High-Level Expression of Dok-1 in Neuronsof the Primate Prefrontal Cortex andHippocampus

A. Smith,1 J. Wang,1 C.M. Cheng,1 J. Zhou,1 C.S. Weickert,2 and C.A. Bondy1*1Developmental Endocrinology Branch, National Institute of Child Health, National Institutes of Health,Bethesda, Maryland2National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland

The docking protein p62Dok-1 (Dok-1) has a central rolein cell signaling mediated by a wide range of proteintyrosine kinases, including intrinsic membrane kinases,such as the insulin-like growth factor-1 (IGF-1) receptor.To elucidate potential IGF signaling mechanisms, weused DNA array technology to investigate novel kinasetargets expressed in the primate dorsolateral prefrontalcortex (DLPFC). Dok-1 transcripts were among the mostabundant found in this structure. Because Dok-1 expres-sion has not been characterized in brain, we evaluated itsexpression pattern using immunoblotting, in situ hybrid-ization, and immunohistochemistry in the rhesus monkeyprefrontal cortex and hippocampal formation. Dok-1 an-tibodies identified a 62-kDa band in lysates from theDLPFC, consistent with the known size for Dok-1. In situhybridization showed that Dok-1 mRNA was expressedin all layers of the DLPFC and in all neuronal subregionsof the hippocampal formation. Immunohistochemicalanalysis showed Dok-1 immunoreactivity concentratedin pyramidal neurons of cortical layers IV–V and through-out Ammon’s horn and in granule neurons of the dentategyrus. Dok-1 expression was also identified in endothe-lial cells of cerebral blood vessels. These expressionpatterns are very similar to those of the IGF-1 receptor andsuggest that Dok-1 could be among the downstream tar-gets of IGF signaling in areas of the primate brain involvedin learning and memory. Published 2003 Wiley-Liss, Inc.†

Key words: prefrontal cortex; neuron; IGF1 receptor;hippocampus; pyramidal neuron; rhesus monkey

The docking protein p62Dok-1 (Dok-1) plays acentral role in cell signaling mediated by a wide range ofprotein tyrosine kinases (Yamanashi and Baltimore, 1997).It is a founding member of a class of docking proteinscontaining an amino-terminal pleckstrin homology do-main, several Src homology 3 (SH3) recognition sites, anda carboxyl terminus with tyrosine residues activated byreceptor-based tyrosine kinases secondary to stimulationby insulin, insulin-like growth factors (IGFs), vascularendothelial growth factor (VEGF), and platelet-derivedgrowth factor (PDGF; Guo et al., 1995; Sanchez-Margalet

et al., 1995; Holgado et al., 1996; Yamakawa et al., 2002).It appears that Dok-1 mediates cell signaling and helps toregulate cell proliferation and migration, principallythrough inhibitory effects on rasGAP activity (Carpino etal., 1997; Bhat et al., 1998; Kashige et al., 2000; Tamir etal., 2000; Di Cristofano et al., 2001; Songyang et al.,2001). In particular, the formation of a Dok-1-rasGAPcomplex results in the down-regulation of rasGAP activityand the inhibition of mitogenic effects of these variousgrowth factors (Kashige et al., 2000; Di Cristofano et al.,2001; Wick et al., 2001; Shah and Shokat, 2002).

Dok-1 expression and effects have been character-ized primarily in hematopoietic tissues and cells (Carpinoet al., 1997; Jones and Dumont, 1998; Yamakawa et al.,2002). Although the expression of other members of theDok family (Dok-2, Dok-4, Dok-5) has been noted inbrain (Grimm et al., 2001), nothing is known concerningthe expression and distribution of Dok-1 in brain. In thecourse of a DNA array study of the effects of estrogen onrhesus monkey prefrontal cortex, we noted that Dok-1was one of the most abundant transcripts detected, al-though it was not altered by estrogen. In this study, wecharacterize Dok-1 gene and protein expression in theprimate prefrontal cortex and hippocampal formation.These expression patterns suggest that Dok-1 may repre-sent an important factor in the modulation of cell signalingby growth factors such as insulin, IGF-1, and VEGF in theprimate brain.

MATERIALS AND METHODS

Animals

Female rhesus monkeys (Macacca mulatta) 5–7 years of age(n � 9) from the NIH Poolesville colony were used in accor-dance with a protocol approved by the NICHD Animal Careand Use Committee and the guidelines published in the NIH

*Correspondence to: C.A. Bondy, Bldg. 10/10N262, 10 Center Dr., NIH,Bethesda, MD 20892. E-mail: bondyc@mail.nih.gov

Received 30 May 2003; Revised 2 September 2003; Accepted 3 September2003

Journal of Neuroscience Research 75:218–224 (2004)

Published 2003 Wiley-Liss, Inc. †This article is a US Government workand, as such, is in the public domain in the United States of America.

