, Ser159 is the principal site for Cdk5 activation andits phosphorylation in the T-loop also acts to further openthe catalytic site and thereby enhance catalytic activity.Finally, in vitro studies with CKI and CKII phosphorylationof recombinant Cdk5, both kinases specifically targetingthe serine/threonine, glutamic residues following Ser159 inthe T-loop, pointed to CKI as a possible in vivo candidatefor the site-specific phosphorylation of Cdk5 [91]. This is


consistent with the studies showing CKI as one of theprincipal neurofilament associated kinases, and leads oneto speculate that in the macromolecular complexes ofcytoskeletal proteins and kinases, such regulatory phos-phorylations would be facilitated (see below).

What about the substrate binding site for Cdk5? What dowe know about its structural basis? As mentioned above,the preferred phosphorylation site for Cdk5/p35 within thetail domain of NF-H are the KSP repeats within the specificKSPXK motif, which explains why human NF-H is a bettersubstrate than rat NFH with the latter having many moreKSPXXXK repeats than KSPXK. To explore the structuralbasis for this specificity, circular dichroism studies coupledto NMR analysis of KSPXK and KSPXXXK peptides werecarried out and revealed that a two KSPXK repeat peptideformed a stable double S-like bend in the central residues[92]. It suggested that a transient loop like structure at thespecific KSPXK motif was essential for phosphate transferby Cdk5. Subsequently, in the absence of any crystalstructure for Cdk5, the structural requirement of Cdk5 for abasic residue at the n13 position in the KSPXK motif wasanalyzed by site directed mutagenesis, enzyme kinetics andmolecular modeling of Cdk5 structure in association withan H1 peptide [93]. Based on known kinase crystalstructures, mutated Cdk5s were prepared by substitutingAla at Asp acidic sites presumed to bind the lysine residuein the substrate peptide to determine its effect on enzymekinetics. Asp86, within the C-lobe of Cdk5, close to theATP binding pocket, was identified as a principal site forbinding the enzyme to the n13 lysine in the KSPXK motif,stabilized by salt bridges between Lys8 and Lys9 of thesubstrate to Asp91 of the Cdk5.

Substrate specificity of cell cycle Cdks is conferreddirectly by the specific cyclins or indirectly, by virtue of thelocation of the cyclin in the cell [14]. Factors controllingwhen and where Cdk5 forms active complexes with itsregulatory proteins, or their isoforms created by proteolyticcleavage also regulate Cdk5 activity. Cdk5 exists in threeforms within bovine brain extracts, as a monomer that canbe activated by p21 (a p35 fragment), an active Cdk5/p25complex, and as part of a large macromolecular complex(650 kDa) containing p35, only activated during gelfiltration, as if an inhibitor was released [94]. To identifythe proteins within the macromolecular complex by yeasttwo-hybrid studies, three smaller proteins (24, 57, and66 kDa) were cloned which bound to the activator domainin p35 at a common site [95]. The site is also within the p35domain essential to Cdk5 activation. The significance ofthese proteins to p35 function is not clear, however,although two of these are phosphorylated by Cdk5.

A yeast two-hybrid analysis has also demonstrated thatp35 is bound at a specific site in the C-terminal region ofthe rod domain of NF-M from human brain, close to theKSP sites phosphorylated by Cdk5 [96]. The Cdk5/p35complex also binds to the same site in vitro and suggeststhat p35 may act as a substrate-targeting protein for Cdk5within the axon [96]. P35 also targets Cdk5 to microtubulesvia interaction with tau, a key microtubular associatedprotein in axons [97]. Cdk5/p35 is bound to isolatedmicrotubule preparations from bovine brain, an associationthat depends on tau. Disassembly of microtubules by tauphosphorylation releases Cdk5/p35 in the form of a 450-kDamacromolecular tau complex. Moreover, Cdk5/p35 cannot

be reconstituted with microtubules in the absence of tau,which targets the kinase to the tau binding site onmicrotubules.

