Topographic Regulation of Phosphorylation in GiantNeurons of the Squid, Loligo pealei: Role ofPhosphatases

Philip Grant, Harish C. Pant

Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, NationalInstitutes of Health, LNC, NINDS, Bldg. 36, Rm. 4D04, Bethesda, Maryland 20892, and the MarineBiological Laboratory, Woods Hole, Massachusetts 02543

Received 6 May 2003; accepted 25 July 2003

ABSTRACT: In previous studies of phosphoryla-tion in squid stellate ganglion neurons, we demonstratedthat a specific multimeric phosphorylation complexcharacterized each cellular compartment. Although theendogenous protein profile of cell body extracts (giantfiber lobe, GFL), as determined by Coomassie staining,was similar to that of axoplasm from the giant axon, inthis study we show that the protein phosphorylationprofiles are qualitatively different. Whereas many axo-plasm proteins were phosphorylated, including most cy-toskeletal proteins, virtually all phosphorylation inperikarya was confined to low molecular weight com-pounds (<6 kDa). Because phosphorylation of exoge-nous substrates, histone and casein, was equally active inextracts from both compartments, failure to detect en-dogenous protein phosphorylation in cell bodies wasattributed to the presence of more active phosphatases.To further explore the role of phosphatases in theseneurons, we studied phosphorylation in the presence ofserine/threonine and protein tyrosine phosphatase(PTP) inhibitors. We found that phosphorylation of ax-

onal cytoskeletal proteins was modulated by okadaicacid-sensitive ser/thr phosphatases, whereas cell bodyphosphorylation was more sensitive to an inhibitor ofprotein tyrosine phosphatases, such as vanadate. Inhibi-tion of PTPs by vanadate stimulated endogenous phos-phorylation of GFL proteins, including cytoskeletal pro-teins. Protein tyrosine kinase activity was equallystimulated by vanadate in cell body and axonal wholehomogenates and Triton X-100 free soluble extracts,but only the Triton X soluble fraction (membranebound proteins) of the GFL exhibited significant ac-tivation in the presence of vanadate, suggesting higherPTP activities in this fraction than in the axon. Thedata are consistent with the hypothesis that neuronalprotein phosphorylation in axons and cell bodies ismodulated by different phosphatases associated withcompartment-specific multimeric complexes. © 2004

Wiley Periodicals, Inc.* J Neurobiol 58: 514 –528, 2004

Keywords: axon; kinase; phosphorylation; squid; ty-rosine phosphatase

INTRODUCTION

The neuron is a polarized cell consisting of morpho-logically and functionally distinct dendritic/soma and

axonal compartments. Each compartment is sustainedby a dynamic cytoskeletal ultrastructure, a network offilamentous and tubular macromolecules, and associ-ated proteins. Although all these molecules are syn-thesized in the nucleated soma, their transport, distri-bution, assembly, and metabolism differ in eachcompartment. For example, neurofilaments (NFs) arefound in some dendrites, but are most extensivelyphosphorylated in axons (Pant et al., 2000). Tau, amicrotubule (MT)-associated protein, is localized inaxons where it bundles microtubules, whereas a re-

Correspondence to: H. Pant (panth@ninds.nih.gov).© 2004 Wiley Periodicals, Inc. *This article is a US Governmentwork and, as such, is in the public domain in the United States ofAmerica.

DOI 10.1002/neu.10305

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lated microtubule-associated protein, MAP2, modu-lates the MT cytoskeleton in dendrites (Craig andBanker, 1994).

The factors controlling the assembly and cellularlocalization of cytoskeletal macromolecules are regu-lated by reversible, multisite phosphorylation by fam-ilies of serine/threonine protein kinases and phospha-tases (Price and Mumby, 1999; Grant and Pant, 2000).Moreover, the functional state of these phosphoryla-tion systems is modulated by several signal transduc-tion systems regulating cell growth, differentiationand survival, which are based, in part, on the activityof families of tyrosine protein kinases and phospha-tases (Hunter, 1998; Cohen, 2000; Keyse, 2000).

Significantly, most kinases and phosphatases areorganized as macromolecular complexes of enzymes,substrates, and regulators, localized within cellularcompartments by specific targeting and scaffoldingmolecules (Pawson, 1995; Faux and Scott, 1996;Whitmarsh and Davis, 1998; Garrington and Johnson,1999; Brazil and Hemmings, 2000). For example,cytoskeletal proteins such as tau, neurofilaments, andtubulin copurify together as multimeric complexeswith various kinases such as CAM KII (Goldenring etal., 1984), cdk5 and Erk1/2 (Lee and Johnston, 1997;Sobue et al., 2000; Veeranna et al., 2000). Many ofthese complexes also contain phosphatases binding toNFs and tau (Saito et al., 1995; Merrick et al., 1997;Strack et al., 1997; Sontag et al., 1999), and arelocalized within different cellular compartments bycytosolic or cytoskeletal-bound targeting proteins(Strack et al., 1997, 1998). Normally, cytoskeletalmolecules such as NFs are phosphorylated in theaxonal compartment, whereas in some neurodegen-erative disorders (such as amyotrophic lateral sclero-sis—ALS), aberrant hyperphosphorylation of thesemolecules occurs in cell bodies. Hence, we see thatthe problem of compartment-specific patterns of phos-phorylation in neurons is complex and may well de-pend on the factors regulating the assembly of phos-phorylation complexes and their interaction withspecific targeting molecules.

To sort out these complex interactions it is essen-tial to separate neuronal compartments to study therepertoire of molecular players, their organizationwithin each compartment, their specific substrate tar-gets, and their compartment-specific patterns of reg-ulation. Although modest separation has beenachieved in phosphorylation studies of cultured dorsalroot ganglia of vertebrates (Giasson and Mushynski,1996, 1997) only enriched, but contaminated cellbody and neurite fractions were analyzed. Our labo-ratory, however, has been studying the giant fibersystem in the squid for several years, where pure,

uncontaminated axoplasm from giant axons was sep-arated from their respective cell bodies in the giantfiber lobe (GFL) of the stellate ganglion. We haveshown that compartment-specific multimeric com-plexes of cytoskeletal proteins, kinases, phosphatases,and their regulators can be extracted from cell bodiesand axoplasm, each with different phosphorylationactivities (Takahashi et al., 1995; Grant et al., 1999).Endogenous phosphorylation of cytoskeletal proteinsin the axonal complex is considerably greater than inthe complex extracted from the large perikarya in theGFL. Although cell bodies of the GFL containedsimilar cytoskeletal substrates and active kinases,phosphorylation of endogenous substrates was inhib-ited. Either inhibitors of endogenous phosphorylationwere present in the GFL extract, or, alternatively, theGFL may contain high levels of endogenous phospha-tases. To further explore this problem, we comparedthe kinase and phosphatase activities of these frac-tions and found that: (1) axonal and perikaryal phos-phorylation systems are distinct, responding differen-tially to high salt, (2) axoplasm expressed higherlevels of okadaic acid-sensitive phosphatase activities(i.e., ser/thr phosphatases), while GFL fractions didexhibit significantly higher levels of vanadate-sensi-tive phosphatase activities, attributable to the proteintyrosine phosphatases (PTPs).

