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An investigation of experimental conditions for studying protein phosphorylation in micro-slices of rat brain by two-dimensional electrophoresis
Journal of Neuroscience Methods, 24 (1988) 27-38 Elsevier
27
NSM 00802
An investigation of experimental conditions for studying protein phosphorylation in micro-slices of rat brain by two-dimensional electrophoresis Richard Rodnight, Reza Zamani and Alastair Tweedale Department of Biochemistry, Institute of Psychiatry, London (U.K.) (Received 8 April 1987) (Revised 23 October 1987) (Accepted 26 October 1987)
Key words: Phosphoprotein; 32p-incorporation; In vitro labelling; Micro-slice of brain; Lability of protein phosphorylation Procedures are described for studying protein pbosphorylation in 1 mm diameter micro-slices of rat brain tissue using two-dimensional electrophoresis as analytical tool The activity of several protein phosphorylatingsystems, including a major system phosphorylating a 40 kDa substrate complex, was highly dependent on the procedures used for micro-slice preparation and on the Ca2+-content of the preparation medium. Under optimal conditions the pattern of phosphorylation observed in micro-slices closely resembled that obtained by in vivo labelling.
Introduction In previous work (Rodnight et al., 1985) we described a simple and economical method for studying protein phosphorylation in vivo in the rat brain, using high resolution two-dimensional electrophoresis as an analytical tool. In the present paper we describe a complementary in vitro procedure applicable to micro-slices of brain. Basically the work had the following aims: (1) to establish a safe and economical procedure suitable for routine application to discrete brain structures; (2) to compare in vivo and in vitro phosphorylation patterns and create incubation conditions that reproduced as far as possible the in vivo pattern; (3) explore further optimum conditions for analysis of the patterns by two-dimensional electrophoresis.
Correspondence: R. Rodnight. Present address: Departamento de Bioquimica, Instituto de Biociencias, UFRGS (Centro), 90.040 Porto Alegre RS, Brazil.
The work is a development of preliminary experiments reported earlier (Rodnight et al., 1986).
Methods
Source of materials [32p]Inorganic phosphate (10 m C i / m l , carrier free in water) was obtained from Amersham International. Acrylamide and Ampholines were purchased from LKB, Servalyts from Serva (Heidelberg, F.R.G.), sodium dodecyl sulphate (SDS, 99%) from B D H Chemicals (Poole, UK). Other chemicals were purest available. Wistar strain rats of either sex and weight 150-250 g were used as a source of brain tissue. Preparation of micro-slices Rats were killed by decapitation (designated as zero time) and the brain removed within 1 min. Macro-slices encompassing several anatomical re-
0165-0270/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
28 gions were first cut with a Mcllwain chopper. To accomplish this some modifications were necessary to the usual procedure for chopping tissue: in particular it was very important to ensure good adhesion of the comparatively large pieces of brain to the filter paper. This was achieved by initially mounting a cut surface of the tissue on dry filter paper, thus avoiding the use of cyanoacrylate adhesive as recommended by Cuello and Carson (1983). The chopper was adjusted to cut 0.4 mm thick slices and two circles of filter paper moistened with ice-cold medium (see below) fixed to the platform. The brain was roughly dissected (e.g. by sagittally dividing the forebrain) and the main cut surface of the required region gently pressed onto the centre of a dry circle of filter paper. This was placed on the chopper platform and immediately wetted by adding medium drop by drop to the periphery of the paper and allowing it to diffuse towards the tissue. It was important to add just sufficient medium to ensure that the paper and tissue remained in position during the cutting process. The surface of the tissue was then moistened with a minimum quantity of medium and the required number of slices cut. With practice it was possible to cut 10 or more hemi-coronal or longitudinal slices of forebrain or cerebellum. It was also possible to cut whole coronal slices of the midbrain through the thalamus and the colliculi to the brainstem by first removing the paleo- and neocortex and most of the cerebellum and pressing the ventral surface of the remaining tissue onto a circle of dry filter paper. However, without a cut surface in contact with the paper adhesion was less satisfactory and more pressure was needed to prevent the tissue from rifting during chopping. The chopped tissue was transferred to a dish containing about 5 ml of ice-cold medium and the slices teased apart with two sable hair brushes. (No attempt was made to maintain the temperature of the medium at 4 ° C during subsequent operations.) Slices containing the structures required were then transferred to a second dish, the bottom of which was lined with polythene. This dish contained just enough medium to cover the polythene. Discs of tissue (micro-slices) were prepared with stainless steel punches by a procedure
essentially similar to that first described by Palkovits (1973; see also Cuello and Carson, 1983) for thinner sections of frozen brain. With the plunger partially withdrawn the punch was pressed firmly through the appropriate area of the macroslice onto the surface of the polythene, using sufficient pressure to just mark the surface of the latter; on withdrawing the punch the micro-slice remained entrapped in the tip of the punch and could be expelled into the medium of the next stage (see below) by gently depressing the plunger. For all of the present work a punch of internal diameter 1 mm was used; larger diameter punches (e.g. 2.0, 2.5 mm) used in an earlier study (Rodnight et al., 1986) are only applicable in the rat brain to regions such as the caudate or neocortex. To facilitate transfer to the radioactive medium the micro-slices were first collected on a rectangulax tray of black acrylic plastic incorporating shallow depressions each containing 100/~1 of medium at room temperature (17-23 ° C). Two 1 mm diameter micro-slices were placed in each depression. In early experiments the bicarbonate-buffered medium was exposed to air at this stage, but for reasons that will become apparent, in later experiments the tray was enclosed in a box that permitted the medium to be saturated with 95% 02/5% CO 2 as illustrated in Fig. 1. In the present study micro-slices were transferred to the radioactive medium by spearing them with the bent tip of a mounted 21 gauge hypodermic needle. An alternative procedure, developed towards the end of the work and applicable to discs of diameter 1 mm, used an automatic micro-pipette. The plastic tip was cut back so as to provide an orifice just wide enough to admit the micro-slices and the pipette was adjusted to deliver 10/~1. With practice it was possible to introduce two micro-slices, together with 10 #1 of pre-incubation medium, into the pipette tip and deliver both liquid and tissue to an incubation tube containing 15 /~1 of labelling medium instead of the usual 25 #1. This procedure has two important advantages: it avoids the damage caused by spearing the micro-slices and it avoids exposing them to the atmosphere during transfer. Recent work suggests that both these circumstances are significant factors affect-
29 c
A
n i
t
I
2cm
b,
b_
Co
B
C
if--Fig. 1. Equipment used to deplete micro-slices of inorganic phosphate prior to transfer to labelling medium. The central part consisted of a sheet of black acrylic plastic (Perspex) incorporating 8 shallow depressions and boxed as indicated. Each depression contained 100 #1 of bicarbonate-buffered medium and was surrounded by 4 holes through which gas was passed from a lower chamber. Two bleed holes were bored in the tightly fitting lid. Black plastic was used in order to facilitate visualisation of the micro-slices during transfer. ing reproducibility in studies using very small discs of tissue. However, problems with the pipetting procedure m a y arise in experiments where the pre-incubation and incubation media are different.
Incubation conditions The micro-slices were labelled at 3 7 ° C in tapered polystyrene tubes (12 × 75 m m ) containing 100 ~Ci of carrier-free [32p]orthophosphate dissolved in 25 #1 of incubation medium, giving a 32p-concentration of 4 m C i / m l . The incubation area was protected with 12 m m thick screens of acrylic plastic (Perspex). The original 32p-solution was dispensed in the tubes the day before the experiment and dried overnight under vacuum over silica gel. The standard incubation medium had the following composition (mM): NaC1 124, KC1 4, MgSO 4 1.2, N a H C O 3 26, glucose 12, saturated with 95% 0 2 / 5 % CO 2. CaCI2, usually to 1 mM, was added after gassing. In a few experiments K H 2 P O 4 was added in the range 10/~M to 1 mM. The incubation tubes were filled with 95%
02//5% CO 2 and stoppered immediately after adding the medium and gassed again after transferring the micro-slices. Each tube contained two 1 m m diameter micro-slices or one of larger diameters. The standard incubation time was 45 min at 37 ° C and the tubes were gently agitated by rotation every 5 min. Labelling was stopped by addition of 50 t~l of 15% trichloroacetic acid, after which the tubes were placed on ice to await transfer to electrophoresis sample buffer. In preliminary experiments the macro- and micro-slices were prepared in the same bicarbonatebuffered medium used for labelling. In later work, in order to avoid exposing the tissue to p H values higher than 7.4, they were prepared in the same medium but buffered with 25 m M Tris-citrate (pH 7.4 at r o o m temperature) instead of bicarbonate/CO2.
