Atypical ϑ-protein kinase c displays a unique developmental expression pattern in rat brain

DEVELOPMENTAL BRAIN RESEARCH

ELSEVIER

Developmental Brain Research 85 (lYY5) 23Y-24X

Research report

Atypical

l-protein

kinase c displays a unique developmental pattern in rat brain

Starla E. Hunter, Department

of Zoology,

Michael L. Seibenhener, 331

Funchess

Hull,

Auburn

Unicwsity,

Marie W. Wooten Auburn,

AL

36839-5414.

expression x

USA

Accepted 13 December 1994

Abstract The expression of atypical c-protein kinase C (PKC) was examined during prenatal and postnatal rat brain dcvclopmcnt. Immunoblot as well as transcript analysis revealed a dramatic increase in expression at 2-3 days post-birth. which declined thereafter and remained at levels observed in the adult brain. The expression of &PKC precedes that of the other PKC isoforms in developing rat brain. Subcellular fractionation of pup and adult brain documented distribution between all three distinct fractions (A,B,C), including the low speed pellet composed of nuclei. In adult brain, the kinase was enriched in the A fraction of the sucrose gradient. Specific substrate proteins of (-PKC were characterized in each of the subccllular fractions from both pup and adult brain. Four predominant proteins pp76, pp60-doublet, pp54 and pp45 were identified as [-PKC endogenous substrates. All four proteins were phosphorylated on serine residues, while the pp60-doublet was also phosphorylated on tyrosine. The pp60-doublet was the most predominant substrate, specifically enriched in the A fraction of a sucrose gradient of adult brain and immunoprecipitated by monoclonal antibody to pp60c-s”. Keywords:

Protein

kinase C; Zeta; Development;

Isoform

1. Introduction Protein kinase twelve homologous

C (PKC) is a family composed of isoforms: (Y, PI, pII, y, 6, E, 6, 8, L,

A, p and 77 [9,10,18,20-24,281. Members of the PKC family have been reported to be involved in differentiation, proliferation, sensory transduction, hormone and neurotransmitter release, gene expression as well as secretion [16,18]. PKC isoforms are ubiquitously expressed among most animal tissues with brain displaying high expression levels, thus suggesting an involvement in neural processes 1301.Activation, as well as, expression of specific PKC isoforms in brain has been linked with modulation of ion channels, enhancement of neurotransmitter release, synaptic plasticity, neurite extension and long-term potentiation [l&27,31]. Additionally, dysregulation of PKC levels has been related to neuronal cell death, degeneration of neurites and neuronal toxicity [ 161.

* Corresponding author. Fax: (1) (205) 844-9234. 0165-3806/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0165-3806(94)00219-3

Members of the PKC family have been subdivided into classical, nonclassical and atypical groups based on their requirement for calcium and their susceptibility to PMA-induced downregulation [I8]. Members of the classical group (cPKC), (Y, PI, j?II and y, are dependent on calcium and diacylglycerol for activation. On the other hand, the non-classical isoforms (nPKC), 6, E, 0 and 77,lack the C2 calcium-binding domain common to the cPKC members and are thus calcium independent. Atypical (aPKC) isoforms, i, L, A and /L, also lack the C2 region,

as well

as, one of the repeated

cys-rich

zinc

finger binding motifs within the Cl domain 1181.Due to the missing cys-rich region, <-PKC is incapable of binding phorbol ester and therefore is resistant to phorbol ester-induced downregulation compared to either cPKC or nPKC isoforms [1X]. The structural variations that exist between subdivisions of PKC isoforms suggestunique cellular functions for each family group. Additionally, it has been suggestedthat each individual isoform may have a unique role in cellular signalling processesas well. Several studies have been conducted to examine the expression of PKC isoforms during central nervous

240

S.E. Hunter

et al. /Del,elopmental

system (CNS) development [7,S,11,12,15,29,32]. In rat brain, expression of LY, /3, y and &PKC isoforms is relatively low early in postnatal development and increases incrementally 2 to 3 weeks postnatally. The rapid increase in expression of these isoforms coincides with the commencement of synaptogenesis and myelination. By comparison, /3-PKC reaches a maximum in thymus shortly after birth which corresponds with early maturation of this tissue [321. These observations suggest that developmental regulation is in part dependant upon tissue specific expression of a given PKC isoform. The abundant expression of PKC isoforms in brain suggests that each of these enzymes are involved in a host of specific neuronal processes. A brief report exists examining the expression of 8 PKC isoforms in hippocampus, which includes the &‘-isoform [13]. However, no detailed study has been conducted to date on the expression of this PKC isoform in brain.