Cellular localization of p35, however, is more complex,depending on developmental state and neuron type [13,98].It now appears that intracellular localization of p35 alsodepends on factors regulating its degradation in a mannersimilar to the proteolytic regulation of cyclins during thecell cycle [14]. The dynamic on-off switches of kinaseactivities that drive the mitotic cycle depend, in large part,on ubiquitin-mediated proteolytic degradation of specificcyclins. A large macromolecular complex known as thecyclosome acts to catalytically transfer ubiquitin to variousmitotic substrates including cyclins [99]. A recent reportsuggests that p35 in neurons is bound to the membranewhereas p25, a proteolytic fragment and more activeregulator, is soluble, found in the cytosol [100]. Thisderegulation of Cdk5 by proteolytic cleavage of p35 to p25correlated with a shift in intracellular localization withinthe neuron, may play a role in some neuro-degenerativedisorders, including Alzheimers. Overexpression of humanp25 in transgenic mice results in hyperphosphorylation ofcytoskeletal proteins tau and neurofilaments, which accu-mulate as filamentous aggregates in cell bodies and axonsin specific brain regions in a pattern resembling neurons inAlzheimer brains [101]. Activation of Cdk5 by cleavingp35 into an abnormally active p25 fragment is one possibleexplanation for these results. As no Cdk5 assays werecompleted in these studies, alternative explanations arepossible including the action of Cdk5 on other kinasesand/or deregulation of phosphatases.

Proteolytic cleavage of p35 to p25 in vivo and in vitrocan be accomplished by calpain, the Ca dependentprotease [102]. The cleavage changed the solubility of theCdk5/p35 complex, from the particulate (membrane)fraction to the soluble compartment, where p25 pre-dominates. But this may be an abnormal response toneuronal stress or lesioning. It should be noted thatincreased cleavage of p35 correlates with neuronal celldeath, consistent with the view that the more active Cdk5/p25 complex can induce pathological lesions in cytoskeletalproteins. Neuronal responses to hypoxic stress, excitotoxinsand calcium influx, conditions leading to neuronal death,are correlated with calpain-mediated cleavage of p35 to p25and relocalization [103,104]. Inhibition of calpain or ofCdk5 catalytic activity reduces the level of cell deathseen in cultures of amyloid-beta peptide treated corticalneurons.

It is uncertain whether the conversion of p35 to p25 is anormal mechanism of Cdk5 deregulation in neurons. p35is much less stable than the p25 derivative; normally, it israpidly degraded in cortical neurons, a process that isinhibited by various proteosome inhibitors [105]. Removalof the amino-terminal p10 domain of p35 results in theremaining more stable p25 fragment. This suggests that p35turnover normally occurs in neurons via an ubiquitin-mediated pathway. Phosphorylation of p35 by Cdk5 atspecific consensus sequences increased the rate of p35degradation, suggesting a negative feedback mechanism ofregulation of Cdk5 phosphorylating activity. As in the cellcycle, does this represent a mechanism for activating/deactivating the Cdk5 complex during such dynamicprocesses as neuronal migration, axon elongation and


growth cone dynamics, where rapid `turn ons' and `shutdowns' are required? Alternatively, the rapid turnover ofp35 may be a normal mechanism to prevent the formationof the more stable, abnormally active Cdk5/p25, whichappears in pathological states. This hypothesis, whichrequires more extensive investigation, predicts the accu-mulation of p25 in various neurodegenerative disorderssuch as AD.

M A C R O M O L E C U L A R C O M P L E X E S A SP H O S P H O R Y L A T I O N ` M A C H I N E S '

Several of the kinases mentioned above (PKA, PKC,CAMKII, CKI), including a Cdc-2 related kinase and aPP2A phosphatase, together with the cytoskeletal proteinstubulin, actin, kinesin, phosphorylated NF220, and MAPshave been extracted from squid axoplasm in the form ofactive multimeric complex(es) by P13suc1 affinity chroma-tography [106,107]. Tubulin, NF220 and MAPs arepreferentially phosphorylated by these endogenous kinasessuggesting that such macromolecular assemblies maypromote conformational changes that facilitate the sequen-tial order of multisite phosphorylations on cytoskeletalproteins by different kinases [60]. Notably, the identicalP13suc1 chromatographic procedure extracted only inactivecomplexes, with lower kinase levels and few cytoskeletalprotein substrates, from the cell bodies of these giant axons[106]. This suggests that cytoskeletal protein phos-phorylation is topographically regulated within the neuronwith cell bodies and axons characterized by compartment-specific multimeric `phosphorylation machines' [106]. Thishypothesis is consistent with the view that phosphorylationof the multiple repeat acceptor sites in tail domains of NFproteins are preferentially phosphorylated within the axonduring transport [38,108]. As indicated previously, most ofthese sites in mammalian and squid NF-H are phos-phorylated in vivo [26,27,145] and occupy sequence motifsspecific for proline-directed kinases. We would thereforeexpect to find such kinases also associated with neuronalcytoskeletal preparations and, indeed, we do.