Guide for the Care and Use of Laboratory Animals (NIH PublicationNo. 85-23). Animals were sacrificed with pentobarbital. Thebrains were removed and cut sagitally at the midline; brainhemispheres were cut into 1.5-cm coronal slabs through theentire cerebrum, rapidly frozen in isopentane chilled on dry ice,and stored at –70°C. The dorsolateral prefrontal cortices (BA46) from one side of the brain were cut into 12-�m-thicksections at –24°C and thaw mounted onto poly-L-lysine-coatedslides and stored at –70°C until use. Anatomically matched tissuefrom the contralateral side was dissected for protein and RNApreparation.

DNA Array

Total RNA was prepared from prefrontal cortex by Trizolreagent (Invitrogen Life Technologies, Carlsbad, CA) and fur-ther purified with RNeasy Midi Kits (Qiagen Inc., Valencia,CA). Total RNA was reverse transcribed into cDNA and la-beled with 33P-dCTP (Amersham Pharmacia, Piscataway, NJ).The protocols for probe labeling and array filter hybridizationare described in detail at http://www.grc.nia.nih.gov/branches/rrb/dna/protocols.htm. NIA Human Neurobiology Arrays con-taining �1,150 brain enriched cDNAs were used in this study,and the cDNA clone listing is available at http://www.grc.nia.nih.gov/branches/rrb/dna/array.htm. Duplicatearrays were hybridized for each animal. Array results werecaptured using a PhosphoImager (Amersham Biosciences,Sunnyvale, CA), and data were analyzed with ImageQuantsoftware (Amersham Biosciences), followed by z-score statisticalanalysis.

In Situ Hybridization

The human Dok-1 complementary DNA was a 1.53-kbfragment inserted into the EcoRI-NotI site of pT7T3D-PacIvector (ATCC 961767, accession No. AA142943; AmericanType Culture Collection, Manassas, VA). The insert sequenceand orientation were verified by DNA sequencing. 35S-labeledRNA probes were synthesized to a specific activity of approx-imately 2 � 108 dpm/�g as previously described (Bondy andLee, 1993). Antisense probe was synthesized by EcoRI plasmidlinearization, followed by T3 RNA polymerase in vitro tran-scription; sense probe was synthesized by T7 RNA polymeraseafter NotI plasmid linearization. A plasmid containing a 256-bpcDNA fragment encoding the IGF-1 receptor (IGF-1R) waslinearized using Not1 and antisense riboprobe transcribed usingT7 RNA polymerase. The sections were fixed; soaked in 0.25%acetic anhydride, 0.1 mol/liter triethanolamine hydrochloride,and 0.9% NaCl for 10 min; washed; and dehydrated in gradedalcohol solutions. 35S-labeled probe (107 cpm/ml) was added tohybridization buffer composed of 50% formamide, 0.2 M NaCl,50 mM Tris-HCl (pH 8), 250 �g transfer RNA/ml, 10%dextran sulfate, 1� Denhardts’ solution, and 10 mM dithiothre-itol. Coverslips were placed over the sections, and the slideswere incubated in humidified chambers overnight (14 hr) at55°C. Slides were washed several times in 4� SSC (NaCl andsodium citrate; Biofluids, Rockville, MD) to remove coverslips.Sections were dehydrated in graded alcohol solutions (30–100%), followed by a 10-min incubation in a 50% formamidebuffer at 60°C. They were then allowed to cool in 2� SSC,then subject to RNase A treatment (20 �g/ml) for 30 min,

starting at 37°C, allowed to cool to room temperature. Slideswere washed in graded salt solutions of 2�, 1�, and 0.5� SSC,then 0.1� SSC at 50°C for 15 min, then allowed to cool in0.1� SSC. Finally, sections were passed through graded alcohols(30–100%), air dried, and exposed to Bio-Max MR film (East-man Kodak, Rochester, NY) for 7 days. Some sections werelater coated using NTB2 photographic emulsion (EastmanKodak), exposed for 7–14 days, and developed before counter-staining with hematoxylin and eosin.

