cdk-5-mediated neurofilament phosphorylation in shsy5y human neuroblastoma cells

CDK-5-Mediated Neurofilament Phosphorylation in SHSY5YHuman Neuroblastoma Cells

Monica Sharma, Pushkar Sharma, and Harish C. Pant

Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health,Bethesda, Maryland, U.S.A.

Abstract: Cyclin-dependent kinase-5 (CDK-5) has beenshown to play important roles in neuronal developmentand neurogenesis. In vitro studies indicate a role ofCDK-5 in phosphorylation of neurofilaments (NFs). In thisstudy, we have chosen the human neuroblastoma cellline SHSY5Y as a model system to study the in vivophosphorylation of NF proteins by CDK-5. Upon differ-entiation of SHSY5Y cells with retinoic acid, we foundthat the phosphorylation of high molecular mass (NF-H)and medium molecular mass (NF-M) NFs increased,whereas the CDK-5 protein level and kinase activity wereunaffected. The role of CDK-5 in the phosphorylation ofcytoskeletal proteins was studied by using antisense oli-gonucleotides (ONs) to inhibit the expression of theCDK-5 gene. We found that inhibition of CDK-5 levels byantisense ON treatment resulted in a decrease in phos-phorylation of NF-H that correlated with a decline inneurite outgrowth. These results demonstrate that CDK-5is a major proline-directed kinase phosphorylating thehuman NF-H tail domain. Key Words: Cyclin-dependentkinase-5—Human high molecular mass neurofilament—Phosphorylation.J. Neurochem. 73, 79–86 (1999).

Cyclin dependent kinase-5 (CDK-5) is found in bothmitotic and postmitotic cells; its kinase activity, how-ever, has been detected only in postmitotic cells such asneurons (Lew et al., 1992a; Shetty et al., 1993; Tsai etal., 1993). Unlike other CDKs, CDK-5 protein is mosthighly expressed in neuronal tissue (Hellmich et al.,1992). CDK-5 activity depends on its association withthe brain-specific activators p67 (Shetty et al., 1995),p35/p25 (Tsai et al., 1993; Ishiguro et al., 1994; Lew etal., 1994), and p39 (Zheng et al., 1998), which distin-guishes this CDK from all others. CDK-5/p35 kinaseactivity increases during CNS neurogenesis, suggestingthat CDK-5/p35 plays an important role in neuronaldifferentiation (Nikolic et al., 1996). CDK-5 is impli-cated in phosphorylation of serine/threonine sites in Lys-Ser-Pro-X-Lys (KSPXK)-type motifs in the C-terminaltail domain of the high molecular mass neurofilament(NF-H) proteins (Lew et al., 1992b; Shetty et al., 1993).The tail domain of rat NF-H subunit contains;52 KSP

repeats. These repeats can be further classified asKSPXK, KSPXXK, or KSPXXX. In human NF-H, thereare 43/44 KSP repeats. Thirty-four of these repeats are inKSPXK motifs. On the other hand, rat and mouse haveonly 10 and 9 KSPXK repeats, respectively. It has beenshown that virtually all KSP sites in the human and ratNF-H tail domain are phosphorylated in vivo (Elhananyet al., 1994; Jaffe et al., 1998). In vitro phosphorylationof rat NF-H by CDK-5 occurs with extremely low effi-ciency and does not produce an electrophoretic mobilityshift, whereas human NF-H phosphorylated by CDK-5exhibited a decreased mobility, close to that of nativephosphorylated NF-H, suggesting that human NF-H is abetter in vitro substrate for CDK-5 (Shetty et al., 1993;Pant et al., 1997).

We used the human neuroblastoma cell line SHSY5Yas a neuronal model system to study the in vivo role ofCDK-5 phosphorylation of human NF proteins duringneurite outgrowth. As this cell line is derived from hu-mans, the NF-H expressed in these cells contains a largenumber of KSPXK sites that are potential targets forCDK-5. The SHSY5Y cell line is derived from a neuralcrest tumor of early childhood, predominantly composedof undifferentiated neuroblast-like cells. These neuro-blastoma cells undergo differentiation when treated withall-trans-retinoic acid (all-trans-RA). The cells developlong neurites, forming a neuronal phenotype, when stim-ulated for 6–7 days in the presence of micromolar con-centrations of RA (Konig et al., 1990; Pahlman et al.,1990). It has been shown that NF proteins are expressedduring axonal elongation in NB2a/dl cells (Shea et al.,

Received January 21, 1999; revised manuscript received March 10,1999; accepted March 10, 1999.