Brain

Research

85 (1995)

2.2. Subcellular

2. I. Protein

fractionation

Brains were excised from 2-day-old rat pups or adult animals and gently homogenized in 8 vol HB containing 0.32 M sucrose. The homogenate (H) was centrifuged at 1500X g for 15 min resulting in a supernatant (LSSl and pellet (LSP). The LSP contains mostly large elements and nuclei [1,25]. The supernatant (LSS) was recovered and layered onto a three-step discontinuous sucrose density gradient, followed by centrifugation at 242,000~ g for 40 min resulting in separation of three distinct fractions (A, B, Cl. Post-centrifugation the individual fractions were recovered. The A-fraction of fetal brain (0.32 M/0.75 M interface) contains 70% growth cones, the B-fraction (0.75 M/1.0 M interface) contains highly electron dense cellular fragments and the C-fraction (1.0 M/2.66 M) contains lysed cell fragments, mitochondria and endoplasmic reticulum [25]. All procedures were carried out on ice or at 4°C. Protein concentration of these fractions along with samples from the original homogenate and both the supernatant and pellet from the low speed centrifugation were determined. The individual fractions were analyzed for relative content of I-PKC and endogenous substrates. Expression of I-PKC within LSS and LSP was normalized relative to expression in H, while expression in A, B and C was normalized relative to LSS [l]. 2.3. Endogenous

2. Materials

239-248

substrate

assay

and methods

expression

and Western blotting

Time impregnated rats were obtained from Sprague-Dawley. Rat pups at developmental ages - 10, -4, 0 (birth), 3. 5, 7, 11, 14, 21, 35, and 49 days were asphyxiated with carbon dioxide, the brain removed and immediately frozen with liquid nitrogen. These times were chosen for comparative purposes based upon previous study of cPKC isoform expression in CNS development [32]. For the early samples, sibs were pooled to obtain sufficient tissue for analysis [29]. The samples were stored at -80°C until all samples were collected. The tissue samples were then homogenized in homogenization buffer (HB: 20 mM Tris, pH 7.5, 50 mM P-mercaptoethanol, 0.1 mM EDTA, 1 mM PMSF, 1 FM leupeptin, 10 mM aprotinin, 100 PM NaF, 0.2 mM NasVO,, 1 mM p-nitrophenyl phosphate) and membrane bound protein was extracted by end-over-end mixing at 4°C for 30 min by addition of 0.1% NP-40 (v/v) and 50 mM EGTA [31]. Protein concentration was determined using the Bio-Rad Protein assay and equalized between individual samples before analysis on 10% SDS-polyacrylamide gels. Following electrophoresis, the separated proteins were transferred overnight to nitrocellulose membranes in 10 mM Tris, pH 7.5, 1 mM glycine, and 20% MeOH. The membrane was stained with 1% Ponceau (w/v) in 5% TCA, marked and destained in PBS, pH 7.6 prior to immunoblot analysis. Thereafter, the membrane was incubated in 7% milk-PBS, pH 7.6 with 0.1% TX-100 and 0.1% Tween-20 at 37°C for 4-6 h followed by overnight incubation at 4°C with gentle rocking in 7% milk/PBS/ TX-lOO/Tween-20 with 1.5 pg/ml affinity purified I-PKC or a-PKC antibody (Gibco-BRL). The membrane was washed for 1 h at room temperature in PBS/TX-lOO/Tween-20 with multiple changes, incubated for 1.5 h at 37°C with donkey anti-rabbit HRP coupled secondary antibody (1:3,000 in PBS/TX-IOO/Tween-20). and washed again for 1.5 h in PBS/TX-lOO/Tween-20 as above. Visualization of bound antibody was accomplished using the ECL chemiluminescent system (Amersham) followed by exposure of the membrane to Hyperfilm-ECL (Amersham). The relative intensity of each band was determined using a computer-interfaced densitometer. The immunoblots were in the linear range of detection for each antisera with respect to protein loaded on the gel and exposure time employed.

In brief, each 100 ~1 assay was performed with 20 wg of sample protein [17]. The assay was conducted in 50 mM PIPES, pH 6.5, 0.4 mM EGTA, 10 mM MgCI,, 0.2 mg/ml phosphatidylserine in the presence or absence of 300 FM i-pseudosubstrate peptide (sequence 113-129: SIYRRGARRWRKLYRAN) [31]. The assay was initiated by the addition of 2 pCi/ml y”‘P-ATP containing 20 yM cold ATP and incubated for 30 min at 30°C. followed by the addition of 100 ~1 of SDS-PAGE sample buffer (125 mM Tris, pH 6.8, 20% glycerol, 1.5 M /3-mercaptoethanol, 4% (w/v) SDS, 0.5% (w/v) bromophenol blue). Thereafter, the samples were boiled for 10 min and separated by electrophoresis on a 10% SDS-PAGE gel. The gels were fiied, dried and exposed to Kodak X-OMAT XAR-5 film at either -80°C or room temperature. as indicated. 2.4. Phosphoamino