Western Blot Analysis

Immunoblotting was performed as previously described(Cheng et al., 1998), with minor modifications. A small portionof frontal cortex (�1 g) from along the principle sulcus adjacentto the region used for histochemical analysis was homogenizedin a boiling solution containing 10 mM Tris (pH 7.4), 1 mMsodium orthovanadate, and 1% SDS at a ratio of 1 g tissue to17.5 ml solution. An equal volume of total-brain homogenatesfrom each sample (10 �l) was loaded and resolved on NuPAGE10% Bis-Tris Gels (Invitrogen Life Technologies) and trans-ferred to nitrocellulose membranes using electrophoretic transfercell (Bio-Rad, Hercules, CA). Membranes were incubated inblocking buffer (Chemicon, Temecula, CA) for 1 hr at roomtemperature, followed by overnight incubation at 4°C withanti-human Dok-1 antibody (sc-6277; epitope M-276; reactiveto mouse, rat, and human; Santa Cruz Biotechnology, Inc.,Santa Cruz, CA) used in a 1:200 dilution, with gentle agitation.After incubation with horseradish peroxidase-linked secondaryantibody used in a 1:2,000 dilution in 1� PBS-Tween plus 1%milk, the membrane was incubated in a solution containingequal volumes of SuperSignal Luminol/Enhancer and StablePeroxide Solutions (Pierce, Rockford, IL) at room temperaturefor 5 min. Protein bands were then visualized on Kodak XOmat-AR film (Amersham, Cleveland, OH).

Immunohistochemistry

Immunohistochemistry for Dok-1 was performed withthe avidin-biotin-immunoperoxidase technique, as describedpreviously (Cheng et al., 1998). The frozen brain sections werefixed in 4% formaldehyde for 10 min. After quenching in 3%H2O2 for 10 min, tissues were blocked in 10% normal serum for30 min, followed by incubation with primary antibodies at 4°Covernight. Two anti-Dok-1 polyclonal antibodies, sc-6374 (C-19) and sc-6277 (M-276; Santa Cruz Biotechnology, Inc.), wereused at a 1:100 dilution (�2 �g/ml) in separate trials. Afterwashing, sections were incubated in biotinylated secondary an-tibodies (1:400) for 30 min. The signal was detected and am-plified using the ABC peroxidase method (Vector, Burlingame,CA) and visualized with 3,3�-diaminobenzidine. Controls forthe immunohistochemistry procedure were performed by omis-sion of primary antibody in the incubation and substitution withblocking solution and were processed in parallel with the ex-perimental groups. Sections were counterstained with 0.2%methyl green for 5 min and briefly washed in distilled water,before dehydration in alcohols and xylene, and mounted withpermount and coverslips.

Dok-1 Expression in Primate Brain 219

RESULTSAnalysis of the microarray data revealed that, among

the approximately 1,150 genes represented in the NIAneuroarray probe set, Dok-1 gene expression was consis-tently within the top 2% of those most highly expressedwithin the primate prefrontal cortex (data not shown).This was confirmed by in situ hybridization, whichshowed high levels of specific Dok-1 mRNA expressionthroughout the prefrontal cortex (Fig. 1A). This mRNA isselectively concentrated in the gray matter in corticallayers II–VI. There is increased intensity of expression intwo bands running parallel to the pial surface, one atmidcortex level and one in deeper cortical areas. There issome positive Dok-1 signal at the pial surface and in layerI. Dok-1 mRNA is also abundant in the hippocampalformation, where it is most concentrated in the dentategyrus (Fig. 1B). Dok-1 mRNA signal was prominent inCA4 and could be found in CA3 and CA1 subfields.Parahippocampal cortex also has a more intense hybrid-ization signal than the cortex alongside the collateral sul-cus. Negative control staining is shown in Figure 3C.

Western blot analysis showed high levels of Dok-1protein expression in lysates from prefrontal cortex fromall nine monkeys (Fig. 2). The single band found at�62 kDa agrees with the known size of Dok-1 andsuggests that the immunodetection, and subsequent im-munohistochemistry, is specific for this protein. No ap-parent differences in Dok-1 protein levels are foundamong individuals.

Fig. 1. Autoradiographs of Dok-1 mRNA expression in primate prefrontal cortex (A) and hippocam-pus (B) confirm the high expression of this gene as suggested by the microarray data. A,B: Antisenseprobe. C: Sense control probe in adjacent section as background control, hippocampus. ps, Principalsulcus. Scale bar � 500 �m.

Fig. 2. Western blot analysis using polyclonal Ab against human Dok-1confirms specificity and abundance of Dok-1 protein in primate pre-frontal cortex. Shown are protein lysates from dorsolateral prefrontalcortex purified from the brain of nine rhesus monkeys. The Dok-1antibody is noncross-reactive with Dok-2, its closest family member.

