Insect Biochera. Vol. 18, No. 4, pp. 351-358, 1988 0020-1790/88 $3.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1988 Pergamon Press plc

THIOPHOSPHORYLATION OF THE REGULATORY SUBUNIT OF THE cAMP-DEPENDENT PROTEIN

KINASE FROM DROSOPHILA BRAIN TISSUE

ULI MOLLER, ZOFIA WOJNA, BERND KONIG and HANNS-CHRISTOF SPATZ Institut fiir Biologic III, der Universit/it Freiburg, Sch/inzlestrasse I, 7800 Freiburg, F.R.G.

(Received 11 November 1986; revised and accepted 15 December 1987)

Abstract---cAMP-dependent thiophosphorylation can be observed in homogenates of Drosophila heads. The reaction is characteristic for nervous tissues and hardly found in muscle tissue. Apparent molecular weight in SDS-PAGE, isoelectric points, peptide mapping and specific binding to cAMP-agarose identify the major substrate as the regulatory subunit of the Drosophila type II cAMP-dependent protein kinase. Since only very few other proteins are thiophosphorylated, [3'-35S]ATP serves as a specific tool to sensitively assay type II protein kinase in Drosophila tissue and to study phosphorylation and dephosphorylation.

Key Word Index: cAMP-dependent protein kinase II, thiophosphorylation

INTRODUCTION

Genetic (Byers et al., 1981; Aceves-Pina et al., 1983) and neuropharmacological evidence (Folkers and Spatz, 1984) points to an essential role of cAMP in the process of learning and memory of Drosophila. Since in eukaryotes cAMP exerts its function mainly, if not exclusively, by activation of protein kinases (Flockhart and Corbin, 1982; Beavo and Mumby, 1982; Nestler and Greengard, 1984), studies of pro- tein phosphorylation (Devay et al., 1984; Hesse and Marine, 1985; Willmund, 1986; Devay et al., 1986) promise to open a route towards a molecular biology of learning in Drosophila (Quinn et al., 1984; Spatz, 1985).

In Drosophila, where biochemical experiments on single neurons are not yet possible, phosphorylation was studied in homogenates of heads, neural tissue, or synaptosomal fractions (Kelly, 1981). Due to the large number of proteins which are phosphorylated with ATP, the autoradiographs after one- dimensional electrophoresis are difficult to evaluate quantitatively and bands are not easily identified without a two-dimensional analysis (Matsumoto and Pak, 1984). To reduce the complexity we replaced D,-32p]ATP by [~-35S]ATP (Eckstein, 1985). This communication describes the cAMP-dependent thio- phosphorylation of the major substrate and its identification as the regulatory subunit of the Droso- phila type II cAMP-dependent protein kinase (Foster et al., 1984). Thiophosphorylation proves to be a specific and sensitive assay for this protein kinase in the nanogram range. It can also be used to study the mechanism of phosphorylation-dephosphorylation processes in homogenates of neural tissue.

MATERIALS AND METHODS

Chemicals [3'-3SS]ATP and [3'-32p]ATP was obtained from Am-

ersham, England. [3'-S]ATP was obtained from Boehringer,

cAMP, 8-Br-cAMP from Sigma, Coomassie Blue R250 from Serva. Pharmacia and Sigma molecular weight cali- bration kits were used. For electrofocusing urea, ultra pure from BRL, bisacrylamide 2 × crystallized and acrylamide 4 x crystallized from Roth and broad band ampholytes, pH 3.5-9.5, from LKB was used. For the purification of the cAMP-dependent protein kinase and its regulatory subunit DEAE-Sephacel (Pharmacia), CM-Sephadex C-50 (Sigma), Sephacryl S-300 (Sigma) and 8-(2-aminohexyl) amino cAMP-Agarose (Sigma) was used. For limited proteolysis protease Staphylococcus aureus, V8 (Sigma) was used. All other chemicals were of analytical grade.

Stocks Drosophila melanogaster wildtype Canton-S was grown

on standard medium at 22°C under a 12:12 light,lark cycle. Flies 4-6 days after emergence were used for all experiments.