Address correspondence and reprint requests to Dr. H. C. Pant atLaboratory of Neurochemistry, Rm. 4D-28, Bldg. 36, NINDS, NIH,Bethesda, MD 20892, U.S.A.

Abbreviations used:BCIP-NBT, 5-bromo-4-chloro-3-indolyl phos-phate/nitro blue tetrazolium; CDK-5, cyclin-dependent kinase-5; FITC,fluorescein isothiocyanate; NF, neurofilament; NF-H, -L, and -M, high,low, and medium molecular mass neurofilament; NGS, normal goatserum; ON, oligonucleotide; PBS, phosphate-buffered saline; RA, reti-noic acid; TBS, Tris-buffered saline; TTBS, 20 mM Tris-HCl (pH 7.4),150 mM NaCl, and 0.2% Tween 20.

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Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 1999 International Society for Neurochemistry

1995). An increase in neurite outgrowth and increasedphosphorylated NF-H and medium molecular mass NF(NF-M) levels were observed as a result of RA-induceddifferentiation of the SHSY5Y cells. We used antisenseoligonucleotides (ONs) to suppress the expression ofCDK-5 levels in SHSY5Y cells and to determine itseffect on the phosphorylation of NF-H and neurite out-growth. We found that upon CDK-5 antisense ON treat-ment, these cells show a decrease in CDK-5 proteinlevels and a subsequent decrease in the phosphorylationof NF proteins and neurite outgrowth.

MATERIALS AND METHODS

MaterialsThe following antibodies were used: CDK-5 (AB-2) mono-

clonal antibody (Oncogene, Cambridge, MA, U.S.A.), humancdk-5 polyclonal antibody (Pharmingen, San Diego, CA,U.S.A.), goat anti-rabbit (polyclonal) and goat anti-mouse(monoclonal) conjugated to alkaline phosphatase (Kirkegaardand Perry Laboratories, U.S.A.), Texas Red-labeled goat anti-mouse and fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit (Sigma), SMI-34 (phosphorylated NF-H) and SMI-33(dephosphorylated NF-H) (Sternberger, Lutherville, MD,U.S.A.), polyclonal antibody to p35 (gift from Dr. Li Tsai),RMO-281 (phosphorylated NF-M) and FNP7 (dephosphory-lated NF-M) monoclonal antibody (Zymed, San Francisco, CA,U.S.A.), N-52 (dephosphorylated NF-H) (Sigma), low molec-ular mass NF (NF-L; polyclonal) antibody (gift from Dr.Thomas Shea),a-internexin monoclonal antibody (gift fromDr. R. K. M. Liem), anti-vimentin monoclonal antibody(Boehringer Mannheim, Germany),a-tubulin monoclonal an-tibody (Calbiochem, Cambridge, MA, U.S.A.), peripherinpolyclonal antibody (gift from Dr. Linda Perysak), and anti-tau-1 monoclonal antibody (Boehringer Mannheim). Poly-clonal antibody to p67 was made in our laboratory (Shetty etal., 1995).

Other materials used were protein (G1 A) Sepharose beads(Oncogene), 5-bromo-4-chloro-3-indolyl phosphate/nitro bluetetrazolium (BCIP/NBT) substrate for alkaline phosphatase(Kirkegaard and Perry Laboratories), aqueous mounting me-dium (Biomedia Corp.), and BCA protein assay reagent kit(Pierce, Rockford, IL, U.S.A.).

Cell cultureSHSY5Y human neuroblastoma cells were cultured in Dul-

becco’s modified Eagle’s medium supplemented with 10%heat-inactivated fetal calf serum and 2 mM L-glutamine. Themedium was changed every second day. To induce differenti-ation, the cells were treated with 10mM RA in the dark for 4–6days.