Two-dimensional electrophoresis The acid-fixed micro-slices were removed from the incubation tubes with a mounted hypodermic needle tip, immersed briefly in about 100/~I of 250 m M N a 2 H P O 4 to wash off excess acid and then
30 dissolved in a volume of electrophoresis sample buffer sufficient to give a protein concentration of 1 ~tg//~l essentially by the two-stage process described previously (Rodnight et al., 1985). The procedure for 1 mm diameter micro-slices was as follows. The two sample solutions were: A 9.5 M urea, 12.5 mM lysine, 1% sodium dodecyl sulphate. 2% (v/v) 2-mercaptoethanol, 0.005% bromophenol blue; B: 9.5 M urea, 12.5 mM lysine, 5% (v/v) Nonidet P-40, 2% (v/v) 2-mercaptoethanol, 0.005 % bromophenol blue. Each micro-slice contained approximately 30 /~g of protein; therefore two micro-slices (from one incubation tube) were initially dissolved with aid of a fine glass rod in 12 /zl of solution A contained in a 0.25 ml capacity Eppendorf centrifuge tube. Sample solution B (48/~1) was then added and the two solutions thoroughly mixed. The solution was frozen, thawed and centrifuged at 12,000 g (Microcentaur) for 5 min. The supernatant was separated from the trace of insoluble material and set aside pending analysis. Glass tubes (i.d. 1.5 mm, o.d. 4 mm) were used for isoelectric focussing; the gel length was 13.5 cm. The basic gel mixture had the following composition: 3.5% (T) acrylamide (Cbi s = 3.6%), 2% ampholytes, 9.5 M urea, 4% (v/v) Nonidet P-40 and 0.08% N,N,N'N'-tetraethy!ene/diamine (TEMED). This mixture was prepared in bulk and stored in volumes of 0.3 ml in conical plastic tubes at - 7 5 ° C under N 2. Individual gels were prepared at about 3 0 ° C by adding 5#1 of 10% ammonium persulphate to each 0.3 ml, briefly mixing and then filling the glass focussing tube to exactly 2 cm from the f o p b y negative pressure supplied by a 1 ml'syringe a n d needle fitted with a length of polythene'~tiabing and a suitable adaptor. The glass tube, with the syringe and tubing still attached, was then left in the residual gel in the plastic tube until gelation had occurred. Several pH gradients were explored of which the following were employed routinely (% concentrations are w/v). (A) 1.64% Ampholine pH 3.5-10, 0.18% Ampholine pH 5-7, 0.18% Servalyt pH 2--4; (B) 1.6% Ampholine pH 3.5-10, 0.32% Ampholine pH 5-7, 0.08% Servalyt pH 2-4; (C) 2% Servalyt pH 2-11. Equilibrium focussing was performed according to O'Farrell (1975) with gradients (A) and (C), but
using 50 mM NaOH as catholyte and 25 mM H3PO 4 as anolyte as recommended by Duiacan and Hershey (1984). After loading 15/~1 of s~nple and overlaying with an equal volume of 8 M urea the gels were focussed for 400 V overnight followed by 1-2 h at 1000 V. For non-equilibrium focussing (NEPHGE; O'Farrell et al.. 1977) the sample (15 /~1) was loaded at the anode and for anolyte and catholyte we used 10 mM H3PO 4 and 20 mM N a O H respectively. In this case the gels were overlaid with 8 M urea containing cytochrome C (approx. 80 /~g/ml) and the gels were run at 400 V until the main red cytochrome band had reached 7.5 cm from the origin. On completion of focussing the gels in their glass tubes were stored at - 2 0 °C pending transfer. The procedures used for the second dimension were based on Gower and Rodnight (1982) and R0dnight and Perrett (1986). Immediately after thawing gels were extruded by air pressure and equilibrated with gentle shaking for 15 min in 4 ml of second dimension upper buffer supplemented with 2% sodium dodecyl sulphate. Because entry and resolution of phosphoproteins in the basic region of equilibrium focussing gels was unsatisfactory, these gels were bisected, the basic halves discarded and two acidic halves mounted on a single second dimension slab gel with the acidic ends placed centrally; 1% agarose dissolved in standard upper buffer was used to secure the gels. In the case of non-equilibrium runs all of the gel below the cytochrome C band was discarded and the two upper sections mounted on one slab gel, usually with the basic ends central. Either gradient slab gels (6-19%, exponential) or 14% gels, both with 4% stacks, were used for the second dimension. Gradient gels were run during the daytime by the schedule used previously (Gower and Rodnight, 1982); 14% gels were run overnight at a constant potential of 55 V. After electrophoresis the gels were fixed, dried and exposed to X-ray film as described previously (Rodnight et al., 1985). Optimum exposure times for gradient gels varied from 12 to 48 h.
Coding and display of phosphopeptides In the figures phosphopeptides are numbered by the same code used in earlier work (Rodnight
31 et al., 1985, 1986). The 45 kDa substrate of protein kinase C (phosphopeptide 4) is designated B50/F1 since both terms are used in the literature for this molecule (e.g. Gispen et al., 1985; Chan et al., 1986); phosphopeptide 2 (M r 82 kDa) corresponds to the 87 kDa substrate of protein kinase C described by Wu et al. (1982). Evidence concerning the provisional identities of other substrates is given in the above papers. In contrast to our previous work, but to conform with current practice, autoradiographs are displayed with the acidic end of the focussing dimension on the left.
Other methods Inorganic orthophosphate was determined by a micro-adaptation of the method of Heinonen and Lahti (1981). Adenine nucleotides (ADP and ATP) were determined by HPLC using a linear ammonium formate gradient (K. Weaver and M. Brammer, in preparation). Results
Electrophoresis procedures Examples of the various procedures explored are given in Figs. 2-6. The separation obtained with equilibrium focussing using either pH gradient (A) or (C) followed by SDS-electrophoresis in gradient gels (Figs. 2B, 3B and 5A) gave excellent separation of most substrates with isoelectric points less than 6, but generally poorer separation of substrates with higher values; pH gradient (C) gave a marginally superior separation and was more convenient to prepare. However, the separation of B50/F1 from a major 40 kDa substrate complex (phosphopeptide 6) was unreliable using either pH gradient (compare Fig. 2B with Fig. 5A). Although we were unable to improve on this by employing other pH gradients, consistent separation of the two phosphopeptides could be achieved by taking advantage of the anomalously low free electrophoretic mobility of the B50/F1 substrate (Gower and Rodnight, 1982) and its resulting relatively higher migration rate, compared with the 40 kDa complex, in the second dimension on 14% gels (Figs. 4, 6F). The 14% gels were unsuitable, except for a few acidic molecules, for studying phosphopeptides with M r values
above 40 kDa, but gave excellent separation in the range 35-14 kDa (Fig. 4). It may be noted that many of these substrates labelled relatively slowly compared with those of higher Mr, and this necessitated long exposure times which left the high M r region uninterpretable. An unsolved problem encountered with equilibrium focussing concerned the high molecular weight phosphopeptide 3, which we provisionally identify as microtubule-associated protein 2 (MAP-2). For reasons we have been unable to determine the focussing of this molecule was very erratic and in most experiments it either failed to penetrate the gel or remained as a streak in the basic region. The result shown in Fig. 2 was an exceptionally good one in that MAP-2 was fully represented; in the equilibrium separations illustrated in Figs. 3 and 5, on the other hand, it was absent and for this reason the relevant parts of the autoradiographs have been omitted. The problem was not related to the porosity of the focussing gel since total acrylamide concentrations lower than the 3.5% used routinely, or wide variations in the bis-acrylamide concentration, failed to improve matters. The solubility and recovery of the other numbered polypeptides was consistently reproducible throughout the study. Non-equilibrium (NEPHGE) focussing employing pH gradient (B), followed by gradient gels for the second dimension, gave a valuable general separation including the highly basic substrate synapsin I, but completely failed to separate the B50/F1 and 40 kDa substrates (Fig. 6C); however resolution of the complex of phosphopeptides coded 8, which probably include a- and fl-tubulin, was consistently better by NEPHGE than by equilibrium focussing (compare Fig. 2B with Fig. 6C).