METHODS

Materials

Radioactive adenosine 5�-phosphate (�32 P-ATP) with aspecific activity of 1 mC/37MBq was purchased from NewEngland Nuclear (Boston, MA). ATP, histone H1, sodiumortho-vanadate, and �-casein were obtained from Sigma (St.Louis, MO). Okadaic acid, and tyrosine kinase substratepeptide were obtained from Gibco BRL (Gaithersburg,MD). Phosphorylated peptide for tyrosine phosphatase as-say was purchased from Cal Biochem (La Jolla, CA). Pro-tein assays were carried out with a BioRad (Hercules, CA)protein assay kit. Monoclonal antibody SMI-31to phosphor-ylated NF-H purchased from Sternberger and Sternberger(Lutherville, MD), tyrosine phosphatase antibodies, PTP�(C-19), PTP1B (N-19), and SHPTP-1 (C-19), polyclonalsfrom Santa Cruz (Santa Cruz, CA), while biotin-labeled andalkaline phosphatase conjugated secondary antibodies, goatantirabbit and rabbit antigoat IgG were obtained fromKirkegaard and Perry (Gaithersburg, MD).

Preparation of Axoplasm and CellBodies of the Giant Fiber Lobe (GFL)

Live squid (Loligo pealei), obtained from the Marine Bio-logical Laboratory, Woods Hole, MA, were decapitated and

Kinase/Phosphatase Activities in Squid Giant Neurons 515

the stellate ganglion and giant axon were removed. Thesmall giant fiber lobe (GFL) was dissected from the stellateganglion, washed in artificial seawater, and frozen at�20°C. The GFL contains the cell bodies of the stellateganglion whose axons fuse to form the giant axon (Young,1939). Note that these cells are extremely large, activelysecreting cells (600–700 �m in some species; Young, 1936)with dense cytoplasm, large nuclei, and nucleoli, sur-rounded by very small glial-like cells. The bulk of theprotein in GFL extracts was derived from the cytoplasm ofthe giant cells. The axoplasm was squeezed from thecleaned axon in the usual manner (Brown and Lasek, 1990)and frozen at �20°C. In some experiments the tissues weredissected and freshly extracted. In several experiments in-tact axons with sheaths were utilized and compared to theirrespective GFL.

Preparation of Tissue Extracts

Essentially, tissue extraction buffers differed with respect tothe presence or absence of phosphatase inhibitors (10 mM�-glycerophosphate and 5–10 mM sodium or potassiumfluoride). Addition of fluoride and �-glycerophosphate pre-serves cytoskeletal integrity and reduces the activity ofser/thr phosphatases. Extraction Buffer A: 20 mM Tris (pH7.4), 100 mM NaCl, 1% Nonidet P-40 (NP 40), with 1 mMDTT, and a protease inhibitor cocktail (Roche AppliedScience, Indianapolis, IN). Extraction Buffer B: For tyrosinekinase assays, 20 mM Tris pH 7.4, 50 mM NaCl, 5 mMEDTA, 30 �M �-glycerophosphate, 50 mM sodium fluo-ride, with or without 1% Triton X-100, and protease inhib-itor cocktail.

For extraction, 25–50 frozen (stored at �20°C) or freshwhole axons (or axoplasm equivalent) and equal numbers ofGFLs were diluted in extraction buffer at a ratio of 1 mLbuffer to 25 axons (or GFL) and homogenized and allowedto sit in ice for 5–10 min. The homogenate was centrifugedfor 10 min at 10,000 rpm at 4°C and the pellet resuspendedin buffer and centrifuged again for 10 min at 10,000 rpm.The supernatants were combined and used for kinase assaysand gel electrophoresis.

For protein tyrosine kinase and protein tyrosine phos-phatase assays, two extracts were prepared. Here, S1, thefirst supernatant was obtained in buffer without 1% TritonX-100 while the second supernatant S2, was obtained byhomogenizing the pellet from the first extract with 1%TritonX. This was designed to separate cytosolic from membranebound kinases and phosphatases. Protein concentrations ofall extracts were assayed with a BioRad kit and the proteinconcentrations normalized in all cases.

Protein Kinase Assays

For assays in the absence of phosphatase inhibitors, KinaseAssay Buffer A: 20 mM Tris, pH 7.4, 5–10 mM MgCl2, 1mM DTT, with 0.5% NP40 and 1 mM EGTA in the pres-ence and/or absence of okadaic acid (0.5–1.0 �M and/orvanadate 0.1 mM) for standard kinase assay. Kinase Assay

Buffer B for tyrosine kinase assays: 8 mM Tris, pH 7.4, 20mM MgCl2, 0.2 mM EGTA, 1 mM MnCl2, 8 mM �-glyc-erophosphate, 1 mM DTT. In some experiments, a peptidesubstrate (Gibco peptide substrate 13124-011) (0.2–0.25mM) was added along with various concentrations of van-adate, while in assays of endogenous activities, only vana-date was added.

All assays were carried out in a final total volume of 50�L containing kinase assay buffer, with or without exoge-nous substrates (final concentration of histone and casein at0.5 mg/mL, Gibco tyrosine kinase peptide at 0.5 mM),with/or without phosphatase inhibitors. The reaction wasinitiated by the addition of 10 �L of 0.01 mCi (�32 P) ATP(100 �M) and incubated for 1 h at room temperature (24°C).For pad assays, a 20–35 �L aliquot of the reaction mixturewas removed and applied to a 1 inch square of P81 phos-phocellulose paper (Whatman). The papers were exten-sively washed in 75 mM phosphoric acid followed by arinse in 95% alcohol and dried. Activity was determined byliquid scintillation counting and background subtractedfrom each.