acid analysis

Analysis of individual phosphorylated amino acids was performed on proteins identified by endogenous phosphorylation reactions. Post-separation by electrophoresis the proteins were transferred to Immobilon-P Transfer Membrane (Millipore) at 50 volts for 3 h in 10 mM Tris, pH 7.5, 1 mM glycine and 20% MeOH. After which, the membrane was stained (0.1% Ponceau in 1.0% glacial acetic acid), marked, destained in 1% glacial acetic acid and dried at 65°C for 5 min. wrapped in plastic, and exposed to Kodak X-OMAT XAR-5 film for 24 h at - 80°C with intensifying screens. Following exposure, the membrane was aligned with the autoradiogram and the individual protein bands were carefully excised. Pieces of Immobilon containing the phosphorylated substrates were pre-wet for 30 s in MEOH followed by 30 set in distilled water and placed in 50 mm culture tubes (Millipore). Fifty ~1 of constant boiling HCI solution (Sigma) was added to individual tubes which were placed in a vacuum sealable vesicle appropriate for acid hydrolysis. After purging with nitrogen, the tubes were incubated at 110°C for 1 h, centrifuged briefly at 14,000~g and the acid, containing partially hydrolysed phosphoamino acids, was removed and placed in a 0.5 ml microcentrifuge tube. The membrane pieces were further washed with distilled water which was added to the acid containing tubes, followed by lyophilization. Thereafter, the sample was washed twice with distilled water and the final dried pellet frozen at -80°C until further analysis.

SE. Hunter

et al. /Developmental

Brain Research

85 (19951 239-248

241

For phosphoamino acid analysis, each sample was resuspended in 5 ~1 of 0.6 M formic acid, 1.4 M glacial acetic acid, pH 1.9 spiked with phosphoamino acid standards (serine, 1:lO; threonine, 1:lO;

c

25,

A

,,I

T

-

-me-

e

--.

--65

LSS

If

0

DAYS

Fig. 2. Subcellular distribution of 5.PKC in brain. The expression of [-PKC in either early (2 day) or adult brain. Subcellular fractions were obtained as indicated (H, whole brain homogenate; LSS, low speed supernatant; LSP, low speed pellet; A. growth cones: B. lysed growth cone particles, neuronal and glial fragments and golgi cisternae: C, neurite shafts, rough ER and mitochondria). Protein (60 pg) from each fraction was analyzed by Western blot analysis with affinity purified anti-[ PKC antibody. Relative levels were obtained by densitometric scanning and are normalized relative to the homogenate for LSS and LSP, while the expression of A,B.C is relative to the level of [-PKC expressed in LSS. These data are the mean? S.E.M. for five separate experiments.

’

tyrosine, 1:20 of a 5 mg/ml stock) and spotted onto a pre-coated cellulose TLC plate (MCB Reagents) in 0.2 @I volume. After the entire sample was applied to the plate, the phosphoamino acids were separated by electrophoresis for 1 h at 1000 volts using the HTLE7000 high voltage thin layer electrophoresis system (C.B.S. Scientific Company) in buffer containing 870 mM glacial acetic acid, 16 mM pyridine, pH 3.5. The plate was dried for 20 min at 65°C. after which the phosphoamino acid standards were visualized by spraying the plate with 0.3% (w/v) ninhydrin in acetone. The plate was exposed to Kodak X-OMAT XAR-5 film at -80°C with intensifying screens for 5-8 days.

, , , , , , , , , ,

-1040

3

7

14

I

I

21

I

I

I

I

35

I 49

25

_ B 20

L

i5

15

% : k

LSP

_

10

-

5-

011 -10

’ 4

’ 0

’ 3

’ 7

’

’ 14

’

’ 21

’

’

’

’

’ 35

’

’

’

11 49

DAYS 25

20

.5. RNA

-1040

3

7

14

21

35

49

DAYS Fig. 1. Expression of PKC in developing rat brain. Expression of cor (Y- PKC was examined during pre- and postnatal periods. A: each sample was immunoblotted with [-PKC isoform specific antibodies. Each data point represents the mean peak area * S.E.M. tn = three separate replicas of this one experiment). Inset: representative autoradiogram of a [-PKC immunoblot. B: relative expression of ru-PKC obtained by immunoblotting a duplicate set of samples. C: relative expression of I-PKC as determined by Northern blotting. Peak area was determined by densitometric scanning of either Western or Northern blot autoradiograms.

extraction

and Northern

blotting

Total RNA was extracted from developing rat brains using the RNAzol methodTM tCinna/Biotecx, Houston. TX) and separated by electrophoresis in a 1% agarose-denaturing gel. RNA was visualized in the gel by ethidium bromide staining, destained in water and then transferred to a Nytran membrane by capillary action using 20 X SSC. Pre-hybridization in buffer containing 1 M NaCI. 0.05 M Tris, pH 8.0. 10% dextran sulfate (w/v), l%, SDS (w/v) and 100 rig/ml salmon sperm DNA was carried out at 65°C overnight followed by hybridization in fresh buffer containing a 32.mer I-PKC specific oligonucleotide (5’ ggATgAAgCTTTgCCACTT TCCCTggTgTTCATTgC 3’ corresponding to a portion of the 3’-untranslated region of I-PKC [2X]) end labeled with 100 PCi y”zP-ATP using T4 kinase and purified over a NENSORB 20 Nucleic Acid Purification Cartridge (NEN-DuPont). Following hybridization, the membrane was washed once at room temperature for 15 min and once at 65°C for 30 min in 2 x SSC + 0.1% SDS and exposed to Kodak X-OMAT XAR-5 film at -80°C with intensifying screens. The resulting autoradiogram was scanned using a laser densitometer with results being expressed as relative peak intensities.