Micro-slice preparation When micro-slices were prepared in bicarbonate-buffered sahne and transferred to medium containing 32Pi within 7 min of zero time the equilibrium focussing pattern shown in Fig. 2B was obtained. From Fig. 2A it can be seen that the main features of this in vitro pattern were very similar to those observed in brain tissue after labelling in vivo by the technique of Rodnight et al. (1985). This suggested that relatively little post-
32
M r x 10 -3 ~.~
Fig. 2. Autoradiographs prepared from cortical tissue labelled in vivo (panel A) by the procedure of Rodrtight et al. (1985) and labelled in vitro (panel B) by the present standard micro-sliceprocedure, except that the micro-sliceswere prepared in bicarbonatebuffered medium and transferred to labelling medium 7 min after zero time. Equilibrium focussing using pH gradient (A); in both cases the section of the pH gradient displayed extends from approximately 7.3 to 4.0. Gradient gel separation in second dimension. Provisional identities of the numbered phosphopeptides are as follows: 2, 82-87 kDa substrate of protein kinase C; 3, microtubuleassociated protein 2; 4, the B-50/F-1 substrate of protein kinase C; 6, the unknown 40 kDa substrate complex; 7, the a-subunit of pyruvate dehydrogenase; 8, polypeptides co-migrating with tubulin sub-units. mortem change in protein phosphorylating capacity had occurred during the 7 min required for slice preparation. However, only about 6 microslices from a major area of the brain such as the caudate or cerebral cortex could be prepared in this period of time and before embarking on more complex experiments involving several discrete anatomical regions it was considered advisable to re-investigate stability during longer time intervals. To our surprise we found that leaving micro-slices in the preparation medium for longer periods before transferring to the incubation medium had two consequences: (a) a progressive decline in the labelling capacity of a system phosphorylating the 40 k D a substrate complex (phosphopeptide 6 in Fig. 2) to the extent that after 40 re_in in preparation medium virtually no label was transferred to it on subsequent incubation and (b) a marked tendency for the great majority of the remaining substrates to incorporate more 32p with increasing time in preparation medium. A typical experiment is shown in Fig. 3, which compares the pattern obtained from a pair of micro-slices of
caudate nucleus transferred to labeUmg medium 7 min after zero time (panel A) with a pair taken from the same macro-slice transferred after 30 min (panel B). The increased incorporation of 32p resulting from delaying transfer appeared to be due to loss of endogenous inorganic phosphate from the tissue and a consequent increase in the specific radioactivity of the intracellular 32pi pool, In support of this conclusion we found that when 1 m m micro-slices were left in medium for 30 min after preparation over 90% of their inorganic phosphate was lost; with 2.5 m m slices the loss was of the order of 60%. The loss of activity in the 40 k D a phosphorylating system was found to be related to the rise in p H of the bicarbonate-buffered medium as it lost CO 2 to the atmosphere, since it did not occur when micro-slices were left in gassed bicarbonate medium or in Tris-buffered medium (Fig. 3C). The loss was also less marked when micro-slices were maintained in relatively large volumes of bicarbonate-buffered medium which retained CO 2 for longer than did the small volumes used to
33
MrxlO -3 12(
8(
4(
Fig. 3. Autoradiographs prepared from caudate nucleus labelled in vitro after various preparation procedures. Panel A: micro-slices prepared in bicarbonate-buffered medium and transferred to radioactive medium 7 min after zero time; panel B: micro-slices prepared in bicarbonate-buffered medium and transferred 35 min after zero time; panel C: micro-slices prepared in Tris-buffered medium, transferred 7 rain after zero time to 100 /xl of bicarbonate-buffered medium (maintained at pH 7.4 by gassing) in the equipment illustrated in Fig. 1 and transferred to radioactive medium 40 mm after zero time. Focussing, separation conditions and display as in Fig. 2. Arrow indicates the position of the labile 40 kDa phosphorylating system; provisional identity of phosphopeptide labelled 5 is DARPP-32 (Ouimet et al., 1984). collect a series of micro-slices f r o m various brain regions. I n the light of these observations the following procedure was a d o p t e d as standard: micro-slices were prepared in Tris-buffered medium, transferred within 15 min of zero time to bicarbonatebuffered m e d i u m (maintained at p H 7.4 with O 2 / C O 2 ) and then left for 30 rain at r o o m temperature (preincubation stage) to allow time for the tissue inorganic phosphate content to reach a stable low content before transferring to labelling m e d i u m at 37 o C. The procedure gave consistently high recoveries of the 40 k D a p h o s p h o r y l a t i n g system and reproducible levels of 32p incorporation. As a measure of metabolic integrity we measured the A T P and A D P content of micro-slices subjected to the routine procedure. In a typical experiment the initial concentration of A T P ( / ~ m o l / g / f r e s h wt) was 0.68, rising to 0.80 after
preincubation for 30 min and to 1.25 after incubation at 37 ° C for 30 min; the corresponding values for A D P were 0.64, 0.22 and 0.21.
Incubation conditions Time course. I n c o r p o r a t i o n into acid-precipitated protein followed a curvilinear course over a 60 min period without reaching a plateau and with easily detectable levels being attained after 10 min. For most experiments in the present work a 45 min period was adopted. There was no obvious variation in the rate of labelling of different substrates. Effect of varying Ca 2 +-content. In initial experiments designed to study this variable we prepared micro-slices in Ca2+-free bicarbonatebuffered m e d i u m and transferred them directly to labelling m e d i u m containing either E G T A or various concentrations of Ca 2+ within 7 min of zero time. With certain exceptions described below
34
M r x 10 -3
Fig. 4. Autoradiographs prepared from cerebellar cortex labelled in vitro by the standard procedure and using a 14% gel for the second dimension. In panels A and B the gel was exposed for 6 days and 18 h respectively. Focussingconditions and display as in Fig 1, except that pH gradient (C) was used. Note the excellent separation of pliospliopeptide 4 (B-50/F-1) from the 40 kDa complex. The diffuse labelled material marked with an arrow in panel A was an occasional feature of all autoradiographs and is not typical of cerebellar cortex. However, the exceptionally high relative labelling of the 82 kDa phosphopeptide is typical of this tissue, as noted elsewhere (Rodnight et al., 1986). there was no detectable difference in the amount of 32p incorporated in the range of Ca 2+concentrations from 0.5 mM to 1.25 mM; higher Ca2+-concentrations were markedly inhibitory. The major exception to this result concerned the 40 kDa phosphorylating system: regardless of the concentration of Ca 2+ in the labelling medium, micro-slices prepared in Ca2+-free medium always failed to phosphorylate significantly the 40 k D a substrate complex. An early experiment in which the micro-slices were prepared in either normal bicarbonate medium or Ca2+-free bicarbonate medium and transferred to labelling medium containing 1 m M Ca 2÷ is shown in Fig. 5. In a later series of experiments using the standard procedure 0.1 mM Ca 2+ was included in the initial Trisbuffered preparation medium and the micro-slices were transferred for the preineubation and labelling stages to media containing either zero Ca 2+ (EGTA), 0.1 m M Ca 2÷ or 1 m M Ca 2÷. From Fig. 6C, F it can be seen that the inclusion of this low
concentration of Ca 2+ in the preparation medium fully preserved the 40 kDa phosphorylating system when the subsequent media contained 1 mM Ca 2÷. However with 0.1 m M Ca 2+ present throughout virtually no phosphorylation of the 40 k D a complex was observed (Fig. 6B, E). Surprisingly, incorporation of phosphate into the great majority of the remaining acceptors was not decreased in medium containing 1 mM EGTA (Fig. 6A, D). Exactly the same type of lability was observed in at least 3 other less prominent phosphorylating systems. The most interesting of these was a system that is uniquely active in micro-slices from the hippocampus. The acceptor for this system is a 50 k D a phosphopeptide with an isoelectric point of about 6.3; it was particularly well displayed by non-equilibrium focussing (Fig. 6C indicated by × ). Phosphorylation of this acceptor can also be detected in micro-slices from certain other brain areas, but at a much slower rate (in preparation). The two other labile systems phosphoryated substrates with M r values of about 17 kDa (Fig. 6 D - F ) ; so far no regional differences in the activity of these systems have been observed. It may
Mr x 10 -3
Fig. 5. Autoradiographsprepared from cerebral cortex showing the effect of omitting Ca2+ from the preparation medium. Panel A: micro-slicesprepared in bicarbonate-buffered medium and transferred to labelling medinra 7 min after zero time; panel B: as in panel A except that the preparation medium lacked Ca2+. Focussing, separation conditions and display as in Fig. 2. Arrow indicates the position of the labile 40 kDa phosphorylating system. B-50/F-1 is poorly separated from the latter in panel A, but is clearlyevident in B.