As indicated in the introduction, cytoskeletal proteinsfrom brain and spinal cord usually copurify with Cdk5during its isolation from neural tissues. For example, partialpurification of Cdk5 from microtubule preparations fromporcine brain were based on its high affinity to tau [109]and recently it has been noted that Cdk5/p35 is targeted tothese preparations by direct association of p35 with tau[97]. Neurofilament preparations from bovine brain are alsofunctionally associated with Cdk5 and its p25 activator[110]. Cdk5/p25 copurifies with neurofilaments andneurofilaments, in turn, are coimmunoprecipitated withCdk5/p25. The neurofilament and tubulin preparationsextracted from rat brain are active multimeric complexescontaining Cdk5 and Erk1/2 together with tau, tubulin, andphosphorylated NF-H which resemble the protein profile ofa P13suc1 extract [111]. This is consistent with the resultcited previously of p35 binding to NF-M in a yeast two-hybrid assay; monomeric p35 as well as Cdk5/p35 bind toNF-M in the C-terminal domain, close to Cdk5 phos-phorylation sites, with no effect on kinase activity. Otherproline-directed kinases may also be included in neuronalcytoskeletal complexes such as MARK, a high molecularmass kinase [112] and a cdc2-like kinase different from

Cdk5 [110]. Such complexes, or `protein machines' arelocalized within cells, bound to membranes or organelles,and facilitate ordered and directional flow of diversecoupled enzyme reactions [113]. For example, signaltransduction systems of the Ras-raf-MAP kinasepathway are macromolecular complexes of sequentiallyinteracting kinases bound by scaffolding proteins [114].Such complexes bring together kinases for specificactivation, sequential signaling and cellular localization ofdifferent signal transduction systems. Similar `phos-phorylation complexes' of substrates, kinases, phosphatasesand their regulators may function in neurons to regulate thedynamics of cytoskeletal protein interactions during axongrowth, synaptogenesis and stabilization. To illustrate, anearly study showed the phosphorylation-dependent bindingof NF-H directly to microtubules [17]. DephosphorylatedNF-H binds to MTs in vitro with relatively high affinity.Phosphorylation of NF-H by Cdk5, however, presumably inthe tail domain, promoted the dissociation of NF-H frommicrotubules. Moreover, binding of NF-H to microtubulescould be reduced by the addition of recombinant taufragments, suggesting that tau and NFs compete for thesame MT C-terminal binding sites. As tau binding tomicrotubules at these same sites is also modulated by tauphosphorylation/dephosphorylation equilibria, we can seehow macromolecular phosphorylation complexes mayoptimize catalytic interactions during assembly of thedynamic axonal cytoskeleton during growth.

P H O S P H A T A S E S R E G U L A T E N FP H O S P H O R Y L A T I O N

The phosphorylated state of cytoskeletal proteins inneurons results from a dynamic equilibrium modulated bykinase and phosphatase activities. Phosphatases do play animportant role in the labile stages of axonal outgrowth andin the more stable phases of mature axon function duringimpulse conduction. We have seen that phosphorylationdisassembles neurofilaments, and dissociates NFs, MAPsand tau from MTs, both in vitro and in vivo. As nativeneurofilaments are abundantly phosphorylated, phospha-tases may be the primary enzymes regulating cytoskeletalprotein interactions, particularly during more labile stagesof axon outgrowth.

An indication of phosphatase regulation of NF assemblyin vivo is seen in the effect of okadaic acid, a specificphosphatase inhibitor on the cytoskeleton of cultured DRGcells [115]. The NFs shifted into the Triton X solublefraction and exhibited a decreased electrophoretic mobilitysuggesting increased phosphorylation. Inhibition of phos-phatase activity enhanced kinase phosphorylation of NF-Land NF-M head (and tail?) domains disassembling andfragmenting neurofilaments, a condition reversed whenokadaic acid was removed. Subsequent studies identifiedphosphatase 2A as the active phosphatase [116]. Thephosphatase is associated with PKA and CAMK incytoskeletal preparations from rat cerebral cortex thatphosphorylate NF-M [117] and is also associated with thecatalytic sub unit of PP1 in NF fractions from bovine spinalcord [118]. More than 75% of the endogenous phosphataseactivity in these fractions was attributed to PP2A. It isspeculated that its function is to preserve the filament


structure of NFs by its regulation of head domainphosphorylation [119].