ImmunofluorescenceCells were plated on poly-L-lysine (5 mg/ml)-coated glass

coverslips (25 mm in diameter) at densities ranging from50,000 to 100,000 cells/ml. Prior to treatment with RA for 4–6days, the cells were allowed to attach properly for 24 h. Thecells were fixed with 4% paraformaldehyde (pH 7.4) for 30 minprior to washing with 13 phosphate-buffered saline (PBS),three times each for 5 min. Blocking was done in 10% normalgoat serum (NGS) containing Triton X-100 (1%) for 1 h,followed by incubation in 10% NGS without Triton X-100 for10 min at room temperature. The cells were incubated inprimary antibody diluted in 10% NGS with Triton X-100 (1%)

overnight at 4°C followed by three washes in PBS as describedabove and incubated in Texas Red-labeled goat anti-mouse orFITC-labeled goat anti-rabbit fluorescent secondary antibodiesfor 2 h in thedark. The coverslips were washed three times inPBS before embedding the cells in aqueous medium. Fluores-cence was observed on a Zeiss confocal microscope or a Nikonfluorescence microscope.

Antisense experimentsFor antisense experiments, the cells were plated on poly-L-

lysine-coated six-well plates at a density of 50,000 cells/ml.The sequences of antisense ONs 59TTCTCGTATTTCTGC39or 59GCAGAAATACGAGAA39 sense ONs (5 and 10mM)were the same as used by Pigino et al. (1997) (commerciallysynthesized and HPLC purified by Biosynthesis). Search of thegene data bank showed no homology with any other protein. Arandom ON, 59TGCTTCTCGTCTTAT39, was also used. ThisON did not show any effect on CDK-5 levels (data not shown).The ONs were preincubated with 10ml of Lipofectin reagent (1mg/ml) in 100ml of serum-free medium (Optimem I; reducedserum medium). The resulting ON suspension was then addedto the cells (5 or 10mM final ON concentration) and incubatedfor 3–5 h. Control experiments with only Lipofectin did notshow any alteration in cell morphology or CDK-5 levels. Themedium was removed and replaced with RA-containing me-dium. The same procedure was repeated for 2–4 days.

Western blot analysisCells were lysed in a urea-containing buffer [50 mM Tris

(pH 7.4), 1 mM EDTA, and 1 mM EGTA, protease inhibitorcocktail (Boehringer Mannheim), and 7M urea] to solubilizeall cytoskeletal proteins. The protein concentrations were esti-mated by Coomassie Blue staining and BCA protein assay kit(Pierce). Ten micrograms of the cell lysate protein was mixedwith an equal volume of 23 Laemmli’s sample buffer (0.25MTris-HCl, pH 6.8, 25% glycerol, 2% sodium dodecyl sulfate,and 2%b-mercaptoethanol) and boiled for 5 min at 100°C in awater bath. After electrophoresis, the proteins were transferredto a polyvinylidene difluoride Immobilon membrane. Thismembrane was blocked by incubating for 1 h atroom temper-ature in a buffer containing 20 mM Tris-HCl (pH 7.4), 150 mMNaCl, and 0.2% (vol/vol) Tween 20 (TTBS). This was followedby incubation in primary antibody diluted in TTBS for 2 h atroom temperature. The membranes were then washed fourtimes in TTBS (15 min/wash). This was followed by incubationin secondary antibody (goat anti-mouse or goat anti-rabbit IgGconjugated to alkaline phosphatase at a dilution of 1:3,000) for2 h at room temperature. After four 15-min wash cycles inTTBS, the immunoreactive bands were visualized using BCIP/NBT phosphatase substrate solution.

ImmunoprecipitationProtein (G1 A) agarose beads (bed volume 60ml) were

suspended in 1 ml of Tris-buffered saline (TBS; pH 7.4) andcentrifuged at 10,000 rpm for 1 min. The supernatant wasdiscarded, and the beads were subjected to two more washcycles with TBS. The beads were suspended in 600ml of TBSand 60ml of CDK-5 polyclonal antibody (Pharmingen). Aftergentle mixing at room temperature for 30 min, antibody-con-jugated beads were centrifuged and washed three times withTBS as described above. One hundred micrograms of the celllysate (prepared in buffer containing 50 mM Tris-HCl, pH 7.4,1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, proteaseinhibitor cocktail, 2mM okadaic acid, 25 mM b-glycerophos-phate, and 0.1% Triton X-100) was mixed with antibody-

J. Neurochem., Vol. 73, No. 1, 1999

80 M. SHARMA ET AL.

conjugated beads and gently agitated for 2–3 h at 4°C followedby centrifugation at 10,000 rpm for 1 min in a refrigeratedcentrifuge. The beads were washed three times with TBS andsuspended in a buffer containing 50 mM Tris-HCl with pro-tease inhibitor cocktail and the phosphatase inhibitors 2mMokadaic acid and 25 mM b-glycerophosphate.