Preparation of head extracts Twenty flies were frozen at liquid nitrogen temperature

and decapitated. The heads were homogenized with 100 #I 50 mM Tris-HCl buffer, pH 7.5, in an ice-cold glass-Teflon homogenizer. For some of the experiments the homogenate was centrifuged at 40,000 g for 15 min. Aliqtiots were used immediately. Homogenates of tissue from defined fractions of a Drosophila head were prepared as described by Miiller and Spatz (1986).

Purification of cAMP-dependent protein kinase from Drosophila heads

Heads of 20 g of flies were separated from the bodies as described by Harris et al. (1976). The frozen heads were homogenized in 20ml of 10mM potassium phosphate, pH 6.8, I mM EDTA and 15 mM mercaptoethanol (buffer A). The holoenzyme of the cAMP-dependent protein kinase was prepared according to a similar procedure as used by Beavo et al. (1974). It consisted of five steps: ammonium sulphate precipitation, DEAE-Sephacel, aluminium hy- droxide C3', CM-Sephadex and Sep.hacel S-300.

Adsorption to cAMP-agarose A 0.5 ml column with 8-(2-aminohexyl) amino

cAMP-agarose containing approx. 5/z M/ml of covalently- bound cAMP was washed with 10 ml buffer A containing

351

352 ULI Mf3LLER et al.

0.1% SDS and subsequently washed extensively with buffer A. This procedure facilitates the elution of the regulatory subunit of the protein kinase. The prepurified material was slowly (5 ml/h) applied to the column. The column was then washed with 10 ml buffer A containing 2 M NaCI followed by a wash with 10 ml buffer A. Elution (1 ml/h) of the bound material was done at room temperature with 4 ml buffer A containing 10 mM cAMP. After 2 h the flow was stopped and the column cooled to 4°C. A second elution (2 ml buffer A, 10mM cAMP) 24h later still released some bound material (~ 30%).

Phosphorylation and thiophosphorylation Usually 10/~1 of a buffer containing 10mM MgCI2,

50mM Tris-HCI pH7.5, 2/~Ci D,32-P]ATP of sp. act. 3000 Ci/mmol or 2 #Ci [~35-S]ATP of sp. act. 650 Ci/mmol and cAMP at appropriate concentrations were mixed with 6 t~l aliquots of head homogenate (1.2 head equivalents) and incubated at room temperature for 3 min. The reaction was stopped by 0°C and addition of 4/~1 (500 mM Tris-HC1, pH 6.8, 5% mercaptoethanol, 5% SDS and 20% glycerol).

SDS-polyacrylamide electrophoresis (SDS-PAGE) and autoradiography

Gels were 90 × 80 mm 2 in size and 0.5 mm in thickness. The slots for sample application were 5 × 8 × 0.5 mm 3. Gels were prepared according to Laemmli (1970) with an acryl- amide concentration of T = 12.5% and a crosslinking of C = 2.7%. After electrophoresis the gels were stained con- ventionally with Coomassie R250. For autoradiography a Kodak X-Omat AR film was exposed to the dried gel at -80°C for several hours. Autoradiographs were scanned with a Kontron spectrophotometer Uvikon 810,

Electro focusing Two-dimensional analysis of Drosophila head proteins

revealed substantial losses of high molecular weight proteins during electrofocusing. The method to circumvent these difficulties will be described in detail (U. Miiller, to be published). In brief, focusing was carried out in poly- acrylamide slabs, T = 4% and C = 2.7%, with 9 M urea, 2% NP40 and 2% ampholytes, pH 3.5-9.5. The size was 90 x 120 mm 2, the thickness 0.4 mm. Slots for sample appli- cation were 6 x 10 x 0.4 mm 3. The gels were initially placed under a slope of 30 ° to allow sample application and later placed horizontally. The lower glass plate was cooled to 10°C. The upper glass plate consisted of several well-fitted parts which could be removed separately. This allowed the electric field to be applied via electrofocusing strips, which were placed across the slabs at the very ends. The strips were soaked in 0.l M NaOH at the negative and 0.05 M phos- phoric acid at the positive pole. The electric field was slowly built up from 15 to 60 V/cm within 1 h. Thereafter, the strip electrode at the side of the slots was moved by 1 cm towards the other electrode, so that the slots were outside the electric field. Electrofocusing was continued at 120V/cm up to 3500 Vh. This procedure avoids distortion of the electric field due to precipitates at the bottom of the slots and leads to well-defined bands. The polyacrylamide slab gels were stained with Coomassie Blue R250 and autoradiographed as described before. The pH gradient was measured by cutting part of the gel into small strips, placing them in doubly- distilled water and measuring the pH and also by calibration with marker proteins.