PP2A was also shown to dephosphorylate the KSP sitesin the tail domain of NF-H that are phosphorylated byCdk5, implicating PP2A in the regulation of sidearmformation and MT interactions [120]. Cdk5 and PP2A maymutually regulate phosphorylation of the KSPXK repeats inNF-M and NF-H, possibly during axonal transport assidearms extend. At the terminals, turnover of NFs isfacilitated by prior dephosphorylation of these sites bycalcineurin, a calcium-calmodulin dependent phosphatasePP2B before digestion by proteases [121].

C D K 5 A N D N E U R O F I L A M E N TF U N C T I O N

Three separate genes code the NF proteins whoseexpression, during development of the nervous system,provides some clues as to NF function. The early stages ofneurogenesis (E10-11) when neurons are actively migratingand extending neurites, are marked by expression of NF-Land nonphosphorylated NF-M in cell bodies and in growingaxons [122]. Only later, after axons have establishedcontact with synaptic targets does NF-H appear followedby robust accumulation of axonal NFs and phosphorylation,usually concomitant with myelination. The extensive phos-phorylation of NF-H and NF-M tail domains by prolinedirected kinases, particularly in large axons, continues intothe adult and is correlated with sidearm formation,stabilization of the axonal cytoskeleton, and radial growthof myelinating axons. The developmental expression ofCdk5 correlates with the expression of phosphorylatedneurofilaments in fasciculated axonal systems [1] whileCdk5 colocalizes with phosphorylated NF-H in axonbundles of the developing rat cerebellum [123]. Differ-entiation of neurons involves a sequence of events,including neuronal migration, neurite outgrowth, axonelongation, guidance, and finally synaptogenesis, radialgrowth and myelination. The question arises as to the roleof NFs in these processes and whether and how Cdk5/p35modulates NF function. In as much as the role of Cdk5/p35in neural migration, axon elongation and guidance arethoroughly reviewed in the accompanying review papers,we shall focus only on those processes in which NFs maybe involved.

N E U R O F I L A M E N T S A N D A X O NO U T G R O W T H

Virtually all NF knockout mice develop into relativelynormal fertile adults with no gross abnormalities inneuronal pathways except for changes in axon number ordiameters [46]. For example, NF-L [48] and NF-M [49] nullmice are phenotypically normal except that axons inNF-L2/± contained no NFs, exhibited reduced radial growthand delayed regeneration. Even NF-M2/± NF-H2/± doubleKO mice showed no gross disturbances in neuronalnetworks which suggests that NFs do not participate inneurite outgrowth, axon elongation or guidance [124]. Onthe other hand, Cdk5 KO and p35 KO mice exhibitmarked defects in neuronal migration, axon elongationand guidance [125,126]. Abnormal aggregates of phos-phorylated NF-H, however, were seen in neuronal perikarya

of the brain stem and ventral spinal cord in Cdk52/± mice[126], and we shall discuss these later. The data suggest thatNFs may not be involved in axon outgrowth and NF taildomain phosphorylation can occur in the absence of Cdk5/p35, probably by the action of other kinases such as MAPKand/or GSK3. Nevertheless, a series of studies support thehypothesis that initial rates and patterns of axon outgrowthdepend upon NF stabilization of the elongating axoncytoskeleton proximal to the actively migrating growthcone. For example, injections of NF-M antibodies into oneblastomere of a two-celled Xenopus embryo restricts theantibody to one half the developing nervous system and onecan compare initial patterns of axon outgrowth betweeninjected and control sides in vivo and in vitro after isolationof injected neural cells [127]. On the injected side, rates ofaxon outgrowth of cranial and motor neurons were delayedand axonal morphologies were altered. No effects ontubulin or actin were noted, however, suggesting that theaxonal support provided by the NF cyto-scaffoldingproximal to the growth cone, supports the elongatingaxon. Although the role of NF phosphorylation was notevaluated in these studies, we can assume, on the basis ofother studies of neurogenesis in vitro, that axonal NFphosphorylation accompanies neurite outgrowth.