Tyrosine kinase assays with peptide substrate were car-ried out in a similar fashion except that the reaction wasstopped by the addition of 43% TCA, followed by centrif-ugation of the pellet and placing an aliquot of the superna-tant on a P81 pad, washing and counting as above. In allassays, activity was measured as % increase in activity overthe endogenous, either in the absence of substrate or phos-phatase inhibitor, or both.

Protein Tyrosine Phosphatase Assays

A chromogenic assay, based on the Malachite green reagentto detect released inorganic phosphate (BioMol Green, Ply-mouth Meeting, PA) was employed (Harder et al., 1994).The extraction buffer was the same as Extraction Buffer Aabove, 20 mM Tris, pH7.4, 100 mM NaCl, 1 mM DTT, with1% NP40 and protease inhibitor cocktail. The tyrosine phos-phatase assay buffer, contained 100 mM Tris, pH 8.0, 5 mMDTT, with 0.1–0.5 mM peptide (Cal Biochem protein ty-rosine phosphatase substrate (Daum et al., 1993). The reac-tion was started by the addition of an enzyme sample andallowing the assay mixture to incubate at room temperature(RT) for 1 h, followed by the addition of the BioMol reagentand incubation for 20–30 min at RT, then reading at 620 nmin a spectrophotometer. A standard phosphate curve was runin each experiment and the activity measured as specificactivity, nmol phosphate/�g protein/h.

SDS-PAGE, Autoradiography, andWesterns

Gel electrophoresis was performed according to the Lae-mmli procedure. Several gel systems were used (e.g., 10%precast Novex Tris-glycine and 8–16% Novex Tris-glycinegradient gels). Ten to 20 �L sample aliquots (10–20 �gprotein) were loaded in each lane. The gels were stainedwith 0.2% Coomassie brilliant blue in 10% acetic acid/50%

516 Grant and Pant

methanol for 1 h and destained several times in changes of10% acetic acid/50% methanol and soaking it 24 h in 10%methanol, 10% acetic acid, and 10% glycerol. After drying,gels were placed in a cassette with Kodak X-ray film,exposed at �80°C, and the films were subsequently devel-oped.

Immunoblots were prepared from the gels as follows.Proteins were transferred from gels to Immobilon mem-branes using an electroblotting apparatus according to man-ufacturer protocols. Membranes were blocked with TTBS/5%BSA for 1 h at RT. They were then incubated in primaryantibody in TTBS/5%BSA (all antibodies at 1:100 dilution)overnight at 4°C, washed at least four times in TTBS, 10min each, followed by incubation in the appropriate alka-line-phosphatase conjugated secondary antibody (1:5000)for 1 h at RT. Membranes were washed at least four timesin TTBS, 10 min each, then rinsed in distilled water, fol-lowed by color development with the BNTP/BCIP reagent.

Immunocytochemistry

Stellate ganglia were fixed in Bouins fixative, washed sev-eral times in water, embedded in paraffin, and 10-�m sec-tions were mounted on slides. After dewaxing, sectionswere incubated in either 10% normal goat or rabbit serum inPBS with 0.1% Triton X, then incubated in primary anti-bodies prepared in PBS (1:10 PTP�, 1:10 PTP�, 1:1000SMI-31) overnight at 4°C. Sections were washed three tofour times in PBS, 10 min each, incubated in 1:1000 biotin-labeled secondary antibodies for 1 h at RT, washed threetimes in PBS. They were then incubated in 1:2000 Strepta-vidin-HRP in PBS/0.1%Triton X for 1 h at RT, washed, andtreated with Diaminobenzidine reagent to react with HRP,washed, counterstained in methyl green, and mounted inPermount.

RESULTS

Phosphorylation Activity of Axoplasmand GFL Extracts in the Presence andAbsence of Phosphatase Inhibitors

In all previous studies we compared phosphorylationactivities of axoplasm and GFL extracts under stan-dard kinase assay conditions in which both extractionand assay buffers contained ser/thr/phosphatase inhib-itors (sodium fluoride and b-glycerophosphate and/�okadaic acid) and tyrosine protein phosphatase inhib-itors (vanadate at 100 �M, a concentration, inciden-tally that has only a 20–40% effect on kinase expres-sion; see later, Fig..4) (Cohen et al., 1987; Takahashiet al., 1995; Grant et al., 1999). By optimizing kinaseactivities we could compare both endogenous andexogenous substrate-specific patterns of phosphoryla-tion. Under these conditions we demonstrated thatphosphorylation of endogenous cytoskeletal proteins

was considerably greater in axons than in cell bodies,suggesting elevated kinase activities (Takahashi et al.,1995; Grant et al., 1999). In the present study, we setout to compare the relative roles of phosphatases aswell as kinases in these neuronal compartments. Con-sequently, we extracted and assayed in buffers withand without the appropriate phosphatase inhibitors.We found, however, that the presence or absence ofphosphatase inhibitors in the extraction buffers hadminimal impact on phosphorylation activity in thefinal assay. Their presence in the assay buffers, how-ever, had a major effect. The results are shown inFigure 1. A Coomassie stained gel of extracts inBuffer A (no phosphatase inhibitors) in Figure 1(A)illustrates that both axoplasm and GFL extracts con-tain a wide range of similar proteins bands, except forthe presence of phosphorylated neurofilament proteinMW 220 (NF220) in the axoplasm extract (arrow).Identification of the protein bands was based on pre-viously published Western blots of similar extracts(Takahashi et al, 1995; Grant et al, 1999). Note thatthe expression profile of protein bands in GFL andaxon lysates is similar, with some bands showingmore expression in the GFL than in the axoplasm.Phosphorylation of these extracts in kinase assaybuffer A (no phosphatase inhibitors) yields the auto-radiograph in Figure 1(B), showing intense phosphor-ylation of several proteins in axoplasm includingNF220, high molecular weight neurofilament protein(HMW), and tubulin, among others. On the otherhand, the phosphorylation of GFL proteins was min-imal; few if any protein bands were phosphorylated,except for higher activity of a low molecular weightfraction (�6 kDa) arrow) compared to the equivalentaxonal band. In fact, the total endogenous level ofaxoplasm phosphorylation was twice that in the GFL(data not shown). Because both lysates contained aprotease inhibitor cocktail, known to effectively in-hibit most cytoplasmic proteases, it is unlikely that thelow molecular weight activity in the GFL is due toprotein breakdown, inasmuch as the low molecularweight Coomassie-stained bands (�6 kDa) in Figure1(A) are similar in each lysate. Nor can we assumethat GFL cytoplasm was diluted by nuclear proteins;except for phosphorylated neurofilament 220, theCoomassie stained gel showed equivalent proteinband patterns in each lysate [Fig. 1(A)]. It is signifi-cant that the patterns of phosphorylation in both frac-tions resembled those obtained previously in the pres-ence of ser/thr phosphatase inhibitors (Cohen et al.,1987; Takahashi et al., 1995), and suggests that axo-plasm contains more active kinases than the GFL.Alternatively, the GFL fraction may contain variouskinase inhibitors.