Assay of kinase activity in CDK-5immunoprecipitate

A total volume of 50ml of kinase assay mixture contained10 ml of washed CDK-5 protein (G1A) beads in 50 mMTris-HCl (pH 7.4) with 1 mM EGTA, 1 mM dithiothreitol, 5mM MgCl2, and 0.5 mM microcystin LR and phosphorylacceptor substrate (10mg of histone H1). The kinase reac-tion was initiated by the addition of [g-32P]ATP (final con-centration 0.1 mM ) and incubated at 30°C for 30 min. Thereaction was terminated by spotting 30ml of the reactionmixture on P81 phosphocellulose pads that were washed fivetimes in 75 mM phosphoric acid followed by rinsing with95% ethanol; the radioactivity was measured in a liquidscintillation counter or the phosphorylated substrate wasassessed by sodium dodecyl sulfate polyacrylamide gel elec-trophoresis and autoradiography.

RESULTS

Effect of RA stimulation on NF expression andcomparison with other intermediate filaments

SHSY5Y cells possess small processes in an undiffer-entiated state, and after treatment with RA, the cellsdeveloped long neurites, resembling a neuronal pheno-type (Fig. 1). A dramatic increase in phosphorylatedforms of NF-H and NF-M was observed after RA-in-duced differentiation (Fig. 2A and C). The undifferenti-ated cells showed no evidence of phosphorylated NF-Hthat could be demonstrated by SMI-34 antibody (Fig.2A, lane 1). Dephosphorylated NF-H (using a phosphate-independent antibody, N-52) and NF-M, however, werepresent mainly in undifferentiated cells (Fig. 2B and D,lane 1), and upon differentiation with RA, the levels ofNF-H were unaffected. The extensive neurite outgrowthof treated cells correlated well with the significant in-crease in the expression levels of phosphorylated NF-Hand NF-M (Fig. 2A and C). In the case of NF-H, thereduction of dephosphorylated NF-H in RA-treated cellscorrelated well with the increase in phosphorylatedNF-H. However, NF-L levels showed no change upondifferentiation (Fig. 2E). The levels of other intermediatefilament family proteins like vimentin,a-internexin, andperipherin remained unchanged (Fig. 3). The level oftubulin was also unaffected by RA treatment; however,tau levels appeared to increase upon differentiation. RAtreatment may have affected the expression of some tauisoforms (Fig. 3B, lanes 3 and 4). In this study, we havenot analyzed this observation further.

Effect of RA on CDK-5 expression and activityThe levels of CDK-5 in differentiated and undifferen-

tiated SHSY5Y cell lysates were measured using aCDK-5 monoclonal antibody. The western blot analysisshowed that the level of CDK-5 was unaffected by RAtreatment (Fig. 4A, lanes 1 and 2). To confirm this

observation, CDK-5 was immunoprecipitated from thecell lysates of the undifferentiated and RA-differentiatedcells using a polyclonal antibody raised against a C-terminal peptide unique to CDK-5. The immunoprecipi-tate was probed for CDK-5 protein using a monoclonalantibody (Fig. 4B, lanes 1 and 2) and also showed nosignificant effect of RA on CDK-5 expression. TheCDK-5 immunoprecipitate kinase activity showed nosignificant difference in histone phosphorylation be-tween control and differentiated cells (Fig. 4C, lanes 1and 2), suggesting that CDK-5 activity is independent ofRA-induced differentiation under these experimentalconditions (see Materials and Methods).

Inhibition of CDK-5 expression by antisense ONsTo determine whether CDK-5 activity plays a role in

NF phosphorylation, we used antisense ONs to inhibit itsexpression. Antisense ONs have been used successfullyfor specific cellular gene targeting (Wagner, 1995). TheCDK-5 antisense, sense, and random ONs were designed

FIG. 1. Effect of RA on morphology of SHSY5Y cells. The cellswere induced to differentiate for 6 days by adding 10 mM (finalconcentration) all-trans-RA in Dulbecco’s modified Eagle’s me-dium containing 10% heat-inactivated fetal bovine serum. Afterevery 24 h, medium was replaced with fresh medium containing10 mM RA. A: Untreated cells; B: RA-treated cells.