Using the SHSY5Y human neuroblastoma cell linewhich expresses human NF-H, the induction of neuriteoutgrowth by retinoic acid correlated with increased phos-phorylation of endogenous NF-H and NF-M without anysignificant change in the endogenous Cdk5 activity [84].However, using an antisense Cdk5 mRNA, which signifi-cantly eliminated Cdk5 activity from retinoic acid treatedcells, also inhibited NF-H phosphorylation and neuriteoutgrowth. An extension of these studies demonstrates thatadhesive interactions between neurons and substrate,mediated via a laminin-integrin signaling pathway, maybe sufficient to activate Cdk5/p35 phosphorylation ofhuman NF-H in SHSY5Y cells [128]. Retinoic acid inducedincreased integrin a1/b1 expression on the surface of thesecells as neurite outgrowth was stimulated. At the same time,phosphorylation of NF-H tail domain KSP sites increasedsignificantly. Both antibody inhibition of integrin expres-sion and specific inhibition of Cdk5 of these cells reducedNF-H phosphorylation and neurite outgrowth. No stimula-tion of MAP kinase activity was observed, suggesting thatCdk5 at least in these human derived cells was the principalkinase phosphorylating KSP tail domain repeats. Thisillustrates a simultaneous expression of the multifunctionalrole of Cdk5/p35 in neurons; it contributes to the phos-phorylation of tail domains of NF proteins, presumably asthey are transported and assembled in the axon into asupporting cytoskeletal lattice, while at the same time, itpromotes neurite outgrowth, presumably acting at growthcones [129]. It is likely that Cdk5/p35 activation and phos-phorylation of MAP1b during laminin-induced axonelongation in primary cultures of rat cerebellar neuronscan also be attributed to MAP1b's role in axon elongationand growth cone motility [130,131]. Evidently, surface±specific adhesive interactions signal cytoskeletal proteinphosphorylation by Cdk5 and, presumably, other kinasessuch as Erk1/2 [82].

The membrane interactions during synaptogenesis asgrowth cones contact their targets has also been shown totrigger NF-H phosphorylation during development in vivo


and in primary neurons in vitro. As has been mentioned, innormal neuronal development, robust phosphorylation ofNF-H in axons seems to occur after they contact theirtargets and synaptogenesis begins [122,132]. A recentin vitro study of rat cortical neurons has confirmed the roleof synaptogenesis and has shown that brain-derivedneurotrophic factor (BDNF) stimulates both Cdk5 activityand NF-H phosphorylation [133]. Cdk5 activity did notincrease during the first few days of neurite outgrowth butcorrelated with conditions promoting the initiation ofsynaptogenesis and NF-H phosphorylation. The neuro-trophic factor BDNF stimulated an earlier activation ofCdk5 kinase activity and induced a transient increase in Erkkinase activity in a manner similar to the induction of NF-Hphosphorylation in PC12 cells by NGF [134]. As NF-Hphosphorylation in these in vitro systems occurs beforeradial growth of axons, it is possible that NF-H phos-phorylation, as suggested above, is involved in thestabilization of elongating axons during growth.

NF phosphorylation may also play a role during axonalguidance, which is consistent with localized stabilization ofthe axonal cytoskeleton. Within axons of chick motorneurons growing to their specific muscle targets in vivo,phosphorylation of NF-M occurred in a `highly stereotypedand spatially heterogeneous pattern of expression' [135]. Inregions of the distal axon and growth cones, where responseto specific guidance cues were presumed to occur, therewas a robust expression of phospho-NF-M, as if thedecision point is stabilized in the axon by modification ofthe cytoskeleton. These localized expressions of phospho-NF-M, presumably in KSP motifs, marked the turningpoints executed by growth cones during axonal trajectoriesto targets. It would be of interest to know which kinaseswere activated at these sites. Clearly NF phosphorylationparticipates in some aspects of cytoskeletal dynamicsunderlying axon elongation and guidance.

C D K 5 / P 3 5 K N O C K O U T S ,N E U R O F I L A M E N T E X P R E S S I O N A N DE M B R Y O N I C L E T H A L I T Y

A review of Cdk5/p35 regulation of migration, axonelongation and growth cone dynamics of neuronal cellsin vitro and during normal development is included in thiscurrent journal (see the other two minireviews in thisseries). Included are references to the role of Cdk5/p35 inneuronal migration during brain development derived fromgene targeting studies [125,126,136]. Here, however, weshall compare differences in the phenotypes between Cdk5and p35 null mice, with particular emphasis on embryoniclethality.