Kinase/Phosphatase Activities in Squid Giant Neurons 517

To test the level of kinases, we assayed exogenoushistone H1 phosphorylation in the presence of phos-phatase inhibitors (a standard kinase assay with 0.5–1�M okadaic acid and 0.1 mM vanadate). The data inFigure 1(C) show that phosphatase inhibitors in assaybuffers enhanced endogenous activity in both frac-tions; but few, if any new phosphorylated proteinbands appeared in the endogenous GFL fraction, aswas seen in the axoplasm. Virtually all endogenoussubstrates phosphorylated in the GFL were confinedto low molecular weight fractions (large arrow). Even

at the level of 0.1 mM vanadate, no additional endog-enous phosphorylated proteins were seen in the GFLextract. However, high levels of histone kinase activ-ity were present in the GFL fraction. Indeed, exoge-nous substrates such as histone and casein were highlyphosphorylated in the GFL and axoplasm when com-pared to endogenous levels [Fig. 1(C) and (D)]; therewere no significant differences in the means. Evi-dently, abundant kinases are present in GFL, as inaxon extracts, suggesting that the low endogenousphosphorylation in the GFL was not due to low kinase

Figure 1 Endogenous protein phosphorylation in axoplasm and GFL is distinctly different. (A)Coomassie stained gel of soluble extracts of axoplasm and GFL. Arrows at squid phosphorylatedNF-220 and tubulin (TUB), respectively. (B) Autoradiograph of endogenous protein phosphoryla-tion of a similar gel after kinase assay without phosphatase inhibitors. Note phosphorylatedcytoskeletal protein bands in axoplasm and the absence of any phosphorylated bands in GFL exceptfor high activity in very low molecular weight region (arrowhead). (C) Protein phosphorylation ofGFL and axoplasm extracts after addition of phosphatase inhibitors (0.5 �M OA and 0.1 mMvanadate) to assay medium in presence or absence of histone. Large arrow, low molecular weightfraction. Axo � axoplasm; GFL � giant fiber lobe; end � endogenous; hist � histone H1. (D)Exogenous substrate kinase activity in assay containing 0.5 �M OA and 0.1 mM vanadate. Histone(black, n � 11), casein (hatched, n � 7). Activity measured as percent increase over endogenousactivity in absence of substrates (means � S.E.). Histone and casein activities are similar inaxoplasm and GFl.

518 Grant and Pant

activities in these extracts. Previous Western blotstudies showed that for the most part, similar kinasesare shared by GFL and axoplasm (Grant et al., 1999).If each compartment contains similar substrates andkinases, what is responsible for the dramatically dif-ferent endogenous phosphorylation profiles? The datasuggest that each neuronal compartment is character-ized by a different set of factors regulating endoge-nous substrate phosphorylation.

NaCl Has Different Effects on Axoplasmand GFL Endogenous Kinase Activity

Further indication of the differences in regulation ofendogenous phosphorylation in the two neuronalcompartments is derived from studies of the effect ofsalt ions on kinase activity [Fig. 2(A]). Differentconcentrations of NaCl have no effect on the lowlevels of endogenous phosphorylation in the GFL butaxoplasm showed enhanced activities at increasingconcentrations of NaCl, reaching a peak at 100 mMNaCl. Autoradiographs of the phosphorylation pro-files in the two preparations are shown in Figure 2(B).Some axoplasm protein bands do exhibit enhancedphosphorylation at higher NaCl concentrations (nota-bly HMW and NF220 at 50 and 100 �M). The phos-phorylation profile in the GFL, however, exhibits nosignificant change, which agrees with the results oftotal kinase activity shown in Figure 2(B). A similarpattern of salt ion-dependent phosphorylation wasalso expressed by a neurofilament-enriched prepara-tion extracted from axoplasm (Pant et al., 1986). Sub-sequently, it was shown that the principal NF-associ-ated kinase was casein kinase I (Floyd et al., 1991).These observations further indicate that the dynamicsof endogenous phosphorylation in the two neuronalcompartments are different.

Effect of Okadaic Acid (OA) on KinaseActivity in Axoplasm and GFL Extracts

The role of phosphatases in these preparations couldbe explored by the independent use of specific phos-phatase inhibitors such as okadaic acid (Brautigan andShriner, 1988; Hardie et al., 1991) and vanadate (Gor-don, 1991). At different concentrations, okadaic acidis assumed to be a highly specific inhibitor of differentserine/threonine protein phosphatases (Cohen et al.,1990; Hardie et al., 1991). Buffer A extracts of axo-plasm and GFL were assayed for kinase activity atdifferent concentrations of OA, with and without hi-stone substrate. The results are shown in Figure 3.Whereas endogenous phosphorylation of axoplasmproteins is significantly stimulated (25–40%) in OA

concentrations from 100 nM to 10 �M, suggestingPP1 and PP2A as key players, endogenous GFL ac-tivity is relatively unaffected [Fig. 3(A)]. The differ-ences between axoplasm and GFL at 5 and 10 �M aresignificant (p � 0.02 and p � 0.0008, respectively).Because the principal endogenous phosphorylationactivity in the GFL was localized in a very lowmolecular weight protein band, these data suggest thatser/thr phosphatases are not playing a role in regulat-ing endogenous protein phosphorylation in the GFLextract. This can be seen in Figure 3(C), a sampleautoradiograph of a GFL gel, in which few if anyendogenous bands responded to the inhibitor. Com-pare this with an axoplasm gel that displayed signif-icant increments of phosphorylation in various endog-enous protein substrates [Fig. 3(D)].