Peptide mapping Bands from dried SDS-gels were cut out and placed in the

sample wells of a second SDS-gel. Limited proteolysis with Staphylococcus aureus V8 protease was carried out in the stacking gel for 20 min and the fragments separated by SDS-PAGE (Cleveland et al., 1977). Alternatively material eluted from a cAMP-agarose column was subjected to

limited proteolysis with S. aureus V8 protease and analysed by SDS-PAGE and autoradiography.

RESULTS AND DISCUSSION

Figure 1 shows an autoradiogram after SDS- electrophoresis of thiophosphorylated head proteins o f Drosophila. A comparison with the banding pat- tern of [y : :P ]ATP of labelled Drosophila head ho- mogenates (Hesse and Marme, 1985; WiUmund, 1986; Devay et al., 1986) reveals that thio- phosphorylation acts preferentially on one substrate. This preference is found in the presence or absence of added cold ATP or [y-S]ATP. Apparently the thio- phosphate is not equally well-accepted by different substrates. If 2 m M E G T A is present in the phos- phorylation assay as in some of our experiments labelling of a 32 kD band at comparable intensities is found. The identification of this substrate will be subject of a later report. Our SDS-polyacrylamide system gives an apparent molecular weight between 55-60 kD for the substrate of thiophosphorylation. A direct comparison with the band phosphorylated with [~-32p]ATP, usually assigned as 57 or 58 kD band (Foster et al., 1984; Hesse and Marme, 1985; Will- mund, 1986; Friedrich, 1985), shows that they have identical apparent molecular weights. Also peptide mapping according to the method of Cleveland et al. (1977) shows the same fragments of the thio- phosphorylated and the phosphorylated band. It therefore seems likely that this substrate is the regu- latory subunit of the cAMP-dependent protein ki- nase. Isoelectric focusing (Fig. 2) gives additional support. In the presence of c A M P during incubation, 4 neighbouring bands at a pH between 5.1-5.3 are found, close to the isoelectric points of type II regulatory subunits from bovine cerebral cortex (Rubin et al., 1979) and bovine cardiac muscle (Rangel-Aldao et al., 1979).

We therefore purified the protein kinase as described in Materials and Methods, using phos- phorylation o f histones as an assay for kinase activ- ity. In the absence of added substrates, a single phosphorylated band at 5 7 k D in S D S - P A G E is obtained in the last stages of the purification. If subsequent to phosphorylation the purified holo- enzyme is adsorbed to a cAMP-agarose column, more than 7 0 0 of the thiophosphorylated or phos- phorylated material is specifically bound to the c A M P residue (Table 1).

The material eluted from the cAMP-agarose col- umn is the regulatory subunit of the cAMP- dependent protein kinase purified to near homoge- neity. The banding pattern obtained by peptide mapping of the 57 kD band from head homogenates is identical to that of the purified regulatory subunit. This corroborates our supposition that the preferred substrate of thiophosphorylation in Drosophila head homogenates is the regulatory subunit of the cAMP-dependent protein kinase. Figure 3 shows a comparison of peptide mapping of the purified thiophosphorylated and phosphorylated regulatory subunit. The identity of the two patterns demonstrate that thiophosphorylat ion and phosphorylat ion occur at the same position in the protein.