A Cdk5 knockout mouse (Cdk52/Cdk52) exhibited anearly embryonic lethality with 64% of KO mice dyingin utero after E16.5 with only 19% surviving to E18.5,while only 9% were born with hyponea and reducedmobility, dying within 12 h [126]. Brain and spinal cordwere the principal tissues showing any gross pathology,with disrupted corticogenesis, abnormal cerebellar foliationand laminar organization, while in the brain stem and spinalcord, many neurons exhibited abnormal perikaryal inclu-sions of phosphorylated neurofilaments. As Cdk5 kinaseactivity was absent from all brain tissues, the abnormallyphosphorylated neurofilaments could have resulted from a

compensatory activation of other kinases which alsoaccounts for phospho-NF-H in axons. As these abnormalneurons resembled other neurons in known neurodegenera-tive disorders, it suggests that lethality in these KO animalsis due either to the defects in laminar organization in cortexand cerebellum and/or the loss of neurons in basal stem andspinal cord which control vital functions.

A p352/± mouse, on the other hand, exhibits markedreduction of brain Cdk5 kinase activity as a consequence ofthe complete absence of p35 [125]. Nevertheless most ofthese animals survive and grow to the adult stage withoutany gross abnormality, only showing evidence of seizuresand lethality in adult stages. Significantly, the pathologymanifested in the brain resembles the cortical disruptionalso seen in the Cdk5 KO mice. The inside-outside neuronalmigration pattern that gives rise to the laminar organizationof the cortex was abnormal, as were axonal trajectories anddendritic patterns. The effects on neuronal migration inthese KO animals do not seem to be the principal factorresponsible for lethality. It suggests, however, that the deathof neurons in specific regions of the brain that control vitalfunctions may be the primary cause of fetal and postnatallethality in Cdk5 KO mice. This was confirmed by a recentstudy demonstrating that overexpression of Cdk5 in Cdk5KO mice, using a transcript that contained the p35promoter, which is expressed only in neurons, successfullyand completely rescued the lethal phenotype [146].Although Cdk5 activity was restored to only half that ofthe wild type, the laminar patterns of cortex and cerebellumwere normal and there was no sign of any abnormal phos-pho-NF-H filled perikarya in brain stem nor spinal cord. Asp35 expression is restricted mainly to many different brainregions, it is evident that the absence of active Cdk5 in mostother tissues, including muscle was not responsible forlethality of the Cdk5 KO. Rather, defects in the phos-phorylation of cytoskeletal proteins including neurofila-ments, in specific populations of neurons, unrelated toneuronal migration, were responsible for the developmentalfailure in these KO mice.

This does not preclude the possibility that defects inneuronal migration and axonal guidance could havecontributed to lethality in the Cdk5 KO mice as contrastedwith the p35 KO mice. Specific abnormal neuronalnetworks, vital to survival, arising in Cdk5 KO mice,could be distinct from those in p35 KO mice. In otherwords, lethality in Cdk5 KO mice could have been due to acombination of defects in two independent processes andseveral different sites modulated by Cdk5.

C O N C L U S I O N S

The establishment of ordered neuronal networks andlaminar organization during brain development dependson neuronal differentiation, migration of neurons alongradial glia, axonal outgrowth and growth cone guidance tothe appropriate targets, and finally, the establishment ofstable synaptic contacts. We have seen that the Cdk5/p35complex, active primarily in the nervous system, may playa role in each of the processes mentioned. Cdk5 and p35knockout studies have demonstrated the importance of theCdk5/p35 complex in development of the nervous systemand survival of the organism. Recent studies of Cdk5overexpression in the Cdk5 knockout clearly target the


action of the complex to neuronal cells rather than glia.Future work is necessary to identify the specific substratetargets of Cdk5 activity that are regulated in each of theseprocesses. Phosphorylation of neurofilaments and othercytoskeletal proteins have been identified as likely targetsin establishing the axonal cytoskeleton but the relationshipbetween this, neuronal migration, axonal elongation andguidance is still not understood.

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Cyclin-dependent protein kinase 5 (Cdk5) and the regulation of neurofilament metabolism

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