Histone phosphorylation in both fractions, how-ever, was activated by okadaic acid [Fig. 3(B)] (100–150% for axoplasm, 50–75% for GFL) and here, too,the data suggest that inhibition of PP1 and PP2A maybe responsible. It is to be noted, however, that, as seenpreviously in Figure 1(D), the presence of phospha-tase inhibitors, in this case okadaic acid, has similareffects on histone total phosphorylation in axoplasmand GFL [Fig. 3(B)]. Although axoplasm activityseems enhanced, the difference between it and GFLactivity was not significant at all concentrations.Again, we see that with respect to exogenous sub-strates, axoplasm and GFL respond equally to ser/thrinhibitors, which further supports the argument thatthe factors regulating endogenous protein phosphory-lation differ in each neuronal compartment. Thisraises the question as to the identity of these factorsand their mechanisms of action.

Effect of Vanadate on Kinase Activity ofAxoplasm and GFL Extracts

If serine/threonine phosphatases are playing no rolein the endogenous phosphorylation of GFL pro-teins, is it possible that other phosphatases areinvolved? To determine whether protein tyrosinephosphatases (PTPs) are involved, we studied theeffect of different concentrations of vanadate, aknown inhibitor of membrane bound and cytosolictyrosine phosphatases (Swarup et al., 1982; Gor-don, 1991; Huyer et al., 1997) on endogenous phos-phorylation. It has been demonstrated that vanadateat various concentrations will preferentially inhibittyrosine phosphatases without affecting serine/thre-onine phosphatases. The results of these assays areshown in Figure 4. In the presence of low vanadateconcentrations (10 –100 �M), the endogenous phos-phorylation activity of both axoplasm and GFL

Kinase/Phosphatase Activities in Squid Giant Neurons 519

extracts was equally stimulated from 20 – 40%.Higher concentrations (200 �M) seemed to down-regulate axoplasm activity, while favoring GFLphosphorylation. The difference between axoplasmand GFL is significant (p � 0.009) at 200 �M. Theautoradiographs reveal moderate stimulation ofphosphorylation in some axoplasm proteins, whileno new phosphorylated protein bands appeared inthe GFL, a pattern similar to Figure 1(C) (GFLendogenous) in the presence of both okadaic acidand vanadate (data not shown). Here, too, moststimulation by vanadate occurred in the low molec-

ular weight proteins, suggesting that protein ty-rosine phosphatases may play a role in regulatingthe activity of these molecules in the GFL. It shouldbe noted that in these assays the specific tyrosineresidues on different proteins that were phosphor-ylated was not determined. In fact, it is possible thatthe phosphorylation profiles in the presence of van-adate result from indirect effects of PTPs on theactivity of other ser/thr kinases and phosphatases.What is significant, however, is that vanadate af-fects endogenous phosphorylation in GFL extractsdifferently from the axoplasm.

Figure 2 Kinase activities in GFL and axoplasm extracts are differentially sensitive to NaCl ions.Kinase activities assayed in the absence of phosphatase inhibitors. (A) Endogenous activity as cpm� 105 of axoplasm (solid squares) and GFL (open squares) extracts at different NaCl concentrations(example of one of two experiments). (B) Autoradiograph of SDS PAGE gel showing patterns ofendogenous protein phosphorylation at different NaCl concentrations in axoplasm and GFL. Arrowat low molecular weight fractions; most intensely phosphorylated in GFL extracts. HMW � highmolecular weight NF complex.

520 Grant and Pant

Tyrosine Kinase Activity of Whole Axonand GFL Extracts

Vanadate stimulation of endogenous protein phos-phorylation, particularly in the GFL fractions, sug-gests the active participation of protein tyrosine ki-nases and phosphatases in cytoskeletal proteinphosphorylation. As indicated above, the mechanismof vanadate action is not clear, because it may alsohave indirect affects on other kinases. To determinewhether tyrosine kinases are involved, we comparedprotein tyrosine kinase activity using a tyrosine pep-tide as substrate. In this case, whole, intact axons withsheaths and GFLs were extracted in Buffer C (serine/

threonine phosphatase inhibitors only) as soluble ex-tracts in the absence of Triton X-100 (S1), and solubleextracts in the presence of Triton-X 100 (S2), andassayed in tyrosine kinase assay buffer without van-adate (see Materials and Methods). The S2 fraction isenriched in membrane-bound kinases and phospha-tases, which is why we extracted whole axons ratherthan axoplasm to compare the relative contributionsof axoplasm and sheath to phosphorylation activity. InFigure 5(A) we see that tyrosine kinase activities ofS1 fractions of axons and GFL were not significantlydifferent. Activity of the GFL S2 fraction, however(approximately 50% increase over endogenous), wassignificantly greater than the axon S2 fraction (p

Figure 3 Effect of okadaic acid (OA) on phosphorylation of endogenous and exogenous (histone)substrates by GFL and axoplasm extracts. (A) Endogenous phosphorylation at different concentra-tions of OA. Axoplasm � black bars, GFL � hatched bars (means � S.E., n � 4). *p � 0.02; **p� 0.0008 (two-tailed unpaired t test). (B) Histone phosphorylation at different OA concentrations(n � 4). (C) Representative sample autoradiograph of OA effects on phosphorylation patterns ofendogenous and histone substrates in GFL. (D) Sample autoradiograph of OA effects on axoplasmextracts. Arrows mark site of histone band.

Kinase/Phosphatase Activities in Squid Giant Neurons 521

� 0.04, two-tailed unpaired t test, n � 4), suggestinghigher protein tyrosine kinase activities in the GFLmembrane-enriched extract.

We also examined the effect of vanadate onendogenous tyrosine kinase activity of axon andGFL fractions. Again, assuming that the principaleffect of vanadate is on PTPs, we reasoned that theextent of vanadate stimulation of endogenous phos-phorylation in the absence of substrate peptide wasan indirect measure of the level of endogenoustyrosine phosphatase activity. Using the identicalconditions of extraction and assay as above for theassay of tyrosine kinase, we studied the effect ofdifferent concentrations of vanadate on whole ho-mogenates (wh), S1 and S2 fractions [Fig. 5(B)–(D)]. In these studies we used higher concentrationsof vanadate than are usually employed as phospha-tase inhibitors in standard kinase assays. In Figure5(B) and (C), the effect of 1 and 2 mM vanadate onextracts is shown. Note that wh and S1 fractions ofaxons and GFL exhibited equivalent levels of acti-vation in the presence of vanadate (150 –300% overendogenous levels), suggesting the presence ofPTPs. At both concentrations, however, only theGFL S2 fractions exhibited significantly higher per-centage increases in activity than the axonal S2fraction, which, showed no activation (p � 0.05and p � 0.007, respectively, two-tailed unpaired ttest). This suggests the presence of higher levels ofmembrane bound protein tyrosine phosphatases inthe GFL extract compared to the axon in its sheath.