84

58

36

3~ S 32p

Fig. 1. Autoradiogram after SDS-PAGE of thiophosphorylated and phosphorylated head proteins of D. melanogaster. Thiophosphorylation (left lane) is carried out in the presence of 0.3 # M cAMP, 3 # M ATP and 2 # Ci [7 :SS]ATP; phosphorylation (right lane) in the presence of 0.3 p M cAMP, 3/~ M ATP and 2 # Ci [7-32p]ATP. Apparent molecular weights are calibrated with three marker proteins as indicated on the left.

353

~.~

.~

~.~

H °

~ ~

,~

o g-

N

~

= ~

'~"

~ _

. ::

:r o

~

'~

= a

g

.~.~

_

J~

D

.11

3pti

caL

½

S

S

f

de

ns

ity

58 kD

:36 kD

24 kO

35 S 32p

Fig. 3. Autoradiogram of the fragments of the regulatory subunit of the cAMP-dependent protein kinase after limited proteolysis with S. aureus protease. Thiophosphorylation (left lane) or phosphorylation (right lane) are carried out in the purified holoenzyme. Thereafter the regulatory subunit is isolated with a cAMP

affinity column and subjected to limited proteolysis.

355

356 ULI MULLER et al.

Table I. Binding of the partially purified phosphorylated 57 kD substrate to cAMP-agarose

Total Unbound cAMP released labelled labelled labelled protein protein protein

Phosphorylated with: (%) (%) (%)

[1,-35S]ATP 100 7 ± 2 73 ± 12 [7-nP]ATP 100 8 ± 2 71 ± 9

Purified cAMP-dependent protein kinase, holoenzyme, is thiophosphorylated or phos- phorylated in buffer A containing 5 mM MgCI 2, in the absence of cAMP. The material is then applied to a cAMP-affinity column. Material released from the column with buffer A, ± 2M NaC1, is listed as unbound, material released in subsequent elutions with buffer A containing 10 mM cAMP as cAMP released. The amounts of labelled regulatory subunit are measured by autoradiography after SDS PAGE.

1.0 =

=o 0 (18-

~ O . 6 -

X

t-- ,~ o . 4 -

r-~

m~l 0 . 2 - ).,.

' , ~ . I I I I I tiott 0.001 0.01 0.1 1 10

cAMP concentration (p.M)

Fig. 4a. Dependence o f the degree of thiophosphorylat ion with [7-35S] ATP of the regulatory subunit in head homogenates on the concentration of cAMP (O) or 8-Br-cAMP ( 0 ) . In comparison the degree of thiophosphorylat ion of the purified holoenzyme in buffer A containing 5 m M MgC12 is independent of the cAMP concentration (A). The radioactivity in the 58 kD band is measured by autoradiography after

S DS -P AGE and normalized with respect to the maximal value.

1.o-

O 0 . 0 - >,

0

g 0 0 . 6 "

f~ 13_ I.--- '~ O.4-

N. 0.2- L-I

; ~ . I I I I , troll 0 .001 0.01 0.1 1 10

cAMP concentration (FM)

Fig. 4b. Dependence of the degree of phosphorylat ion with [T-32p]ATP of the regulatory subunit in head homogenates on the concentration of cAM P (O) and in the purified holoenzyme phosphorylated in buffer

A containing 5 m M MgCI 2 (A) or in buffer A containing 100mM NaC1 and 5 m M MgCI 2 (*).

cAMP-dependent thiophosphorylation 357

~ma. O.5"

1.0,

0 10 30 60 300 600

T i m e (s )

Fig. 5. Time course of dephosphorylation of the regulatory subunit of the cAMP-dependent protein kinase in head homogenates. Thiophosphorylation (&) or phosphorylation (O) with [~, ::'P]ATP was carried out in head homogenates in the presence of 0.3/zM cAMP. After 2min l mM unlabelled ATP and 10/~M cAMP were added to the reaction mixture and the remaining radioactivity in the 58 kD band

measured by autoradiography after SDS-PAGE.