35
MrxlO-3 C 120'
80
40
|
÷
f
I
I
m
m
80-
4030"
O
O
20--
O O
17-
I
÷
Fig. 6. Autoradiographs prepared from hippocampus showing the effect of EGTA or a low concentration of Ca z+ in the labelling medium. The micro-slices were prepared in a medium containing 0.1 mM CaC12 by the standard procedure. Ca 2+ concentrations in the preincubation and labelling media were as follows: panels A and D, zero (1 mM EGTA); panels B and E, 0.1 mM; panels C and F, 1 mM. Panels A-C: non-equilibrium focussing using pH gradient (B) and gradient gel separation for second dimension; panels D - F : equilibrium focussing using pH gradient (C) and 14% gels for second dimension. Note that in A - C the pH gradient displays the full range of phosphopeptides (with M r values greater than 35 kDa), whereas in D - F the display is restricted to phosphopeptides with isoelectric points in the range 7.5 to 4.0 as in Figs. 2-5. The arrows indicate the position of the labile phoshorylating systems; the labelling of the phosphopeptide marked × in panels A - C is relatively very high in micro-slices prepared from the hippocampus. Note that the migration of phosphopeptides in panel F was faster than in panels D and E.
also b e n o t e d that, like t h e 40 k D a system, t h e 3 o t h e r p h o s p h o r y l a t i n g s y s t e m s w e r e l a b i l e in unbuffered bicarbonate-medium. T h e r e was a c o n s i s t e n t t e n d e n c y for t h e r e l a t i v e l a b e l l i n g o f s y n a p s i n 1 a n d B - 5 0 / F - 1 to b e h i g h e r
in m e d i a c o n t a i n i n g l o w C a 2+ o r E G T A t h a n in the standard incubation medium containing 1 mM C a 2+ ( c o m p a r e Figs. 6A, B w i t h Fig. 6C, a n d F i g 6 D , E w i t h Fig. 6F). B y c o n t r a s t the o p t i m a l p h o s p h o r y l a t i o n o f t h e 82 k D a s u b s t r a t e o f p r o -
36 tein kinase C always required 1 mM labelling medium (Fig. 6A-F).
C a 2+
in the
Inclusion of inorganic phosphate in the medium. Normal Krebs-Ringer medium contains 1-2 mM KH2PO4, but since inclusion of this anion greatly decreases the specific radioactivity of the 32Pi, it is almost universally omitted in labelling studies with intact tissue preparations. However, phosphatefree media are less physiological than normal media and we therefore decided to investigate the extent to which KH2PO4 could be added without necessitating an unacceptable increase in the amount of radioactivity added or in exposure times. Addition of as little as 0.1 mM phosphate to the preparation and preincubation media as well as to laboring media, resulted in a very low level of incorporation of radioactivity. However, after depletion of tissue phosphate by preparation and preincubation in phosphate-free media, it was possible to include 0.25 mM KH2PO 4 in the labelling medium without needing any increase in exposure; even with 1 mM phosphate in the labelling medium optimum exposure times were only increased threefold. One possible explanation of this observation is that the K m for the system transporting phosphate ions to the sites of ATP synthesis is higher than the actual concentration of phosphate derived from the carrier-free 32Pi in the medium; addition of cold phosphate may therefore be stimulating uptake by saturating the transport system. However it must be pointed out that phosphorylation patterns were unaffected by addition of KH2PO4 to the labelling medium.
Discussion
Our results demonstrate the feasibility of studying protein phosphorylation in relatively small cell-containing preparations of the rat brain. The 1 mm diameter discs used enable phosphorylation patterns to be obtained for structures such as the substantia rtigra, globus pallidus and amygdaloid nuclei. No attempt was made in the present work to label smaller discs of tissue applicable to more discrete nuclei. The main problem likely to be encountered in scaling down the procedure still further is not so much a matter of analytical scale
as of the integrity of the tissue: with decreasing size the ratio of the area of the cut surface of a disc to its volume increases, thus increasing the proportion of damaged tissue and the change of loss to the medium of essential constituents. This consideration may partly explain why in our preliminary study in which micro-slices of 2.5 mm diameter were used (Rodnight et al., 1986) the lability of the 40 kDa phosphorylating system was not noticed. The main analytical problems encountered in this work were the inconsistent focussing of MAP-2 and the generally poor resolution of the the B50/F1 substrate from the 40 kDa complex. Neither problem was encountered when we commenced work in this area ~ d we are inclined to ascribe current difficulties tO reagent variation. However a reliable separation of B-50/F1 could always be obtained by using 14% gels for the second dimension. With respect to the main numbered phosphopeptides, the in vitro phosphorylation patterns obtained from cerebral cortex and caudate nucleus were similar to those obtained from the same areas labelled in vivo under anaesthesia. Further work is needed, however, to establish the responsiveness of phosphorylating systems in the micro-slices to physiological activators of protein kinases. It is noteworthy that neither in vivo nor in vitro were we able to detect any labelling of the 50 kDa and 60 kDa subunits of the Ca2+/cal modulin-dependent protein kinase II, even after long exposures. This suggests that this phosphorylation event is only triggered by molecular processes that are not operating under anaesthesia or in the intact tissue in vitro. It has been proposed that the autophosphorylation of this enzyme constitutes a Ca2 +-triggered molecular switch operating in vivo (Miller and Kennedy, 1986). The lability of several phosphorylating systems during micro-slice preparation contrasts strikingly with the robust stability of the majority of systems. The quantitatively outstanding labile system phosphorylates a 40 kDa complex consisting of two phosphopeptides with identical isoclectric points and very similar M r values. Since both components exhibit exactly the same lability characteristics, they are probably both phosphorylatcd
37
by the same kinase system; however there is no evidence as to whether they are subunits of the same protein. The 40 kDa substrate complex appears to be highly characteristic of cell-rich regions of nervous tissue since significant labelling of phosphopeptides occupying the same position on two-dimensional separations could not be detected in micro-slices from spleen, kidney, liver or adrenal cortex; however in micro-slices of adrenal medulla a similar doublet was labelled (in preparation). On the basis of subcellular labelling studies we tentatively suggested (Rodnight et al., 1985) that the 40 kDa substrate complex is phosphorylated by a Ca2+/calmodulin-dependent kinase. Later work has confirmed this conclusion by showing that in subcellular preparations of cytosol the calmodulin antagonist, calmidazolium, inhibits the labelling of the complex by [-/-32P]ATP in the presence of Ca 2+ and calmodulin (unpublished data). The sensitivity of the 40 kDa and other systems to Ca 2÷ lack appears to involve two processes: irreversible inactivation and reversible inhibition. The former only occurs in the complete absence of Ca 2÷ in the preparation medium and can be prevented by inclusion of a low concentration of Ca 2÷ (0.1 mM). Reversal of the latter process, however, requires a higher concentration of Ca 2÷ (1 mM in the present study) and is presumably distinct. It may be relevant that Dunkley and Robinson (1981) found that preincubation of subcellular fractions resulted in an irreversible loss of Ca2+/calmodulin-dependent protein kinase activity. By contrast the remaining systems are remarkably insensitive to Ca 2÷ lack. This includes the B-50/F1 substrate of the Ca 2÷ and lipiddependent protein kinase C and synapsin 1 which is a substrate for Ca2+/calmodulin-dependent protein kinase II. Presumably in the intact tissue both these systems retain sufficient tightly bound native Ca 2÷ even in the presence of EGTA in the medium. A similar low Ca2+-sensitivity of protein phosphorylating systems in synaptosomes was observed by Robinson and Dunkley (1985).
Acknowledgements We are grateful to the Medical Research Council in the U.K. and to Research Fund of the Bethlem Royal and Maudsley Hospitals for support.
References Chan, S.Y., Murakami, K. and Routtenberg, A. (1986) Phosphoprotein FI: purification and characterization of a brain kinase C substrate related to plasticity, J. Neurosci., 6: 3618-3627. Cuello, A.C. and Carson, S. (1983) Microdissection of fresh rat brain tissue sfices. In A.C. Cuello (Ed.), Brain Microdissection Techniques, Wiley, Chichester, pp. 37-125. Duncan, R. and Hershey, J.W.B. (1984) Evaluation of isoelectric focussing running conditions during two-dimensional isoelectric focussing/sodium dodecyl sulphate polyacrylamide gel electrophoresis: variation of gel patterns with changing conditions and optimized isoelectric focussing conditions, Anal. Biochem., 138: 144-155. Dunkley, P.R. and Robinson, P. (1981) Calcium-stimulated protein kinases from rat cerebral cortex are inactivated by preincubation, Biochem. Biophys. Res. Commun., 102: 1196-1202. Gispen, W.H., Leussissen J.L.M., Oestreicher, A.B., Verkleij, A.J. and Zwiers, H. (1985). Presynaptic loealisation of B-50 phosphoprotein--the (ACTH)-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metablism, Brain Res., 328: 381-385. Gower, H. and Rodnight, R. (1982) Intrinsic protein phosphorylation in synaptic plasma membrane fragments from the rat. General characteristics and migration behaviour on polyacrylamide gels of the main phosphate acceptors, Biochim. Biophys. Acta, 716: 45-52. Heinonen, J.K. and Lahti, R.J. (1981) A new and convenient determination of inorganic orthophosphate and its application to the study of inorganic pyrophosphate, Anal. Biochem., 113: 313-317. Miller, S.G. and Kennedy, M.B. (1986) Regulation of brain type II CaE+/calmodulin-dependent protein kinase by autophosphorylation: a Ca 2 +-triggered molecular switch, Cell, 44: 861-870. O'FarreU, P.H. (1975) High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem., 250: 4007-4021. O'Farrell, P.Z., Goodman, H.M. and O'Farrell, P.H. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins, Cell, 12: 1133-1142.