The assays above were carried out on frozen tis-sues stored at �20°C. We also extracted freshly pre-

pared tissues in buffer C and assayed in the presenceand absence of 5 mM vanadate [Fig. 5(D)]. The pat-tern of vanadate stimulation of all axon fractions wasrelatively similar to the frozen/thawed axon extracts.The GFL whole homogenate showed even greaterstimulation (more than 600%), while stimulation ofendogenous phosphorylation in S1 and S2 fractionswas similar to the results at 2 mM vanadate, with theGFL S2 activity significantly higher than the axonalS2 fraction (p � 0.0004). The data suggest that GFLextracts, particularly those containing Triton X solu-ble enzymes, are enriched in protein tyrosine phos-phatases.

To determine which endogenous proteins arephosphorylated in the presence of 1–2 mM vana-date, gels and autoradiographs were prepared fromfresh tissue assay mixtures. An example of a typicalassay is shown in the autoradiograph in Figure 6.What is conspicuous in this figure is that endoge-nous phosphorylation patterns of axon and GFL areremarkably similar. In this assay system, we see forthe first time, that similar proteins in all fractions ofaxon and GFL are phosphorylated, including a pu-tative tubulin fraction. The endogenous axonal pro-file of phosphorylation (first lane in all panels)exhibited more robust phosphorylation, particularlyin TUB, NF-220, and HMW bands than in the GFL.In the axon, however, stimulation of protein phos-phorylation by vanadate was noted primarily in thewh and S1 fractions but not in the S2, where van-adate seemed to inhibit instead, which is consistentwith results shown earlier in Figure 5. On the otherhand, vanadate in all GFL fractions did have astimulatory effect for most proteins, particularlytubulin and the low molecular weight molecules atthe bottom of the gel (arrow). It should be stressedthat it is not possible to determine which residueswere phosphorylated under these conditions. Ty-rosines on some proteins may have been phosphor-ylated, but we cannot exclude the possibility thatser/thr kinases were also activated indirectly. Nev-ertheless, the differential phosphorylation profilesof the S2 fractions of axon and GFL at these van-adate concentrations is consistent with the hypoth-esis that protein phosphorylation is regulated lo-cally within each neuronal compartment.

Protein Tyrosine Phosphatase Activitiesof Axon and GFL Extracts

The vanadate data suggested greater levels of tyrosinephosphatase activity in GFL fractions. To explore thisfurther we used a colorimetric assay for a more directassay of protein tyrosine phosphatase activity based

Figure 4 Effect of vanadate on endogenous kinase activ-ity of frozen-thawed axoplasm and GFL extracted in BufferA (no phosphatase inhibitors). Activity measured as %increase in the presence of different concentrations of van-adate (means � S.E.). Black bar � axoplasm, hatched bar� GFL. Numbers in parentheses � n; **p � 0.009 (two-tailed unpaired t test).

522 Grant and Pant

on a Malachite green reagent (BioMol) to measurephosphate released from appropriate substrates(Harder et al., 1994). The results are shown in Figure7. Using a specific tyrosine-phosphopeptide as sub-strate (Daum et al., 1993) we compared fresh extractsof axons and GFL. Note that the extraction buffercontained 1% NP 40, which should solubilize mem-brane bound proteins and should be equivalent to theS2 fractions assayed above (Figs. 5 and 6) The GFLextracts showed more than two times the axonal PTPactivity (p � 0.02, two-tailed unpaired t test). Theseresults are consistent with the hypothesis that GFL S2extracts contain higher levels of tyrosine phosphatasesand, accordingly, are more sensitive to vanadate asseen in Figure 5. In general, the tyrosine phosphataseactivities in these assays were considerably lowerwhen compared to phosphatase activities of mostmammalian tissues.

Figure 5 Effect of vanadate on tyrosine kinase activity.(A) Tyrosine kinase activity of axon and GFL supernatantfractions in the absence (s1) and presence of 1% Triton-X(s2). Activity measured as % increase over endogenous inpresence of peptide (n � 4). Black bar � axon; white bar� GFL. *p � 0.04 (two-tailed unpaired t test). (B) Endog-enous kinase activity of axon and GFL as determined in thepresence of 1.0 mM vanadate. No peptide substrate in assay.Activity measured as % increase over endogenous (means� S.E.). *p � 0.05 (n � 2, two-tailed unpaired t test). (C)Activity at 2 mM vanadate. **p � 0.007 (n � 4, two-tailedunpaired t test). (D) Activity in presence of 5 mM vanadate.**p � 0.004 (n � 3, two-tailed unpaired t test) Black� axon; hatched � GFL; wh � whole homogenate; s1�first supernatant; s2 � 2nd supernatant in 0.1% TritonX-100.

Figure 6 Sample of autoradiograph of tyrosine kinaseassays of whole (wh), S1, and S2 extracts of axons and GFLfrom freshly prepared tissues in presence of vanadate. Smallarrow in axon panel at high molecular weight band (HMW)disappears in vanadate. Note phosphorylation at NF-220and TUB in whole (wh) and S1 fractions. Large arrow atlow molecular-weight fraction in GFL. Note that variousprotein bands in GFL are phosphorylated as strongly as inthe axon, particularly in vanadate.