220 Smith et al.

In the dorsolateral prefrontal cortex (DLPFC),Dok-1 immunoreactivity was detected in pyramidal andnonpyramidal neurons (Fig. 3). Dok-1-positive cells aredistributed in all layers of the monkey DLPFC (Fig. 3A).Dok-1 containing cells can be found in layer I and may

correspond to nonpyramidal neurons known to be presentthere and/or glial cells. Within layers III and V (Fig. 3B),Dok-1 protein is found in the majority of neurons; how-ever, there are some neurons that appear not to be Dok-1immunoreactive. Negative control staining is shown in

Fig. 3. Dok-1 immunoreactivity in the primate dorsolateral prefrontal cortex. A: Section throughoutprefrontal cortex (�33). B: Cortical layer V. C: Higher magnification (�66) of layer V. D: Controlimmunostaining with ommision of Dok-1 primary antibody from incubation, suggesting that immu-noreactivity is specific. Brown deposits are positive Dok-1 antibody staining against cytoplasmicmethyl green counterstaining.

Dok-1 Expression in Primate Brain 221

Figure 3D. Almost 80% of pyramids in layers IV and Vwere Dok-1 immunoreactive, a lower percentage of py-ramidal neurons being Dok-1 positive in the supragranularlayers (Fig. 4). Dok-1 immunoreactivity was concentratedin the nuclear compartment of pyramidal neurons, non-pyramidal neurons, and some glia. This staining patternwas identical for the two Dok-1 primary antibodies.

Dok-1 expression was also found in the hippocampalformation (Fig. 5). Dok-1 immunoreactivity was particu-larly pronounced in the large pyramidal neurons of Am-mon’s horn (Fig. 5A), with similar numbers of positivecells from subfield CA3 to subfield CA1 (Fig. 4). Underhigher magnification, it is clear that Dok-1 localization ispredominantly nuclear, with possible nucleolar sparing(Fig. 5B). Dok-1 immunoreactivity was also evident in ahigh percentage of dentate granule cells (Fig. 5C). Finally,intense Dok-1 immunostaining was evident in endothelialcells in cerebral blood vessels (Fig. 5D).

Because Dok-1 is a major target for growth factor-stimulated receptor kinases, we investigated the localiza-tion of the important neurotrophic IGF-1R with relationto Dok-1 expression in the primate brain. Heavy IGF-1RmRNA expression was found in parallel with Dok-1 inboth the DLPFC and the hippocampus (Fig. 6).

DISCUSSIONWith this study, we provide the first evidence that

Dok-1 is expressed in the primate brain and show that itsexpression is predominantly neuronal, notably in pyrami-dal neurons of the prefrontal cortex and pyramidal andgranule neurons of the hippocampal formation, two re-gions of the brain important for “higher” functions, suchas cognition and memory. Dok-1, is the prototype mem-ber of a family of adaptor proteins that has at least fivemembers (Dok-1–5), all of which are phosphorylated by awide range of protein and receptor tyrosine kinases (DiCristofano et al., 1998; Jones and Dumont, 1998; Grimmet al., 2001). Previous studies suggested that expression ofDoks-1–4 has been found mainly in hematopoietic and

visceral tissues (Carpino et al., 1997; Lemay et al., 2000;Grimm et al., 2001). However, some limited Dok-4 ex-pression has been reported for the spinal cord and dorsalroot ganglia, and Dok-5 expression has been reported forbrain, spinal cord, and dorsal root ganglia in the mouse(Grimm et al., 2001). Surprisingly, little or nothing isknown of the family’s founding member, Dok-1, in any-thing but hematopoeitic tissues and cells.

Our data from microarray studies, in situ hybridiza-tion, Western blotting, and immunohistochemistry allconfirm substantial expression of Dok-1 in the primatebrain. The only previous study investigating Dok-1 ex-pression in primate brain involved Northern analysis of ahuman multitissue blot, which suggested that Dok-1 ex-pression was found in pancreas and heart, with only veryweak expression in brain (Carpino et al., 1997). To verifyour array data, we first performed in situ hybridization.Using a riboprobe specific for Dok-1 mRNA, we foundan abundant and widespread signal, first throughout thesame prefrontal cortex and then in a further brain region,the hippocampus. At the protein level, immunohisto-chemistry also confirmed specific Dok-1 protein expres-sion throughout the cortex as well as in the hippocampalformation. At high magnification, Dok-1 immunoreactiv-ity appeared to be present at the pial surface, in bloodvessels, but predominantly in neurons, insofar as the in-tense signal found in pyramidal neurons was mostly absentfrom glial cells. This agrees with data for Dok-5, whichalso appeared to be preferentially expressed in neurons ofthe dorsal root ganglia but apparently was absent in glia(Grimm et al., 2001). Positive Dok-1 immunoreactivitywas present in a large subset of neurons in all cortical layersas well as in hippocampal neurons and granule cells of thedentate gyrus. At least three Dok family members, there-fore, appear to be present in the central nervous system,namely, Dok-4 and Dok-5, and in this study we have nowadded Dok-1 to this list.