81CDK-5-MEDIATED NEUROFILAMENT PHOSPHORYLATION

and transfected in cells as described in Materials andMethods. An optimal 10mM dose of ONs was standard-ized for transfecting the cells. Treatment of RA-stimu-

lated cells at this concentration of antisense ONs for 2–4days resulted in;70% suppression in CDK-5 proteinlevels as compared with sense ON-treated cells (Fig.5A). Sense-treated cells exhibited similar morphologyand CDK-5 levels as untreated controls (data not shown).

The CDK-5 immunoprecipitate was used in assessingthe associated kinase activity with histone as a substrate(Fig. 5B). Expectedly, CDK-5 immunoprecipitate fromantisense ON-treated cells showed only a little kinaseactivity as compared with sense control. These datasuggest that inhibition in CDK-5 levels and activity wasachieved using specific antisense ONs. Treatment ofthese RA-differentiated cells with CDK-5 antisense ONsalso resulted in decreased neurite outgrowth (see below).In addition to CDK-5, SHSY5Y cells also express

FIG. 2. Increase in phosphorylated NF-H and NF-M upon RAdifferentiation. The cell lysates from RA-treated and -untreatedcells were probed with various phospho (p) and dephospho (dp)antibodies. A: SMI-34 (pNF-H); B: N-52 (dpNF-H); C: RMO-281(pNF-M); D: FNP7 (dpNF-M); E: NF-L. In each case, lane 1 is thelysate of untreated and lane 2 of treated cells.

FIG. 3. Cytoskeletal proteins indifferentiated and undifferenti-ated cells. Most of the cytoskel-etal proteins are present in similaramounts in both RA-differentiatedand -undifferentiated cells. A:Lanes 1 and 2, a-internexin; lanes3 and 4, vimentin; lanes 5 and 6,a-tubulin. Lanes 1, 3, and 5 arelysates from undifferentiated andlanes 2, 4, and 6 are lysates fromdifferentiated cells. B: Lanes 1and 2, peripherin; lanes 3 and 4,tau-1. Lanes 1 and 3 are from un-differentiated and lanes 2 and 4are from differentiated cells.

FIG. 4. Identification of CDK-5in SHSY5Y cells by western blotanalysis and demonstration ofhistone kinase activity of theCDK-5 immunoprecipitate. A:Western blot analysis of CDK-5of untreated cells (lane 1) andRA-treated cells (lane 2). B: Im-munoprecipitates of CDK-5show equal amount of CDK-5.C: Autoradiogram shows his-tone phosphorylation by theCDK-5 immunoprecipitate fromuntreated cells (lane 1) and RA-treated cells (lane 2).

FIG. 5. A: Western blot analysis of antisense and sense ON-treated cells. The RA-differentiated cells were treated with anti-sense/sense ONs as described in Materials and Methods. Lane1, 10 mM sense ON-treated cells; lane 2, 10 mM antisenseON-treated cells. B: CDK-5 activity in immunoprecipitate fromantisense and sense ON-treated cells.


82 M. SHARMA ET AL.

CDC2; its expression was not affected by antisense ONtreatment (data not shown).

Effect of CDK-5 antisense on NF phosphorylationThe neurite outgrowth upon RA-induced differentia-

tion of these cells correlated well with the expression ofNF-H proteins and their increased phosphorylation lev-els. To determine the role of CDK-5 in the phosphory-lation of KSP motifs of NF proteins, we examined thephosphorylation of NF-H proteins by western blottingusing phospho-epitope-specific antibodies followed bydetection with immunofluorescence.

Whole-cell lysates were prepared in a urea-containingbuffer by extracting SHSY5Y cells treated with antisenseand sense ONs to solubilize all the cytoskeletal proteins(see Materials and Methods). The immunoblot analysisrevealed a significant decrease in staining of phosphor-ylated NF-H (Fig. 6A, lane 2) from antisense-treatedcells compared with the sense and untreated cells, usingSMI-34 antibody. This antibody recognizes phospho-epitopes of NF-H and recently has been shown to bespecific for KSPXK sites (Bajaj and Miller, 1997). Therewas no detectable change in the staining for the dephos-phorylated NF-H when probed with phosphate-indepen-

dent antibody SMI-33 (Fig. 6B). The phospho-NF-Mantibody also showed a slight decrease in staining in theantisense-treated cells (Fig. 6C), which is consistent withthe smaller number of KSPXK sites in human NF-M.There was no significant difference in the staining ofother cytoskeletal proteins like vimentin (Fig. 6D) andtubulin (data not shown).