Kinase/Phosphatase Activities in Squid Giant Neurons 523

Western Blots of Axoplasm and GFLExtracts to Identify Protein TyrosinePhosphatases

Because no squid-specific antibodies to tyrosine phos-phatases were available, we resorted to commerciallyderived antibodies to mammalian specific phospha-tases to explore the possibility that axoplasm and GFLextracts expressed different tyrosine phosphatases.The tyrosine phosphatase antibodies used, a receptorPTP� and two cytosolic PTPs, PTP1B and SHPTP-1,were prepared against peptides in the conserved PTPcatalytic domain of the respective human PTPs (car-boxy terminus of PTP� and SHPTP-1, the amino-terminus of PTP1B). At best, assuming that catalyticdomains are evolutionarily conserved, we reasonedthat despite the evolutionary divergence betweenmammals and cephalopods, these antibodies mightreveal different patterns of expression in each neuro-nal compartment. The results for GFL, axon, andstellate ganglion (SG) extracts are shown in Figure 8.Note that the SG contains the large cell bodies of theGFL, glia and many afferent axons converging on thegiant synapse. The PTP� receptor antibody reacts inmammals with a 250-kDa protein, and as seen in thefigure, protein bands at slightly less than 250 kDareact only in the GFL and SG-soluble S1 fractions.Reactivity at 250 kDa is much more diffuse in theTriton soluble (membrane bound) S2 fractions, how-ever. The cytosolic PTP1B phosphatase should be

detected at 50 kDa, and is expressed in the S1 and S2fractions of GFL and SG, but is absent from theaxoplasm. Finally, the SHPTP-1 cytosolic phospha-tase which, in mammals, is approximately 65 kDa, isexpressed in the S1 and S2 fractions of GFL and SG.It is to be noted that additional protein bands in theGFL and SG fractions were expressed and were alsoabsent in the axoplasm (data not shown). Because theantibodies were prepared against the conserved cata-lytic domain of the PTPs in all cases, it is possible thatsome squid phosphatases in the GFL, may have cross-reacted, showing more expression in cell bodies thanin axons. Until these proteins are identified, the datasuggest that GFL and axons differ with respect to theexpression of epitopes related to catalytic domains oftyrosine phosphatases.

Immunocytochemical (ICC) Localizationof Putative Protein TyrosinePhosphatases in Squid Stellate Ganglion

With the same qualifications mentioned above aboutthe specificity of these antibodies, we used them todemonstrate the cellular localization of expression inthe squid stellate ganglion by immunocytochemicalassay (Fig. 9). What is of interest is that each mem-brane-bound tyrosine phosphatase antibody, PTP�and PTP� showed a similar cell body localization inthe form of cytoplasmic granular clusters that wereabsent from giant axons [Fig. 9(B)–(D)]. There wasno evidence of any membrane specific expression,however. A strong axon-specific antibody that reactswith phosphorylated NF220 in the squid (Takahashi etal., 1995) is illustrated to contrast axon staining with cell

Figure 7 Tyrosine phosphatase activity of Buffer A(w/1% NP 40) extracts (S2 equivalent) of fresh axon (black)and GFL (hatched) tissues as assayed with a specific phos-phopeptide substrate using the Malachite green colorimetricassay. Specific activities as nmols PO4/ug protein/h (means� S.E.). *p � 0.02 (n � 6, two-tailed unpaired t test).

Figure 8 Western blots of axon and GFL extracts assayedfor the expression of PTPs. S1 ans S2 fractions compared.Note that for each PTPase antibody, the S1 and S2 fractionsof the GFL and the whole stellate ganglion exhibited higherexpression than axons in bands that corresponded to thespecific molecular weights of the mammalian proteins, re-spectively, 250 kDa for the receptor PTP� , 50 kDa for thecytoplasmic PTP1B and 65 kDa for the cytoplasmicSHPTP-1.

524 Grant and Pant

Figure 9 Immunocytochemical localization of putative protein tyrosine phosphatases in sectionsof stellate ganglion. (A) Section stained with secondary antibody only, (rabbit antigoat) as a negativecontrol for (B), (C), and (D); 100 u bar for (A) and (B). (B) Section stained with 1:100 PTP�. Noteintense labeling of large cell bodies and neuropil in center of ganglion while sections of giant axonsare unstained. (C) An enlarged view of the same section to illustrate the nature of the condensedgranular staining pattern within cell bodies (arrowheads). Giant axon is relatively nonreactive; 100 ubar for (C), (E). Staining of giant cell bodies in ganglion with 1:100 PTP�. Here, too, giant axonstained as in (B) above; 100 u bar for (D), (F). (E) Negative control for (F), stained with secondaryantibody (rabbit antimouse). (F) Section stained with SMI31 which is expressed on phosphorylatedNFs seen in giant axon and small axons in nerve cross-section. Note no staining in cell bodies.

Kinase/Phosphatase Activities in Squid Giant Neurons 525

body staining [Fig. 9(F)]. Because we cannot identifythe reactive epitope in these sections, nor do we knowanything about crossreactivity with squid proteins, wecan only conclude that the tyrosine phosphatase anti-bodies did show specific reactivity with cell bodyepitopes rather than axonal proteins. These results areconsistent with the Western blot data above suggest-ing localization of tyrosine phosphatases in cell bod-ies more so than in giant axons.

DISCUSSION

The squid giant axon system has made it possible tostudy some of the factors that regulate the reversiblephosphorylation of proteins in the cell body and ax-onal compartments of large neurons. The complexityof this regulation is manifest in the diversity of ki-nases and phosphatases involved, their reciprocal ac-tivation/inactivation interactions, and the factors spe-cifically assembling and targeting these players tointracellular compartments. Our results are consistentwith the hypothesis that each cellular compartment ischaracterized by a unique “phosphorylation machine”(Takahashi et al., 1995; Grant et al., 1999). The dif-ferential effects of high salt on endogenous proteinphosphorylation activities in axoplasm and GFL dosuggest that the association of kinases, substrates, andphosphatases in each compartment complex is alsodifferent. Although we do not understand the natureof these complexes, nor the role of salt ions in regu-lating their activity, our data suggest that differentphosphatases do play a key role in regulating thecompartment-specific patterns of phosphorylation.

Over the years of studying phosphorylation ofsquid giant axons and cell bodies, the most remark-able and reproducible observation was the signifi-cantly higher patterns of cytoskeletal protein phos-phorylation in the axonal compartment compared tothe GFL (Pant et al., 1986; Cohen et al., 1987; Taka-hashi et al., 1995; Grant et al., 1999). Despite thecomplete absence of phosphatase inhibitors, severalendogenous proteins, including cytoskeletal proteinssuch as NF-220 and tubulin, were extensively phos-phorylated in axons while phosphorylation of a sim-ilar range of diverse endogenous proteins in the GFL[see Fig. 1(A)], was extremely low or virtually absentin vivo and in vitro. All GFL phosphorylation activitywas confined to a low molecular weight (�6 kDa)fraction instead, which raises the possibility that theperikaryal cytoskeletal proteins were degraded byproteases from the lysosomes present in cell bodiesbut absent in axons. We believe this is unlikely be-cause (1) a concentrated protease inhibitor cocktail

was present in the extraction buffer, which also lackedcalcium, essential for calpain activity, a principal Ca-dependent protease in axoplasm and cell bodies (Pantet al., 1982); (2) the Coomassie stained gels [Fig.1(A)] indicate that the profile of expressed proteins issimilar in both preparations except for the phosphor-ylated NF 220. If high molecular proteins in the GFLwere degraded by lysosomal proteolysis, we wouldhave expected a signifcant downward shift in molec-ular weights of GFL proteins, which was not the case.Finally, (3) the GFL phosphorylation profile in Figure6 and its similarity to the axonal profile indicates thathigh molecular weight proteins are present in the GFLextracts and have not been degraded.