38 Ouimet, C.C., Miller, P.E., Hemmings, H.C., Walaas, S.I. and Greengard, P. (1984) DARPP-32, a dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein enriched in dopamine innervated brain regions, J. Neurosci., 4: 111-124. Palkovits, M. (1973) Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Res., 59: 449-450. Robinson, P.J. and Dunldey, P.R. (1985) Depolarisation-dependent protein phosphorylation and dephosphorylation in rat cortical synaptosomes is modulated by calcium, J. Neurochem., 44: 338-348. Rodnight, R. and Perrett, C. (1986) Protein phosphorylation and synaptic transmission: receptor-mediated modulation
of protein kinase C in a rat brain fraction enriched in synaptosomes, J. Physiol. (Paris), 81: 340-348. Rodnight, R., Trotta, E.E. and Perrett, C. (1985) A simple and economical method for studying protein phosphorylation in vivo in the rat brain, J. Neurosci. Methods, 13: 87-95. Rodnight, R., Perrett, C. and Soteriou, S. (1986) Two-dimensional patterns of neural phosphoproteins from the rat labelled in vivo under anaesthesia and in vitro in slices and synaptosomes, Prog. Brain Res., 69: 373-382. Wu, W.C-S., Walaas, S.I., Nairn, A.C. and Greengard, P. (1982) Calcium phospholipid regulates the phosphorylation of a M r '87K' substrate protein in brain synaptosomes, Proc. Natl. Acad. Sci. U.S.A., 79: 5249-5253.
27
NSM 00802
An investigation of experimental conditions for studying protein phosphorylation in micro-slices of rat brain by two-dimensional electrophoresis Richard Rodnight, Reza Zamani and Alastair Tweedale Department of Biochemistry, Institute of Psychiatry, London (U.K.) (Received 8 April 1987) (Revised 23 October 1987) (Accepted 26 October 1987)
Key words: Phosphoprotein; 32p-incorporation; In vitro labelling; Micro-slice of brain; Lability of protein phosphorylation Procedures are described for studying protein pbosphorylation in 1 mm diameter micro-slices of rat brain tissue using two-dimensional electrophoresis as analytical tool The activity of several protein phosphorylatingsystems, including a major system phosphorylating a 40 kDa substrate complex, was highly dependent on the procedures used for micro-slice preparation and on the Ca2+-content of the preparation medium. Under optimal conditions the pattern of phosphorylation observed in micro-slices closely resembled that obtained by in vivo labelling.
Introduction In previous work (Rodnight et al., 1985) we described a simple and economical method for studying protein phosphorylation in vivo in the rat brain, using high resolution two-dimensional electrophoresis as an analytical tool. In the present paper we describe a complementary in vitro procedure applicable to micro-slices of brain. Basically the work had the following aims: (1) to establish a safe and economical procedure suitable for routine application to discrete brain structures; (2) to compare in vivo and in vitro phosphorylation patterns and create incubation conditions that reproduced as far as possible the in vivo pattern; (3) explore further optimum conditions for analysis of the patterns by two-dimensional electrophoresis.
Correspondence: R. Rodnight. Present address: Departamento de Bioquimica, Instituto de Biociencias, UFRGS (Centro), 90.040 Porto Alegre RS, Brazil.
The work is a development of preliminary experiments reported earlier (Rodnight et al., 1986).
Methods
Source of materials [32p]Inorganic phosphate (10 m C i / m l , carrier free in water) was obtained from Amersham International. Acrylamide and Ampholines were purchased from LKB, Servalyts from Serva (Heidelberg, F.R.G.), sodium dodecyl sulphate (SDS, 99%) from B D H Chemicals (Poole, UK). Other chemicals were purest available. Wistar strain rats of either sex and weight 150-250 g were used as a source of brain tissue. Preparation of micro-slices Rats were killed by decapitation (designated as zero time) and the brain removed within 1 min. Macro-slices encompassing several anatomical re-
0165-0270/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
28 gions were first cut with a Mcllwain chopper. To accomplish this some modifications were necessary to the usual procedure for chopping tissue: in particular it was very important to ensure good adhesion of the comparatively large pieces of brain to the filter paper. This was achieved by initially mounting a cut surface of the tissue on dry filter paper, thus avoiding the use of cyanoacrylate adhesive as recommended by Cuello and Carson (1983). The chopper was adjusted to cut 0.4 mm thick slices and two circles of filter paper moistened with ice-cold medium (see below) fixed to the platform. The brain was roughly dissected (e.g. by sagittally dividing the forebrain) and the main cut surface of the required region gently pressed onto the centre of a dry circle of filter paper. This was placed on the chopper platform and immediately wetted by adding medium drop by drop to the periphery of the paper and allowing it to diffuse towards the tissue. It was important to add just sufficient medium to ensure that the paper and tissue remained in position during the cutting process. The surface of the tissue was then moistened with a minimum quantity of medium and the required number of slices cut. With practice it was possible to cut 10 or more hemi-coronal or longitudinal slices of forebrain or cerebellum. It was also possible to cut whole coronal slices of the midbrain through the thalamus and the colliculi to the brainstem by first removing the paleo- and neocortex and most of the cerebellum and pressing the ventral surface of the remaining tissue onto a circle of dry filter paper. However, without a cut surface in contact with the paper adhesion was less satisfactory and more pressure was needed to prevent the tissue from rifting during chopping. The chopped tissue was transferred to a dish containing about 5 ml of ice-cold medium and the slices teased apart with two sable hair brushes. (No attempt was made to maintain the temperature of the medium at 4 ° C during subsequent operations.) Slices containing the structures required were then transferred to a second dish, the bottom of which was lined with polythene. This dish contained just enough medium to cover the polythene. Discs of tissue (micro-slices) were prepared with stainless steel punches by a procedure
essentially similar to that first described by Palkovits (1973; see also Cuello and Carson, 1983) for thinner sections of frozen brain. With the plunger partially withdrawn the punch was pressed firmly through the appropriate area of the macroslice onto the surface of the polythene, using sufficient pressure to just mark the surface of the latter; on withdrawing the punch the micro-slice remained entrapped in the tip of the punch and could be expelled into the medium of the next stage (see below) by gently depressing the plunger. For all of the present work a punch of internal diameter 1 mm was used; larger diameter punches (e.g. 2.0, 2.5 mm) used in an earlier study (Rodnight et al., 1986) are only applicable in the rat brain to regions such as the caudate or neocortex. To facilitate transfer to the radioactive medium the micro-slices were first collected on a rectangulax tray of black acrylic plastic incorporating shallow depressions each containing 100/~1 of medium at room temperature (17-23 ° C). Two 1 mm diameter micro-slices were placed in each depression. In early experiments the bicarbonate-buffered medium was exposed to air at this stage, but for reasons that will become apparent, in later experiments the tray was enclosed in a box that permitted the medium to be saturated with 95% 02/5% CO 2 as illustrated in Fig. 1. In the present study micro-slices were transferred to the radioactive medium by spearing them with the bent tip of a mounted 21 gauge hypodermic needle. An alternative procedure, developed towards the end of the work and applicable to discs of diameter 1 mm, used an automatic micro-pipette. The plastic tip was cut back so as to provide an orifice just wide enough to admit the micro-slices and the pipette was adjusted to deliver 10/~1. With practice it was possible to introduce two micro-slices, together with 10 #1 of pre-incubation medium, into the pipette tip and deliver both liquid and tissue to an incubation tube containing 15 /~1 of labelling medium instead of the usual 25 #1. This procedure has two important advantages: it avoids the damage caused by spearing the micro-slices and it avoids exposing them to the atmosphere during transfer. Recent work suggests that both these circumstances are significant factors affect-
29 c
A
n i
t
I
2cm
b,
b_
Co
B
C
if--Fig. 1. Equipment used to deplete micro-slices of inorganic phosphate prior to transfer to labelling medium. The central part consisted of a sheet of black acrylic plastic (Perspex) incorporating 8 shallow depressions and boxed as indicated. Each depression contained 100 #1 of bicarbonate-buffered medium and was surrounded by 4 holes through which gas was passed from a lower chamber. Two bleed holes were bored in the tightly fitting lid. Black plastic was used in order to facilitate visualisation of the micro-slices during transfer. ing reproducibility in studies using very small discs of tissue. However, problems with the pipetting procedure m a y arise in experiments where the pre-incubation and incubation media are different.