The function of Dok-1 in the central nervous systemremains unclear. However, based on previous studies ofthis protein, a variety of important modulatory effects mayexist. Dok-1 avidly associates with the ras GTPase-activating protein (rasGAP) after phosphorylation by non-receptor protein tyrosine kinases, such as v-Abl, c-Kitv-Src, and Nck (Carpino et al., 1997; Yamanashi andBaltimore, 1997), and receptor tyrosine kinases by includ-ing IGF, insulin, and VEGF reeptors (Guo et al., 1995;Sanchez-Margalet et al., 1995; Holgado et al., 1996; Ya-manashi and Baltimore, 1997). If Dok-1 expression inbrain is linked with receptor tyrosine kinases, one partic-ularly strong candidate that has also been extensively stud-ied in our laboratory is IGF-1R. Stimulation of rat HTChepatoma cells with IGF-1 induces phosphorylation ofDok-1 and its association with rasGAP (Sanchez-Margaletet al., 1995). The most well-characterized substrate ofIGF-1R is the insulin receptor substrate-1 (IRS-1). Inter-estingly, Dok-1’s structure is very similar to that of IRS-1(Yamanashi and Baltimore, 1997), a known insulin andIGF-1R docking protein (White, 1998). Dok-1 has also

Fig. 4. Dok-1-immunopositive cells in DLPFC cortical layers andhippocampal regions in primate brain. Data shown are mean percentageof cells showing positive staining among 100 cells counted in eachregion in each of three individual animals (with SD).

222 Smith et al.

Fig. 5. Dok-1 immunoreactivity in the primate hippocampus. Brown deposits are positive antibodystaining against cytoplasmic methyl green counterstaining. A: Ammon’s horn, CA1 field. B: High-power magnification; large pyramidal cells of CA1. C: Dentate gyrus granule cells. D: Dok-1immunoreactivity in blood vessel of primate cerebral cortex. Scale bars � 20 �m in A,C, 50 �m inB,D.

Fig. 6. In situ hybridization. IGF-1R and Dok-1 mRNA expression in consecutive sections ofprimate hippocampal formation and DLPFC. A: IGF-1R antisense probe; hippocampal formation.B: Dok-1 antisense probe; hippocampal formation. C: Sense control, IGF-1R; hippocampal forma-tion. Magnification �3.6

Dok-1 Expression in Primate Brain 223

been shown to be a direct substrate for the insulin receptortyrosine kinase and may play an essential role in regulatingthe insulin signaling pathway (Wick et al., 2001). Further-more, Dok-4 and Dok-5, which have been localized inbrain (Grimm et al., 2001), have also recently been iden-tified as substrates involved in insulin signaling (Cai et al.,2003). In this study, we have shown overlapping patternsof Dok-1 and IGF-1R expression in the adult primatebrain, indicating the potential for Dok-1 participation inneuronal IGF-1 signaling. The intense Dok-1 immuno-reactivity found in some blood vessels may also be linkedto hematopoeitic factors, such as VEGF. Stimulation ofthe VEGF receptor in endothelial cells has been shown tocause phosphorylation of Dok-1 similar to that observedduring IGF-1R activation (Guo et al., 1995). The recep-tors for IGF1 and VEGF also share structural similarities,and possible interactions have been suggested. AlthoughDok-1 immunoreactivity was found in cytoplasm, it wasmost notably concentrated in neuronal nuclei. This sug-gests that Dok-1 may be involved in novel signalingpathways in the brain. In conclusion, these studies showthat Dok-1 is present in important regions of the primatecentral nervous system and suggest that it may be animportant mediator of the actions of various protein ty-rosine kinases and growth factors, including the IGF re-ceptor in the brain.

ACKNOWLEDGMENTWe thank R.V. Dreyfuss of the Medical Photogra-

phy Branch, NIH, for excellent photomicroscopy work.

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