242 2.6. Immrnoprrcipitat~on

brane and the pp60 amino acid analysis.

Immunoprecipitation was conducted using protein obtained from the A fraction. The endogenous substrate assay was scaled by a factor of five to contain 200 pg of protein. The assay was terminated on ice by addition of 1% deoxycholate (v/v). 1% NP-40 (v/v), and 0.1% SDS (w/v). To each 1 ml assay mixture. 100 ~1 of a 1Oc: Pansorbin solution in IP Buffer (50 mM Tris. pH 7.5, 0.5 M NaCI, 1% NP-40 (v/v), 0.5% deoxycholate (w/v). O.lC;; SDS (w/v), IO mM aprotinin, 1 PM leupeptin, 1 mM PMSF, 0.1 mM EDTA, 100 PM NaF. 1 mM Na,VO,) was added, mixed at 3°C for 20 min. followed by centrifugation for 2 min at 14,000~ g. To the resulting supernatant, 2 Kg of monoclonal antibody 327 to c-WC was added per mg of protein and incubated for 2 h at 4°C with constant end-over-end mixing. Thereafter, 50 ~1 of a 10% mixture of rabbit anti-mouse (H+ L) (Jackson Immunoresearch)/Pansorbin in IP Buffer was added followed by incubation for 1 h at 4°C with constant mixing. The immunoprecipitate was pelleted at 14,000~ g and washed three times in excess IP Buffer. To the final pellet 100 ~1 SDS-sample buffer was added, vortexed, boiled, centrifuged and electrophoresed on a 10% SDS-PAGE gel. The separated proteins were transfered to Immobilon-P membrane (Millipore) at 50 volts for 3 h and exposed to Kodak X-OMAT XAR-5 film overnight at ~80°C. Following exposure, the film was aligned with the corresponding transfer mem-

bands

were

excised

and subjected

to phospho-

3. Results 3.1. Der>elopmental and subcellular localization of I-PKC Initial studies were undertaken to determine whether the expression of I-PKC was developmentally regulated in rat brain. The specificity of the affinitypurified antibodies used in the present study has been previously verified in our laboratory [31]. Immunoreactivity to the - 74 kDa protein band detected with the (-PKC antisera was blocked by preincubation of the antibody with specific peptide antigen [34]. Western blot analysis of whole brain homogenates obtained from embryonic-fetal, perinatal and post-natal rats revealed peak expression of I-PKC within the 2-day period after birth (Fig. 1A). Three peaks of expression

A

106 Kd

1 )

pp76

4 )

PP54

5 b

pp45

80

-

H-

H+

LSS-

LSS+

LSP- LSP+

A-

A+

B-

B+

c-

32.5

c+

Fig. 3. Endogenous [-PKC substrates. Shown is a representative autoradiogram of endogenous substrates identified in subcellular fractions of 2-day-old rat pup brain (A) or adult rat brain (B). The brain samples were separated by differential sucrose gradients and protein (20 pg) obtained from an individual fraction (H, LSS. LSP, A. B. C) + i-pseudosubstrate was incubated in an endogenous substrate reaction. The samples were resolved on 10% SDS-polyacrylamide gels. The arrows indicate individual bands which consistently decreased in the presence of pseudosubstrate peptide.

SE. Hunter

et al. / DrL~elopmental

Brain

Research

85 (1995)

243

239-248

106Kd

lb

PP76

4b

PP54

5)

Pp45

80

49.5

-

H-

H+

LSS-

LSS+

LSP- LSP+

A-

A+

B-

B+

c-

32.5

c+

Fig. 3 (continued).