How, then, do we explain the absence of endoge-nous protein phosphorylation in the GFL? Amongseveral possible explanations, two obvious ones arise:(1) axons have more serine/threonine kinase activity;(2) there are more active serine/threonine phospha-tases in the GFL. As to the first, we find that kinaseactivities for exogenous substrates, casein and his-tone, in the presence of phosphatase inhibitors, aresimilarly active in each cell compartment, in agree-ment with a previous study (Grant et al., 1999). More-over, GFL and axoplasm lysates share many kinasesas determined by Western blots (Grant et al., 1999).As for ser/thr phosphatases, they also seem to be moreabundant in axons because activities of endogenousaxonal kinases were stimulated eightfold by okadaicacid, while GFL extracts (except in the presence ofexogenous substrates) were unaffected (Fig. 3). Theresponse to okadaic acid is consistent with previousobservations showing axoplasm lysates and P13 mul-timeric complexes expressing higher levels of PP2Aand calcineurin (PP2C) than the GFL (Grant et al,1999).

One explanation for the different endogenousphosphorylation patterns in cell body and axoplasm isthat the protein substrates in axonal multimeric com-plexes are in structural conformations with manymore ser/thr sites accessible for phosphorylation. NFproteins, for example, though synthesized in cell bod-ies, undergo assembly and transport within the axonwhere their KSP enriched tail domains are extensivelyphosphorylated in vivo (Cohen et al., 1987; Nixon andShea, 1992; Pant, 1995; Pant et al., 2000; Jaffe et al.,2001). These sites are not phosphorylated in cell bod-ies except in neurodegenerative disorders such asALS (Miller et al., 2002). Other proteins, many asso-ciated with cytoskeletal proteins, also undergo con-formational changes as they are transported from cellbody into axon to assemble into a stable axonal frame-work, and they too, may expose phosphorylation sites.Because the axonal compartment is enriched in as-

526 Grant and Pant

sembled cytoskeletal proteins, we assume the kinase/substrate complexes form a more active multimericcomplex than in the GFL (Grant et al., 1999). Hence,the greater endogenous protein phosphorylation ofaxonal complexes, even in the absence of phosphataseinhibitors, may be due to greater affinity of kinases toendogenous substrates than in GFL fractions.

The differential effects of vanadate on GFL and ax-ons also suggest that tyrosine phosphatases may play arole in regulating endogenous phosphorylation. Vana-date has been employed as an inhibitor of PTPs instandard assays of kinase activity, but its specificity andits mode of action are still not well understood (Gordon,1991). Vanadate also inhibits ATPases, and in someinstances may stimulate certain PTPs. Its chemistry iscomplex; it undergoes polymerization, depending onpH, and it has a variety of biological effects in vivo andin cell lysates (Gordon, 1991). Nevertheless, others, andwe have used it as a probe into the activities of PTPs andPTKs, assuming that its effects are similar in extractsfrom each neuronal compartment.

With these qualifications about the specificity of van-adate inhibition, we have shown that the GFL is moresensitive to this inhibitor than the axon; that is, in dif-ferent concentrations of vanadate, endogenous phos-phorylation of GFL proteins was significantly stimulatedcompared to the axon. This was particularly true for theTriton X soluble membrane bound S2 extract suggestingthat inhibition of membrne-bound PTPs, stimulates theactivity of protein kinases in the GFL. Moreover, in thepresence of high vanadate concentrations, a more exten-sive pattern of GFL endogenous phosphorylation wasseen, similar to that in axoplasm, with phosphorylationof higher molecular weight cytoskeletal proteins (seeFig. 6). This profile of phosphorylation was never beforeseen in previous studies of GFL extracts in standardkinase assays, even in the presence of okadaic acid andlow vanadate concentrations [see Fig. 1(B) and (C)]. Itappears that vanadate, by virtue of its inhibition of PTPsin the GFL, may have affected the overall activities ofser/thr kinases as well as protein tyrosine kinases therebystimulating endogenous phosphorylation.

The higher PTP activity of the GFL extract and thehigher tyrosine kinase activity of the GFL S2 fractionsuggests that tyrosine kinases and/or tyrosine phos-phatases may play a more critical role in the regula-tion of endogenous protein phosphorylation inperikarya than in the giant axon, where the activitiesof ser/thr kinases and phosphatases predominate. Al-though neither the Western blot assays nor the ICClocalization studies identify squid-specific PTPs, thegreater crossreactivities of these epitopes in the GFLare at least consistent with the hypothesis that PTPsplay a more important regulatory role in endogenous

phosphorylation of GFL than in the axon. Because ofthe complex network of reciprocal interactions regu-lating kinases and phosphatases in various signalingcascades, however, the mechanisms of action are notunderstood.

Various kinases in the MAP kinase signal transduc-tion pathways are activated by phosphorylation at morethan one site, usually a serine and a tyrosine residue. Forexample, the Erk1/2 kinases are activated by phosphor-ylation at T183 and Y185 by MEK1, itself activated byphosphorylation at a serine 217 or S221 (Cohen, 2000).Dephosphorylation at any of these sites, either by classesof ser/thr phosphatases, tyrosine-specific phosphatasesor dual specificity phosphatases that act at serine andtyrosine sites, inactivates the pathway (Keyse, 2000).These phosphatases are presumed to act as negativefeedback regulators of the various MAP kinase signaltransduction pathways, and a preferential targeting ofthese to cell bodies would be consistent with the obser-vations that cytosolic and membrane bound vanadate-sensitive PTPs are more prevalent in the GFL than inaxons. We believe that future studies of the selectivelocalization and relative roles of squid specific ser/thrphosphatases and PTPs in GFL and axons should pro-vide further insight into the compartment-specific regu-lation of cytoskeletal protein phosphorylation in largeneurons.

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