Incubation conditions The micro-slices were labelled at 3 7 ° C in tapered polystyrene tubes (12 × 75 m m ) containing 100 ~Ci of carrier-free [32p]orthophosphate dissolved in 25 #1 of incubation medium, giving a 32p-concentration of 4 m C i / m l . The incubation area was protected with 12 m m thick screens of acrylic plastic (Perspex). The original 32p-solution was dispensed in the tubes the day before the experiment and dried overnight under vacuum over silica gel. The standard incubation medium had the following composition (mM): NaC1 124, KC1 4, MgSO 4 1.2, N a H C O 3 26, glucose 12, saturated with 95% 0 2 / 5 % CO 2. CaCI2, usually to 1 mM, was added after gassing. In a few experiments K H 2 P O 4 was added in the range 10/~M to 1 mM. The incubation tubes were filled with 95%
02//5% CO 2 and stoppered immediately after adding the medium and gassed again after transferring the micro-slices. Each tube contained two 1 m m diameter micro-slices or one of larger diameters. The standard incubation time was 45 min at 37 ° C and the tubes were gently agitated by rotation every 5 min. Labelling was stopped by addition of 50 t~l of 15% trichloroacetic acid, after which the tubes were placed on ice to await transfer to electrophoresis sample buffer. In preliminary experiments the macro- and micro-slices were prepared in the same bicarbonatebuffered medium used for labelling. In later work, in order to avoid exposing the tissue to p H values higher than 7.4, they were prepared in the same medium but buffered with 25 m M Tris-citrate (pH 7.4 at r o o m temperature) instead of bicarbonate/CO2.
Two-dimensional electrophoresis The acid-fixed micro-slices were removed from the incubation tubes with a mounted hypodermic needle tip, immersed briefly in about 100/~I of 250 m M N a 2 H P O 4 to wash off excess acid and then
30 dissolved in a volume of electrophoresis sample buffer sufficient to give a protein concentration of 1 ~tg//~l essentially by the two-stage process described previously (Rodnight et al., 1985). The procedure for 1 mm diameter micro-slices was as follows. The two sample solutions were: A 9.5 M urea, 12.5 mM lysine, 1% sodium dodecyl sulphate. 2% (v/v) 2-mercaptoethanol, 0.005% bromophenol blue; B: 9.5 M urea, 12.5 mM lysine, 5% (v/v) Nonidet P-40, 2% (v/v) 2-mercaptoethanol, 0.005 % bromophenol blue. Each micro-slice contained approximately 30 /~g of protein; therefore two micro-slices (from one incubation tube) were initially dissolved with aid of a fine glass rod in 12 /zl of solution A contained in a 0.25 ml capacity Eppendorf centrifuge tube. Sample solution B (48/~1) was then added and the two solutions thoroughly mixed. The solution was frozen, thawed and centrifuged at 12,000 g (Microcentaur) for 5 min. The supernatant was separated from the trace of insoluble material and set aside pending analysis. Glass tubes (i.d. 1.5 mm, o.d. 4 mm) were used for isoelectric focussing; the gel length was 13.5 cm. The basic gel mixture had the following composition: 3.5% (T) acrylamide (Cbi s = 3.6%), 2% ampholytes, 9.5 M urea, 4% (v/v) Nonidet P-40 and 0.08% N,N,N'N'-tetraethy!ene/diamine (TEMED). This mixture was prepared in bulk and stored in volumes of 0.3 ml in conical plastic tubes at - 7 5 ° C under N 2. Individual gels were prepared at about 3 0 ° C by adding 5#1 of 10% ammonium persulphate to each 0.3 ml, briefly mixing and then filling the glass focussing tube to exactly 2 cm from the f o p b y negative pressure supplied by a 1 ml'syringe a n d needle fitted with a length of polythene'~tiabing and a suitable adaptor. The glass tube, with the syringe and tubing still attached, was then left in the residual gel in the plastic tube until gelation had occurred. Several pH gradients were explored of which the following were employed routinely (% concentrations are w/v). (A) 1.64% Ampholine pH 3.5-10, 0.18% Ampholine pH 5-7, 0.18% Servalyt pH 2--4; (B) 1.6% Ampholine pH 3.5-10, 0.32% Ampholine pH 5-7, 0.08% Servalyt pH 2-4; (C) 2% Servalyt pH 2-11. Equilibrium focussing was performed according to O'Farrell (1975) with gradients (A) and (C), but
using 50 mM NaOH as catholyte and 25 mM H3PO 4 as anolyte as recommended by Duiacan and Hershey (1984). After loading 15/~1 of s~nple and overlaying with an equal volume of 8 M urea the gels were focussed for 400 V overnight followed by 1-2 h at 1000 V. For non-equilibrium focussing (NEPHGE; O'Farrell et al.. 1977) the sample (15 /~1) was loaded at the anode and for anolyte and catholyte we used 10 mM H3PO 4 and 20 mM N a O H respectively. In this case the gels were overlaid with 8 M urea containing cytochrome C (approx. 80 /~g/ml) and the gels were run at 400 V until the main red cytochrome band had reached 7.5 cm from the origin. On completion of focussing the gels in their glass tubes were stored at - 2 0 °C pending transfer. The procedures used for the second dimension were based on Gower and Rodnight (1982) and R0dnight and Perrett (1986). Immediately after thawing gels were extruded by air pressure and equilibrated with gentle shaking for 15 min in 4 ml of second dimension upper buffer supplemented with 2% sodium dodecyl sulphate. Because entry and resolution of phosphoproteins in the basic region of equilibrium focussing gels was unsatisfactory, these gels were bisected, the basic halves discarded and two acidic halves mounted on a single second dimension slab gel with the acidic ends placed centrally; 1% agarose dissolved in standard upper buffer was used to secure the gels. In the case of non-equilibrium runs all of the gel below the cytochrome C band was discarded and the two upper sections mounted on one slab gel, usually with the basic ends central. Either gradient slab gels (6-19%, exponential) or 14% gels, both with 4% stacks, were used for the second dimension. Gradient gels were run during the daytime by the schedule used previously (Gower and Rodnight, 1982); 14% gels were run overnight at a constant potential of 55 V. After electrophoresis the gels were fixed, dried and exposed to X-ray film as described previously (Rodnight et al., 1985). Optimum exposure times for gradient gels varied from 12 to 48 h.
Coding and display of phosphopeptides In the figures phosphopeptides are numbered by the same code used in earlier work (Rodnight
31 et al., 1985, 1986). The 45 kDa substrate of protein kinase C (phosphopeptide 4) is designated B50/F1 since both terms are used in the literature for this molecule (e.g. Gispen et al., 1985; Chan et al., 1986); phosphopeptide 2 (M r 82 kDa) corresponds to the 87 kDa substrate of protein kinase C described by Wu et al. (1982). Evidence concerning the provisional identities of other substrates is given in the above papers. In contrast to our previous work, but to conform with current practice, autoradiographs are displayed with the acidic end of the focussing dimension on the left.
Other methods Inorganic orthophosphate was determined by a micro-adaptation of the method of Heinonen and Lahti (1981). Adenine nucleotides (ADP and ATP) were determined by HPLC using a linear ammonium formate gradient (K. Weaver and M. Brammer, in preparation). Results
Electrophoresis procedures Examples of the various procedures explored are given in Figs. 2-6. The separation obtained with equilibrium focussing using either pH gradient (A) or (C) followed by SDS-electrophoresis in gradient gels (Figs. 2B, 3B and 5A) gave excellent separation of most substrates with isoelectric points less than 6, but generally poorer separation of substrates with higher values; pH gradient (C) gave a marginally superior separation and was more convenient to prepare. However, the separation of B50/F1 from a major 40 kDa substrate complex (phosphopeptide 6) was unreliable using either pH gradient (compare Fig. 2B with Fig. 5A). Although we were unable to improve on this by employing other pH gradients, consistent separation of the two phosphopeptides could be achieved by taking advantage of the anomalously low free electrophoretic mobility of the B50/F1 substrate (Gower and Rodnight, 1982) and its resulting relatively higher migration rate, compared with the 40 kDa complex, in the second dimension on 14% gels (Figs. 4, 6F). The 14% gels were unsuitable, except for a few acidic molecules, for studying phosphopeptides with M r values
above 40 kDa, but gave excellent separation in the range 35-14 kDa (Fig. 4). It may be noted that many of these substrates labelled relatively slowly compared with those of higher Mr, and this necessitated long exposure times which left the high M r region uninterpretable. An unsolved problem encountered with equilibrium focussing concerned the high molecular weight phosphopeptide 3, which we provisionally identify as microtubule-associated protein 2 (MAP-2). For reasons we have been unable to determine the focussing of this molecule was very erratic and in most experiments it either failed to penetrate the gel or remained as a streak in the basic region. The result shown in Fig. 2 was an exceptionally good one in that MAP-2 was fully represented; in the equilibrium separations illustrated in Figs. 3 and 5, on the other hand, it was absent and for this reason the relevant parts of the autoradiographs have been omitted. The problem was not related to the porosity of the focussing gel since total acrylamide concentrations lower than the 3.5% used routinely, or wide variations in the bis-acrylamide concentration, failed to improve matters. The solubility and recovery of the other numbered polypeptides was consistently reproducible throughout the study. Non-equilibrium (NEPHGE) focussing employing pH gradient (B), followed by gradient gels for the second dimension, gave a valuable general separation including the highly basic substrate synapsin I, but completely failed to separate the B50/F1 and 40 kDa substrates (Fig. 6C); however resolution of the complex of phosphopeptides coded 8, which probably include a- and fl-tubulin, was consistently better by NEPHGE than by equilibrium focussing (compare Fig. 2B with Fig. 6C).