Thiophosphorylation of the regulatory subunit is most prominant in homogenates of neural tissue. If retinae, optical lobes and protocerebrum (neural tissue) and proboscis (mainly muscle tissue) are pre- pared separately (Miiller and Spatz, 1986), radio- active labelling of the 57 kD band for comparable amounts of protein from [protocerebrum with optical lobes] to [retinae] to [proboscis] is found in ratios 100:10:1. Thiophosphorylation of the regulatory sub- unit occurs predominantly in the supernatant of head homogenates. Only 10% of it is found in resuspended particulate material after low speed centrifugation of homogenized heads. Also the thiophosphorylated material remains soluble. Less than 10% binds to particulate material added to the mixture after incu- bation with [y:SS]ATP. The reaction is quite fast. After the handling time of 20 s 80% of a plateau value, reached in less than 2 min, is found. No change of the degree of thiophosphorylation is found if by appropriate EGTA-CaC12 mixtures the concen- tration of free Ca 2+ is varied over a range of 10-SM-10-3M, both in the presence and in the absence of cAMP.

cGMP stimulates thiophosphorylation of the 57 kD band only at concentrations above 10/~M. In contrast the degree of thiophosphorylation of the regulatory subunit depends strongly on the concen- tration of cAMP added to the head homogenate with an optimum at 0.3/~M cAMP or 8-Br-cAMP (Fig. 4a).

The near 10-fold increase of thiophosphorylation at cAMP concentrations below 0.3/~M is much less pronounced in phosphorylation (Fig. 4b) and not at all present in thiophosphorylation or phos- phorylation of the purified protein kinase. This can be understood in terms of a kinase-phosphatase equilibrium in the head homogenates: in the un- dissociated cAMP-dependent protein kinase phos- phatidyi groups are protected from the attack by phosphatases. If cAMP is added to the homogenate

the protein kinase dissociates into two catalytic sub- units and a dimer of the regulatory subunit (Kennedy, 1987). If phosphatases are present, as in head homogenates but not in the purified holoenzyme, the highly phosphorylated regulatory subunits are dephosphorylated and can be re- phosphorylated. The reaction can adequately be de- scribed as a dynamic equilibrium of kinase and phosphatase action, connected in series with a cAMP-dependent dissociation reaction. If [y-S]ATP is present, this dynamic equilibrium is changed. The thiophosphate is equally well-accepted by the protein kinase as can be shown by [y-32P]ATP isotope dilu- tion experiments. Different, however, is the rate of dephosphorylation as measured in a pulse chase experiment (Fig. 5). The thiophosphate group is remarkably stable against phosphatase action (Gratecos and Fischer, 1974; Cassel and Glaser, 1982; Eckstein, 1985).

The decrease of thiophosphorylation and phos- phorylation above I pM cAMP (Ueda et al., 1973) in head homogenates can be accounted for by the salt concentrations in the preparation. Figure 4b shows a corresponding decrease of phosphorylation of the purified holoenzyme at a salt concentration of 100mM NaC1, which is not observed at low salt concentrations and in the absence of divalent cations other than Mg 2÷ (Maeno et al., 1974).

Thus thiophosphorylation allows to measure the degree to which the regulatory subunit of the cAMP- dependent protein kinase can be phosphorylated even in crude homogenates. This is not possible using [y-32P]ATP, unless all phosphatases were inhibited. Due to its virtue as an almost irreversible reaction thiophosphorylation can be used as a tool to in- vestigate the equilibrium of kinase and phosphatase action. A comparison of phosphorylation and thio- phosphorylation under standardized conditions could also be used to determine cAMP concen- trations in homogenates. Thus it may prove to be a

358 ULI Mi.~LLER et al.

useful tool to investigate molecular processes believed to be constitutive in the mechanism of learning and memory (Kandel and Schwartz, 1982; Kennedy, 1987).

Acknowledgements--We would like to thank Drs P. Friedrich, D. Marine and R. Willmund for helpful sug- gestions. The work was supported through stipends from the DAAD, GFPS and Freiburger Stiftungsverwaltung to Z.W. and funds from the Deutsche Forschungs- gemeinschaft.

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