were observed, however due to the variability in expression among the individual samples the most consistent level of peak expression was noted during a period of 2-3 days immediately after birth. Thereafter, I-PKC levels gradually decline to those observed in adult brain. Recent studies have documented that commercially available [-PKC antibodies cross-react with a cPKC isoform [2]. Additionally, we find that I-PKC antisera will react weakly with recombinant a-PKC (M.L. Seibenhener and M.W. Wooten, unpublished findings). Thus to further validate our observations, as control, the expression of a-PKC was examined in a duplicate set of samples (Fig. 1B). The expression pattern for this isoform was low during embryonic-fetal, perinatal periods and began to increase N day 7 commencing with synaptogenesis and myelination. The expression pattern for cu-PKC is similar, if not identical, to that previously characterized in rat brain for this particular isoform 1321. Thus, compared with previous study of either cPKC or nPKC isoforms [7,8,11,12,29,321, I-PKC possess a unique developmentally regulated pattern of expression. However, to further characterize the ex-

pression of J-PKC, we also undertook transcript analysis using an oligonucleotide probe specific to the 3’ untranslated region of this particular isoform [28]. Northern blot analysis specific for I-PKC transcript (Fig. 1C) documented a similar expression pattern as to that observed by immunoblotting (Fig. IA). Low expression of the l-transcript was observed 10 days prior to birth, steadily increasing to peak levels in the 2-day-old pup, then declining to basal levels by day 14 and remaining constant throughout postnatal development. Subcellular distribution studies of I-PKC in brain employing discontinuous sucrose gradient centrifugation was conducted. This well established protocol [1,251 allows for the isolation of several discrete subcellular fractions. In both pup and adult brain fractions, I-PKC was roughly equally distributed between LSS: small cellular elements and LSP: large elements, including cell perikarya and nuclei (Fig. 3). Further fractionation of LSS into three fractions A,B,C led to enrichment of l-PKC in the A fraction obtained from the adult brain. In contrast, the protein was equally distibuted between all three fractions of the samples recovered from pup

244

S.E. Hunter

Table 1 Titration of [-PKC pseudosubstrate purified from rat brain through previously described [ 171

concentration. [-PKC was semithe step of Heparin-Sepharose as

+ Pseudosubstrate [ccMl

%Inhibition activity

0 9 18 37 75 150 300

0 38 46 51 6.5 74 80

Enzyme activity pseudosubstrate PRKRQGSVRRRV)

et al. / Del,elopmental

of phosphotransferase

was measured using E peptide (sequence site in l -PKC substituting Ser for Ala’s”: and 20 PM y’“P-ATP [17].

brain and not specifically enriched this stage of brain development. 3.2. Endogenous tion of pp60

149-164: ERMR-

in any fraction

at

Brain Research

85 11995) 239-248

the protein band corresponding to the pp60-doublet was excised from each lane and the amount of y32PATP associated with the pp60-doublet was quantitated by liquid scintillation counting (Fig. 5B). Addition of &‘-PKC pseudosubstrate peptide diminished the phosphorylation of pp60, although not completely. Using the cpm of the plus and minus containing reactions, a I-PKC specific substrate phosphorylation profile for the pp60-doublet was obtained (Fig. 50. The most abundant form of the phosphorylated protein was localized within fraction (A), which coincided with the abundance of the kinase (Fig. 2). Since pp60 was expressed as a doublet and phosphorylated on tyrosine residues, we speculated that the identity of this protein might be STC. Immunoprecipitation with monoclonal antibody to src resulted in precipitation of the pp60doublet (Fig. 6A) which was phosphorylated on tyrosine residues (Fib. 6B).

I-PKC substrate proteins und identifica-

To initiate a characterization of I-PKC specific substrates, a specific pseudosubstrate inhibitor of l-PKC activity was employed. Each PKC isoform contains a unique and specific pseudosubstrate site. A synthetic peptide corresponding to amino acids 113-129 of lPKC was synthesized and tested for its ability to inhibit semi-purified 1171 I-PKC activity (Table 1). Complete inhibition of in vitro phosphotransferase activity was not observed, which is likely due to other kinases which copurify with l-PKC and which are capable of utilizing the exogenous protein as a substrate. Protein from each subcellular fraction of pup or adult brain was assayed in an endogenous substrate reaction in the presence or absence of [-pseudosubstrate peptide. Each sample was preincubated with peptide inhibitor prior to conducting the endogenous assay. Analysis of phosphorylation reactions by autoradiography revealed the presence of several proteins whose phosphorylation diminished upon inclusion of peptide inhibitor (Fig. 3). Several predominant proteins pp76, pp60-doublet, pp54 and pp45 were common to both pup and adult brain samples (Fig. 3A and B); although, the pp60-doublet was the major substrate phosphorylated within these fractions. Phosphoamino acid analysis was performed on the four excised phosphoprotein bands (Fig. 4). pp76, pp54 and pp45 were phosphorylated on serine residues; however, both upper and lower bands of the pp60 doublet were phosphorylated on serine and tyrosine residues (Fig. 4). To further characterize the predominant I-PKC phosphoprotein substrate present in adult brain, the exposure time required to visualize the predominant endogenous substrate was optimized (Fig. 5A). Upon careful alignment of the dried gel and autoradiogram

4. Discussion In this study, we document that 5-PKC displays a unique developmentally regulated pattern of expres-

(+) Pi w

Ser Thr +

Tyr -

origin t-1

~~76

~~60

~~50

PP54

PP45

Fig. 4. Phosphoamino acid analysis. Individual endogenous substrates, as indicated, were recovered from an adult sample which had been subjected to phosphorylation/separation and the phosphoamino acid content analyzed.