Micro-slice preparation When micro-slices were prepared in bicarbonate-buffered sahne and transferred to medium containing 32Pi within 7 min of zero time the equilibrium focussing pattern shown in Fig. 2B was obtained. From Fig. 2A it can be seen that the main features of this in vitro pattern were very similar to those observed in brain tissue after labelling in vivo by the technique of Rodnight et al. (1985). This suggested that relatively little post-
32
M r x 10 -3 ~.~
Fig. 2. Autoradiographs prepared from cortical tissue labelled in vivo (panel A) by the procedure of Rodrtight et al. (1985) and labelled in vitro (panel B) by the present standard micro-sliceprocedure, except that the micro-sliceswere prepared in bicarbonatebuffered medium and transferred to labelling medium 7 min after zero time. Equilibrium focussing using pH gradient (A); in both cases the section of the pH gradient displayed extends from approximately 7.3 to 4.0. Gradient gel separation in second dimension. Provisional identities of the numbered phosphopeptides are as follows: 2, 82-87 kDa substrate of protein kinase C; 3, microtubuleassociated protein 2; 4, the B-50/F-1 substrate of protein kinase C; 6, the unknown 40 kDa substrate complex; 7, the a-subunit of pyruvate dehydrogenase; 8, polypeptides co-migrating with tubulin sub-units. mortem change in protein phosphorylating capacity had occurred during the 7 min required for slice preparation. However, only about 6 microslices from a major area of the brain such as the caudate or cerebral cortex could be prepared in this period of time and before embarking on more complex experiments involving several discrete anatomical regions it was considered advisable to re-investigate stability during longer time intervals. To our surprise we found that leaving micro-slices in the preparation medium for longer periods before transferring to the incubation medium had two consequences: (a) a progressive decline in the labelling capacity of a system phosphorylating the 40 k D a substrate complex (phosphopeptide 6 in Fig. 2) to the extent that after 40 re_in in preparation medium virtually no label was transferred to it on subsequent incubation and (b) a marked tendency for the great majority of the remaining substrates to incorporate more 32p with increasing time in preparation medium. A typical experiment is shown in Fig. 3, which compares the pattern obtained from a pair of micro-slices of
caudate nucleus transferred to labeUmg medium 7 min after zero time (panel A) with a pair taken from the same macro-slice transferred after 30 min (panel B). The increased incorporation of 32p resulting from delaying transfer appeared to be due to loss of endogenous inorganic phosphate from the tissue and a consequent increase in the specific radioactivity of the intracellular 32pi pool, In support of this conclusion we found that when 1 m m micro-slices were left in medium for 30 min after preparation over 90% of their inorganic phosphate was lost; with 2.5 m m slices the loss was of the order of 60%. The loss of activity in the 40 k D a phosphorylating system was found to be related to the rise in p H of the bicarbonate-buffered medium as it lost CO 2 to the atmosphere, since it did not occur when micro-slices were left in gassed bicarbonate medium or in Tris-buffered medium (Fig. 3C). The loss was also less marked when micro-slices were maintained in relatively large volumes of bicarbonate-buffered medium which retained CO 2 for longer than did the small volumes used to
33
MrxlO -3 12(
8(
4(
Fig. 3. Autoradiographs prepared from caudate nucleus labelled in vitro after various preparation procedures. Panel A: micro-slices prepared in bicarbonate-buffered medium and transferred to radioactive medium 7 min after zero time; panel B: micro-slices prepared in bicarbonate-buffered medium and transferred 35 min after zero time; panel C: micro-slices prepared in Tris-buffered medium, transferred 7 rain after zero time to 100 /xl of bicarbonate-buffered medium (maintained at pH 7.4 by gassing) in the equipment illustrated in Fig. 1 and transferred to radioactive medium 40 mm after zero time. Focussing, separation conditions and display as in Fig. 2. Arrow indicates the position of the labile 40 kDa phosphorylating system; provisional identity of phosphopeptide labelled 5 is DARPP-32 (Ouimet et al., 1984). collect a series of micro-slices f r o m various brain regions. I n the light of these observations the following procedure was a d o p t e d as standard: micro-slices were prepared in Tris-buffered medium, transferred within 15 min of zero time to bicarbonatebuffered m e d i u m (maintained at p H 7.4 with O 2 / C O 2 ) and then left for 30 rain at r o o m temperature (preincubation stage) to allow time for the tissue inorganic phosphate content to reach a stable low content before transferring to labelling m e d i u m at 37 o C. The procedure gave consistently high recoveries of the 40 k D a p h o s p h o r y l a t i n g system and reproducible levels of 32p incorporation. As a measure of metabolic integrity we measured the A T P and A D P content of micro-slices subjected to the routine procedure. In a typical experiment the initial concentration of A T P ( / ~ m o l / g / f r e s h wt) was 0.68, rising to 0.80 after
preincubation for 30 min and to 1.25 after incubation at 37 ° C for 30 min; the corresponding values for A D P were 0.64, 0.22 and 0.21.
Incubation conditions Time course. I n c o r p o r a t i o n into acid-precipitated protein followed a curvilinear course over a 60 min period without reaching a plateau and with easily detectable levels being attained after 10 min. For most experiments in the present work a 45 min period was adopted. There was no obvious variation in the rate of labelling of different substrates. Effect of varying Ca 2 +-content. In initial experiments designed to study this variable we prepared micro-slices in Ca2+-free bicarbonatebuffered m e d i u m and transferred them directly to labelling m e d i u m containing either E G T A or various concentrations of Ca 2+ within 7 min of zero time. With certain exceptions described below
34
M r x 10 -3
Fig. 4. Autoradiographs prepared from cerebellar cortex labelled in vitro by the standard procedure and using a 14% gel for the second dimension. In panels A and B the gel was exposed for 6 days and 18 h respectively. Focussingconditions and display as in Fig 1, except that pH gradient (C) was used. Note the excellent separation of pliospliopeptide 4 (B-50/F-1) from the 40 kDa complex. The diffuse labelled material marked with an arrow in panel A was an occasional feature of all autoradiographs and is not typical of cerebellar cortex. However, the exceptionally high relative labelling of the 82 kDa phosphopeptide is typical of this tissue, as noted elsewhere (Rodnight et al., 1986). there was no detectable difference in the amount of 32p incorporated in the range of Ca 2+concentrations from 0.5 mM to 1.25 mM; higher Ca2+-concentrations were markedly inhibitory. The major exception to this result concerned the 40 kDa phosphorylating system: regardless of the concentration of Ca 2+ in the labelling medium, micro-slices prepared in Ca2+-free medium always failed to phosphorylate significantly the 40 k D a substrate complex. An early experiment in which the micro-slices were prepared in either normal bicarbonate medium or Ca2+-free bicarbonate medium and transferred to labelling medium containing 1 m M Ca 2÷ is shown in Fig. 5. In a later series of experiments using the standard procedure 0.1 mM Ca 2+ was included in the initial Trisbuffered preparation medium and the micro-slices were transferred for the preineubation and labelling stages to media containing either zero Ca 2+ (EGTA), 0.1 m M Ca 2÷ or 1 m M Ca 2÷. From Fig. 6C, F it can be seen that the inclusion of this low
concentration of Ca 2+ in the preparation medium fully preserved the 40 kDa phosphorylating system when the subsequent media contained 1 mM Ca 2÷. However with 0.1 m M Ca 2+ present throughout virtually no phosphorylation of the 40 k D a complex was observed (Fig. 6B, E). Surprisingly, incorporation of phosphate into the great majority of the remaining acceptors was not decreased in medium containing 1 mM EGTA (Fig. 6A, D). Exactly the same type of lability was observed in at least 3 other less prominent phosphorylating systems. The most interesting of these was a system that is uniquely active in micro-slices from the hippocampus. The acceptor for this system is a 50 k D a phosphopeptide with an isoelectric point of about 6.3; it was particularly well displayed by non-equilibrium focussing (Fig. 6C indicated by × ). Phosphorylation of this acceptor can also be detected in micro-slices from certain other brain areas, but at a much slower rate (in preparation). The two other labile systems phosphoryated substrates with M r values of about 17 kDa (Fig. 6 D - F ) ; so far no regional differences in the activity of these systems have been observed. It may
Mr x 10 -3
Fig. 5. Autoradiographsprepared from cerebral cortex showing the effect of omitting Ca2+ from the preparation medium. Panel A: micro-slicesprepared in bicarbonate-buffered medium and transferred to labelling medinra 7 min after zero time; panel B: as in panel A except that the preparation medium lacked Ca2+. Focussing, separation conditions and display as in Fig. 2. Arrow indicates the position of the labile 40 kDa phosphorylating system. B-50/F-1 is poorly separated from the latter in panel A, but is clearlyevident in B.