Immunofluorescence detection of phospho- anddephospho-NFs

The effects of CDK-5 suppression seen in the immu-noblot (Fig. 6) suggest that CDK-5 catalyzes the phos-phorylation of NF-H in these cells. To study these effectsat a cellular level, immunofluorescence experimentswere performed using SMI-34 and anti-CDK-5 as pri-mary antibodies and Texas Red- or FITC-labeled fluo-rescent secondary antibodies. Fluorescence was detectedby confocal microscopy (Fig. 7) and revealed that thestaining for both SMI-34 and CDK-5 is reduced uponCDK-5 antisense treatment, as expected from the immu-noblot analysis. A significant decrease in neurite lengthwas also observed. The sense-treated cells did not differsignificantly from the untreated cells (Fig. 7B and D);untreated or sense ON-treated cells (50–60%) had neu-rites. However, only 14% of antisense ON-treated cellshad neurites (Fig. 7E). These data suggest that CDK5plays an important role, directly or indirectly, in neuriteoutgrowth of the SHSY5Y human neuroblastoma cells,which was also evident when they were probed withanti-vimentin antibody (Fig. 8). This also showed thatcell viability is not altered upon CDK-5 antisense ONtreatment (;80%).

DISCUSSION

NF phosphorylation at the C-terminal domain is con-sidered to be responsible for increasing the axonal cali-ber (de Waegh et al., 1992). This phosphorylation takesplace at S-residue sites in KSP motifs of the C-terminaldomain of NF-H and NF-M. There have been a numberof kinases like CDK-5, mitogen-activated protein ki-nases, and stress-activated protein kinases (Shetty et al.,1993; Giasson et al., 1997; Veeranna et al., 1998) thathave been reported in phosphorylation of these sites. Toprobe the in vivo role of CDK-5 in this event, we firstestablished SHSY5Y human neuroblastoma cells as acellular model. These neural crest-derived cells respondto RA treatment and differentiate and achieve neuronalphenotype, developing long neurites as a result of dif-ferentiation. In these differentiated neuronal cells, nosignificant change in the expression of cytoskeletal pro-teins like vimentin, peripherin,a-internexin, anda-tu-bulin was observed. The western blot analysis showeddramatic increase in levels of phosphorylated NF-M andNF-H proteins. There was a strong correlation betweendifferentiation, neurite outgrowth, and increase in NF-Mand NF-H phosphorylation. These studies were per-formed using antibodies that specifically recognize theKSP sites in the tail domain of NF-H and NF-M (Stern-

FIG. 6. Effect of antisense ON treatment on NF proteins. West-ern blot analysis of CDK-5 antisense and sense ON-treated celllysates was performed using the following antibodies: A: SMI-34(pNF-H); B: SMI-33 (dpNF-H); C: RMO-281 (dpNF-M); D: vimen-tin. In all cases, lane 1, control cells; lane 2, antisense ON-treated cells; lane 3, sense ON-treated cells. P-, phospho; dP-,dephospho.



berger and Sternberger, 1983; Lee et al., 1987). Mainlyunphosphorylated forms of NF-H or NF-M were de-tected in undifferentiated cells. Different dephosphory-lated states of NF-M were seen in unstimulated cells,when the cells have only small neurites as shown in Fig.2D (lane 1).

In the last few years, various groups have identifiedkinases involved in the phosphorylation of the KSP sitesin the tail portion. These include protein kinases such asCDK-5 (Hisanaga et al., 1993; Shetty et al., 1993),GSK-3 (Guan et al., 1991; Roder and Ingram, 1991),stress-activated protein kinases (Giasson et al., 1997),and mitogen-activated protein kinases (Veeranna et al.,1998). However, in vivo evidence related to this questionhas been lacking. CDK-5/p35 has been shown to phos-phorylate KSPXK sites of NF-H when overexpressed inCOS cells (Sun et al., 1996). Interestingly, analysis ofNF proteins from CDK-5 “knock-out” mice did not showany change in the phosphorylation state (Ohshima et al.,

1996). It is possible that the phospho-NF antibodies usedin this work were not sensitive enough to recognize thefew phosphorylated epitopes in the mouse NF-H, whichhas only 10 KSPXK (Julien et al., 1988) sites that can bephosphorylated by CDK-5. This represents 20% of thetotal KSP sites, the rest of which can be phosphorylatedby mitogen-activated protein kinases and stress-activatedkinases (Giasson et al., 1997; Veeranna et al., 1998).