SE. Hunter

et al. /Det~rlopmental

Brain Research

sion. The expression of this isoform peaks in early perinatal life compared to the expression of either (Y, p, y, or &PKC isoforms which is low during embryonic and perinatal development and progressively increases 2-3 weeks after birth [32]. No study exists which details the expression pattern for l-PKC, although a preliminary study of I-PKC during hippocampal development likewise documented an increase in early perinatal life and decreased expression thereafter [13]. Our own findings would be consistent with this pattern of ex-

H -

LSS

245

239-248

pression. Additionally, expression of this kinase early in development, as compared to late, for other PKC isoforms indicate that PKC signalling pathways can be selectively acquired or deleted during neuronal differentiation. Thus, the selective expression of particular PKC isoforms may play a role in modulating neuronal sensitivity or insensitivity for CaZt during development. The mechanism by which the expression of l-PKC is regulated is unknown. Information regarding the ge-

LSP

+-+

X5 (1995)

B

C

-+-+-+-+

-

66.2 Kd

-

45Kd

2,500

E 8 a

H

LSS

LSP

A

H

LSS

LSP SAMPLE

A

C

1,000 600 600

s

C

Fig. 5. Characterization of pp60. Endogenous substrate reactions were conducted on protein (20 yg) obtained from subcellular fractions (H, LSS, LSP, A,B,C) of adult rat brain. The reactions were conducted in the absence or presence of l-PKC pseudosubstrate peptide. A: shown is a representative autoradiogram exposed for 2 h at -80°C to optimize the exposure time required to visualize pp60 substrate protein. B: the protein band corresponding to the pp60-doublet was aligned with the autoradiogram and excised from the gel and counted by liquid scintillation counting. Shown are the cpm incorporated into the pp60-doublet in the absence or presence of pseudosubstrate peptide. C: the difference (A) in cpm for J-specific phosphotylation of pp60 is shown

246

SE. Hunter

et al. / DrL~elopmental

Brain Research

nomic structure of the various PKC genes is lacking. Recently, analysis of the P-PKC gene has documented that potential sites exist which play a role in autoregulation at the transcriptional level [19]. In addition, y-PKC contains a novel site identified by DNase I footprint analysis to which protein binding is inversely related to the levels of y-PKC expression at different stages of development [3]. Our data documents that &‘-PKC is upregulated, as well as, downregulated during development. It is therefore likely that transcription of (-PKC is under the control of positive as well as negative regulatory molecules. It will be of interest to deduce the genomic structure of the I-PKC, identify possible proteins which play a role in transcriptional regulation and to understand the role which [-PKC itself may have on the expression of other PKC isoforms. Our data are consistent with the localization of c-PKC in neurons. In rabbit brain, l-PKC has previously been detected in nuclei of nerve cells [6]. In rat cerebellum, I-PKC is uniformly distributed within

85 (19951 239-248

Purkinje, interneurons, granule cells and deep cerebellar neurons [4]. The localization of (-PKC to neuronal nuclei and developing processes parallels the localization observed in NGF-differentiated PC12 cells [31]. In these cells, J-PKC is concentrated in growth cones of extending neurites and axonal varicosities. Our data suggest that it is likely that I-PKC may play a role in early phases of neuronal development. The subcellular distribution of this kinase in both pup and adult brain indicates the potential for multiple subcellular sites: nucleus, growth cone, synaptosomes as well as other subcellular organelles. Further characterization of the A, B and C fractions as well as immunohistochemical studies using adult brain material will be necessary to specifically pinpoint the subcellular sites to which this kinase localizes. Fractionation of adult brain compared to fetal brain results in slightly different separation and enrichment of cellular components [l]. In fetal brain I-PKC is equally distributed between all three fractions. Colocalization of I-PKC and specific substrates in fraction A would suggest that proximal localization

B.

(+) Pi

*

A. -

107 Kd

-

76

Ser + Thr * Tyr

*

origin

*

52

a

b

C

(-) Fig. 6. Identification of pp60. An endogenous substrate reaction was conducted on protein (200 pg) obtained from the A fraction recovered from subcellular fractionation of adult brain. A: the reactions were subsequently immunoprecipitated with MAb 327 and separated by SDS-PAGE, followed by autoradiographic visualization. a, represents the endogenous substrate reaction. b, represents the immunoprecipitate of the endogenous substrate reaction. c. represents the supernatant recovered post-immunoprecipitation. B: the immunoprecipitate (b) was subjected to phosphoamino acid analysis.

SE.