35
MrxlO-3 C 120'
80
40
|
÷
f
I
I
m
m
80-
4030"
O
O
20--
O O
17-
I
÷
Fig. 6. Autoradiographs prepared from hippocampus showing the effect of EGTA or a low concentration of Ca z+ in the labelling medium. The micro-slices were prepared in a medium containing 0.1 mM CaC12 by the standard procedure. Ca 2+ concentrations in the preincubation and labelling media were as follows: panels A and D, zero (1 mM EGTA); panels B and E, 0.1 mM; panels C and F, 1 mM. Panels A-C: non-equilibrium focussing using pH gradient (B) and gradient gel separation for second dimension; panels D - F : equilibrium focussing using pH gradient (C) and 14% gels for second dimension. Note that in A - C the pH gradient displays the full range of phosphopeptides (with M r values greater than 35 kDa), whereas in D - F the display is restricted to phosphopeptides with isoelectric points in the range 7.5 to 4.0 as in Figs. 2-5. The arrows indicate the position of the labile phoshorylating systems; the labelling of the phosphopeptide marked × in panels A - C is relatively very high in micro-slices prepared from the hippocampus. Note that the migration of phosphopeptides in panel F was faster than in panels D and E.
also b e n o t e d that, like t h e 40 k D a system, t h e 3 o t h e r p h o s p h o r y l a t i n g s y s t e m s w e r e l a b i l e in unbuffered bicarbonate-medium. T h e r e was a c o n s i s t e n t t e n d e n c y for t h e r e l a t i v e l a b e l l i n g o f s y n a p s i n 1 a n d B - 5 0 / F - 1 to b e h i g h e r
in m e d i a c o n t a i n i n g l o w C a 2+ o r E G T A t h a n in the standard incubation medium containing 1 mM C a 2+ ( c o m p a r e Figs. 6A, B w i t h Fig. 6C, a n d F i g 6 D , E w i t h Fig. 6F). B y c o n t r a s t the o p t i m a l p h o s p h o r y l a t i o n o f t h e 82 k D a s u b s t r a t e o f p r o -
36 tein kinase C always required 1 mM labelling medium (Fig. 6A-F).
C a 2+
in the
Inclusion of inorganic phosphate in the medium. Normal Krebs-Ringer medium contains 1-2 mM KH2PO4, but since inclusion of this anion greatly decreases the specific radioactivity of the 32Pi, it is almost universally omitted in labelling studies with intact tissue preparations. However, phosphatefree media are less physiological than normal media and we therefore decided to investigate the extent to which KH2PO4 could be added without necessitating an unacceptable increase in the amount of radioactivity added or in exposure times. Addition of as little as 0.1 mM phosphate to the preparation and preincubation media as well as to laboring media, resulted in a very low level of incorporation of radioactivity. However, after depletion of tissue phosphate by preparation and preincubation in phosphate-free media, it was possible to include 0.25 mM KH2PO 4 in the labelling medium without needing any increase in exposure; even with 1 mM phosphate in the labelling medium optimum exposure times were only increased threefold. One possible explanation of this observation is that the K m for the system transporting phosphate ions to the sites of ATP synthesis is higher than the actual concentration of phosphate derived from the carrier-free 32Pi in the medium; addition of cold phosphate may therefore be stimulating uptake by saturating the transport system. However it must be pointed out that phosphorylation patterns were unaffected by addition of KH2PO4 to the labelling medium.
Discussion
Our results demonstrate the feasibility of studying protein phosphorylation in relatively small cell-containing preparations of the rat brain. The 1 mm diameter discs used enable phosphorylation patterns to be obtained for structures such as the substantia rtigra, globus pallidus and amygdaloid nuclei. No attempt was made in the present work to label smaller discs of tissue applicable to more discrete nuclei. The main problem likely to be encountered in scaling down the procedure still further is not so much a matter of analytical scale
as of the integrity of the tissue: with decreasing size the ratio of the area of the cut surface of a disc to its volume increases, thus increasing the proportion of damaged tissue and the change of loss to the medium of essential constituents. This consideration may partly explain why in our preliminary study in which micro-slices of 2.5 mm diameter were used (Rodnight et al., 1986) the lability of the 40 kDa phosphorylating system was not noticed. The main analytical problems encountered in this work were the inconsistent focussing of MAP-2 and the generally poor resolution of the the B50/F1 substrate from the 40 kDa complex. Neither problem was encountered when we commenced work in this area ~ d we are inclined to ascribe current difficulties tO reagent variation. However a reliable separation of B-50/F1 could always be obtained by using 14% gels for the second dimension. With respect to the main numbered phosphopeptides, the in vitro phosphorylation patterns obtained from cerebral cortex and caudate nucleus were similar to those obtained from the same areas labelled in vivo under anaesthesia. Further work is needed, however, to establish the responsiveness of phosphorylating systems in the micro-slices to physiological activators of protein kinases. It is noteworthy that neither in vivo nor in vitro were we able to detect any labelling of the 50 kDa and 60 kDa subunits of the Ca2+/cal modulin-dependent protein kinase II, even after long exposures. This suggests that this phosphorylation event is only triggered by molecular processes that are not operating under anaesthesia or in the intact tissue in vitro. It has been proposed that the autophosphorylation of this enzyme constitutes a Ca2 +-triggered molecular switch operating in vivo (Miller and Kennedy, 1986). The lability of several phosphorylating systems during micro-slice preparation contrasts strikingly with the robust stability of the majority of systems. The quantitatively outstanding labile system phosphorylates a 40 kDa complex consisting of two phosphopeptides with identical isoclectric points and very similar M r values. Since both components exhibit exactly the same lability characteristics, they are probably both phosphorylatcd
37
by the same kinase system; however there is no evidence as to whether they are subunits of the same protein. The 40 kDa substrate complex appears to be highly characteristic of cell-rich regions of nervous tissue since significant labelling of phosphopeptides occupying the same position on two-dimensional separations could not be detected in micro-slices from spleen, kidney, liver or adrenal cortex; however in micro-slices of adrenal medulla a similar doublet was labelled (in preparation). On the basis of subcellular labelling studies we tentatively suggested (Rodnight et al., 1985) that the 40 kDa substrate complex is phosphorylated by a Ca2+/calmodulin-dependent kinase. Later work has confirmed this conclusion by showing that in subcellular preparations of cytosol the calmodulin antagonist, calmidazolium, inhibits the labelling of the complex by [-/-32P]ATP in the presence of Ca 2+ and calmodulin (unpublished data). The sensitivity of the 40 kDa and other systems to Ca 2÷ lack appears to involve two processes: irreversible inactivation and reversible inhibition. The former only occurs in the complete absence of Ca 2÷ in the preparation medium and can be prevented by inclusion of a low concentration of Ca 2÷ (0.1 mM). Reversal of the latter process, however, requires a higher concentration of Ca 2÷ (1 mM in the present study) and is presumably distinct. It may be relevant that Dunkley and Robinson (1981) found that preincubation of subcellular fractions resulted in an irreversible loss of Ca2+/calmodulin-dependent protein kinase activity. By contrast the remaining systems are remarkably insensitive to Ca 2÷ lack. This includes the B-50/F1 substrate of the Ca 2÷ and lipiddependent protein kinase C and synapsin 1 which is a substrate for Ca2+/calmodulin-dependent protein kinase II. Presumably in the intact tissue both these systems retain sufficient tightly bound native Ca 2÷ even in the presence of EGTA in the medium. A similar low Ca2+-sensitivity of protein phosphorylating systems in synaptosomes was observed by Robinson and Dunkley (1985).
Acknowledgements We are grateful to the Medical Research Council in the U.K. and to Research Fund of the Bethlem Royal and Maudsley Hospitals for support.
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38 Ouimet, C.C., Miller, P.E., Hemmings, H.C., Walaas, S.I. and Greengard, P. (1984) DARPP-32, a dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein enriched in dopamine innervated brain regions, J. Neurosci., 4: 111-124. Palkovits, M. (1973) Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Res., 59: 449-450. Robinson, P.J. and Dunldey, P.R. (1985) Depolarisation-dependent protein phosphorylation and dephosphorylation in rat cortical synaptosomes is modulated by calcium, J. Neurochem., 44: 338-348. Rodnight, R. and Perrett, C. (1986) Protein phosphorylation and synaptic transmission: receptor-mediated modulation
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