Because human NF-H has a large number of KSPXKsites (Shetty et al., 1993), we hypothesized that its phos-phorylation state may be heavily dependent on the ac-tivity of CDK-5 in the cells. As CDK-5 phosphorylatesmany proteins other than NF-H/M with XS/TPXK mo-tifs, phosphorylation of these proteins will be affected byCDK-5 antisense ON treatment. The available inhibitorsof CDK-5 are nonspecific because they inhibit mostCDKs with similar efficiency; therefore, antisense meth-odology was used to specifically inhibit CDK-5 levels.SHSY5Y cells were differentiated with RA as described

FIG. 7. Immunofluorescence detection of CDK-5 and phosphorylated NF-H of RA in sense and antisense ON-treated differentiatedSHSY5Y cells. Confocal images of antisense ON-treated cells were stained with SMI-34 (A) and cdk-5 (C). Sense-treated cells werestained with SMI-34 (B) and CDK-5 (D). The number of cells with neurites relative to the total number of cells is also shown (E). A cellwith neurite size twice the diameter of the cell body was considered to possess neurites.


84 M. SHARMA ET AL.

earlier and treated with antisense ONs for 2–4 days.Significant decreases in CDK-5 levels were observed inthe antisense-treated cells, whereas sense and untreatedcells showed no difference, confirming the specificity ofthe ONs. Moreover, the sequences did not contain fourcontiguous guanosine residues, which increases the non-specific effects (Wagner, 1995). Additionally, the anti-sense ON treatment does not reduce the levels of otherproteins such as tubulin, vimentin, and tau.

A decrease in neurite outgrowth also correlates withsuppression of CDK-5 levels of kinase activity, suggest-ing that CDK-5 plays a role in neurite growth. Pigino etal. (1997) found a similar decrease in neurite outgrowthand MAP1B phosphorylation upon CDK-5 suppressionin cerebellar macroneurons. On suppression of CDK-5,these cells were not able to develop axons or sustain

elongation. Therefore, Pigino et al. concluded thatCDK-5 is a specific modulator of axonal outgrowthrather than having an essential role in neurite outgrowth.Nikolic et al. (1996) reported a similar observation whenrat cortical neurons were treated with p35 antisense.However, the CDK-5 targets involved in this processwere not identified. A wide variety of processes affectneurite outgrowth and elongation. This study suggeststhat among these processes, the phosphorylation of neu-ronal cytoskeletal molecules (e.g., NF proteins) byCDK-5 may be one contributing to neurite outgrowth inhuman neuroblastoma SHSY5Y cells.

CDK-5 and its regulator p35 have been shown to playan important role in normal corticogenesis (Ohshima etal., 1996; Chae et al., 1997). Mutant cdk-5 (2/2) micedie in the perinatal period with severe defects in thecerebral and cerebellar cortices and hippocampus. Asimilar phenotype is observed in reeler mutants (Oh-shima et al., 1996). Mutant CDK-5 regulator p35 (2/2)mice have an inverted layering neurogenic gradient andabnormal axonal patterns (Chae et al., 1997). This studyfurther supports that CDK-5 is important for modulationof the neuronal cytoskeleton. In a recent study, Nikolic etal. (1998) have shown that phosphorylation of Pak1 byactive CDK-5 (CDK-5/p35 complex) affects the dynam-ics of the reorganization of the actin cytoskeleton inneurons and thus promotes neuronal migration and neu-rite outgrowth.

Acknowledgment: The authors thank Dr. Thomas Shea forthe human neuroblastoma cell line SHSY5Y. We also thankDrs. Harold Gainer, Philip Grant, and Wayne Albers for givingcritical scientific suggestions. Dr. Carolyn Smith’s help withconfocal microscopy is appreciated. Monica Sharma thanks Dr.D. K. Jain (Zoology Department, Bareilly College, India) forencouragement with this work.

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86 M. SHARMA ET AL.

cdk-5-mediated neurofilament phosphorylation in shsy5y human neuroblastoma cells

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