Hunter

et ul. / Der~elopmerztul

between the two may play a significant role in facilitating regulatory interactions. At this time we cannot exclude the possibility that these phosphoproteins are indirect substrates of I-PKC. It remains to be determined whether any of these endogenous substrates are directly phosphorylated within an intact cell by direct activation of this specific kinase. However, this study provides the first step in describing which proteins may be likely targets of the kinase in vivo. The role of c-src within neurons is not clearly understood however, it has been observed that its expression plays a role in the regenerative capacity of nerves [14]. Additionally, a-tubulin is phosphorylated in response to NGF and src [5]. Thus, it has been suggested that src may play a role in cytoskeletal reorganization which occurs during neurite extension [51 and it is possible that 5-PKC may regulate the interaction of src with its target substrate(s). Possible interactions between src and iPKC may be necessary to transduce signals for process extension, axonal outgrowth and guidance. A likely function for (-PKC might be related to intracellular signalling induced by activation of surface receptors for neurotrophic factors. I-PKC is involved in NGF-signalling [31] and dependant in part upon trk (M.W. Wooten, unpublished findings). Since members of the trk family are also developmentally regulated [26], we speculate that I-PKC might play a role in mediating signals necessary for neurotrophic factor responses during brain ontogeny. Although it is slightly premature to ascribe a precise function for l-PKC in brain, we hypothesize that development of CNS function at the specific period of peak I-PKC expression is correlated with. and dependant upon, the expression of this isoform.

Acknowledgements This work was supported in part by funding from the Howard Hughes Life Science Scholars Program, Auburn University. I-PKC pseudosubstrate peptide was synthesized by the M. Russ at the Dept. of Biochemistry, Univ. Kentucky, Lexington, KY. We wish to thank Dr. D. Michael Payne for src MAb 327 and assistance with phosphoamino acid analysis and members of our laboratory for discussion and review of draft versions of this manuscript. All three authors contributed equally to this manuscript.

References [l]

Bare, D.J., Lauder, J.M., Wilkie. M.B. and Maness, P.F., p59fyn in rat brain is localized in developing axonal tracts and subpopulations of adult neurons and glia, Oncogene. 8 (1993) 1429-1436.

Brain

Research

El

85 (1995)

2.19-248

247

Battle, E., Fabre. M. and Garcia de Herreros. A., Antipeptide antibodies directed against the C-terminus of protein kinase C { react with a Ca”and TPA-sensitive PKC in HT.29 human intestinal epithelial cells. FEBS Lett.. 344 (1994) 161-165. K.-H., Widen. S.G.. Wilson. S.H. and Huang, K.-P.. [31 Chen. Identification of a nuclear protein binding element within the rat brain protein kinase y promoter that is related to the developmental control of this gene. FEBS Lett.. 325 (1993) 210-214. D.E., Compartmentalization of the cere[41 Chen, S. and Hillman. bellar cortex by protein kinase C delta, Neuroscience. 56 (1993) 177-18X. of 01[51 Cox. M.E. and Maness, P.F., Tyrosine phosphorylation tubulin is an early response to NGF and ~~60’.‘” in PC12 cells, J. Mol. Neurosci.. 4 (1993) 63-72. M.. Uchida, C., Usuda, N., Nagata, T. and Hidaka, [hl Hagiwara. H.. (‘-Related protein kinase C in nuclei of nerve cells. &o&em. Bioph~~s. Res. Commun.. 168 (1990) 161-168. T.. Ase. K.. Sawamura, S.. Kikksawa, U.. Saito. N.. [71 Hashimoto, Tanaka, C. and Nishizuka. Y.. Postnatal development of a brain-specific subspecies of protein kinase C in rat, J. Neurosci. Rex. 8 (1088) 1678-1679. [Xl Hirata. M., Saito. N., Kono. M. and Tanaka. C.. Differential expression of the PI-and pII- PKC subspecies in the postnatal developing rat brain: an immunocytochemical study. Del,. Bruin Rex, 62 (1991) 229-238. G.M.. O’Brian, C.A.. Johnson, M.D., Kirschmeier. P. [91 Housey. and Weinstein, LB., Isolation of cDNA clones encoding protein kinase C: evidence for a protein kinase C-related gene family. Proc. Nutl. Acad. Sci. USA. 84 (1987) 1065-1069. L.F., Yoshida, Y., Nakabayashi, H.. Knopf, J.L., Young [lOI Huang. Ill. W.S. and Huang. K.P., Immunichemical identification of protein kinase C isozyme as products of discrete genes. Biochem. Biophys. Rex Comm., 149 (1987) 946-953. [ill Huang. F.L., Young III. W.S., Yoshida. Y. and Huang, K., Developmental expression of protein kinase C isozymes in rat cerebellum, Del,. Brain Rex, 52 (1990) 121-130. [121 Kim. H., Hirota. S.. Onoue. H., Hirata, T., Suzuki. K., Ohno, S.. Kuroki, T.. Kitamura. Y. and Nomura. S., Localization and developmental expression of a novel protein kinase C ci gene, Del,. Brain Rex, 70 (1992) 239-244. Hrabe. J. and Sacktor, T.C., Develop[I31 Jiang. X.. Naik. M.U., mental expression of the protein kinase C family in rat hippocampus. Der,. Brain Rex, 78 (1994) 291-295. 1141LeBeau. J.M., Tedeschi. B. and Walter, G., Increased expression of pp60’~“” protein-tyrosine kinase during peripheral nerve regeneration, J. Neurosci. Rex, 28 (1991) 299-309. [ISI Mangoura, D.. Sogos, V. and Dawson. S., Protein kinase C-2 is a developmentally regulated, neuronal isoform in the chick embryo central nervous system, J. Neurosci. Rex, 35 (1993) 48X-498. of protein kinase [IhI Mattson, M.P.. Evidence for the involvement C in neurodegenerative changes in cultured human cortical neurons, Exp. Neural., 112 (1991) 95-103. H. and Exton. J.H.. Purification and characterization [171 Nakanishi. of the [-isoform of protein kinase C from bovine kidney, .I. Biol. Chem.. 267 (1992) 16347-16354. [181 Nishizuka. Y., Intracellular signalling by hydrolysis of phosholipids and activation of protein kinase C, Science, 258 (1992) 607-614. [19] Obeid. L.M.. Blobe. G.C.. Karolak, L.A. and Hannun, Y.A.. Cloning and characterization of the major promoter of the human protein kinase C p gene, L Biol. Chem., 267 (1992) 20804-20810. [20] Ohno, S.. Kawasaki. H., Konno, Y., Inagaki, M., Hidaka. H. and Suzuki, K., A fourth type of rabbit protein kinase C, Biochemistry. 27 (1988) 2083-2087.

248

SE. Hunter

et al. / Der~elopmentai

[21] Ono, Y., Fujii, T., Ogita, K., Kikkawa. U., Igarashi, K. and Nishizuka, Y., Identification of three additional members of rat protein kinase C family: 6.. t-, and <-subspecies, FEBS Left.. 226 (1987) 125-128. [22] Ono, Y., Fujii, T., Ogita, K., Kikkawa, U.. Igarashi, K. and Nishizuka, Y., Protein kinase C-zeta subspecies from brain: its structure, expression, and properties, Proc. Natl. Acad. Sci. USA, 86 (1989) 3099-3103. 1231 Osada, S.I., Mizuno, K., Saido, T., Akita, Y., Suzuki, K., Kuroki, T. and Ohno. S., A phorbol ester receptor/protein kinase. nPKC, a new member of the protein kinase C family predominantly expressed in lung and skin, J. Biol. Chem., 265 (1990) 22434-22440. [24] Parker, P.J., Coussens. L., Totty, N.. Rhee. L.. Young, S., Chen, E., Stabel. S., Waterfield, M.D. and Ullrich. A., The complete primary structure of protein kinase C. The major phorbol ester receptor, Science, 233 (1986) 853-858. 1251 Pfenninger, K.H., Ellis, L.. Johnson, M.P., Friedman, L.B. and Somio. S., Nerve growth cones isolated from fetal rat brain: Subcellular fractionation and characterization, Ccl/, 35 (1983) 573-584. 1261 Ringstedt, T., Lagercrantz, H. and Persson, H., Expression of members of the trk family in the developing postnatal brain, Deu. Brain Rex, 72 (1993) 119-131.

Brain Research

85 (1995)

239-248

[27] Sacktor, T., Osten, P., Valsamis, H., Jiang, X., Naik, M. and Sublette, E., Persistent activation of the l-isoform of protein kinase C in the maintenance of long-term potentiation, Proc. Natl. Acad. Sci. USA, 90 (1993) 8342-8346. [28] Selbie, L.A.. Schmitz-Peiffer, C., Sheng, Y. and Biden, T.J., Molecular cloning and characterization of PKC L, an atypical isoform of protein kinase C derived from insulin-secreting cells, J. Biol. Chem.. 268 (1993) 24296-24302. 1291 Sposi, N.M.. Bottero. L., Cossu, G., Russo, G., Testa, U. and Peschle, C., Expression of protein kinase C genes during ontogenie development of the central nervous system, Mol. Cell. Biol.. 9 (1989) 2284-2288. [30] Wetsel. W.C., Khan, W.A., Merchenthaler, I., Rivera, H., Halpern, A.E.. Phung, H.M., Vilar-Negro. A. and Hannun, Y.A., Tissue and cellular distribution of the extended family of protein kinase C isoenzymes, J. Cell Biol., 117 (1992) 121-133. [31] Wooten. M.W., Zhou, G., Seibenhener, M.L. and Coleman, ES., A role for [-protein kinase C in nerve growth factor-induced differentiation of PC12 cells, Cell Growth Different., 5 (1994) 395-403. 1321 Yoshida, Y., Huang, F.L., Nakabayahi, H. and Huang, K.P., Tissue distribution and developmental expression of protein kinase C isozymes, J. Biol. Chem.. 263 (1988) 98689873.