Journal ¢~f the Neurological Sciences, 1976, 29 : 185-193
185
C~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
E R Y T H R O C Y T E SPECTRIN PEAK II P H O S P H O R Y L A T I O N IN D U C H E N N E MUSCULAR DYSTROPHY
ALLEN D. ROSES and STANLEY H. APPEL Division c~/ Neurology, Duke Medical Center, Durham, North Carolina 27710 (U.S.A.)
(Received 23 January, 1976)
SUMMARY Duchenne muscular dystrophy (DMD) is a rapidly progressive crippling disease of young boys that is inherited as an X-linked recessive trait. Previous studies have demonstrated the usefulness oferythrocyte studies in exploring membrane abnormalities in inherited muscular dystrophy. Erythrocyte spectrin peak II protein (m.w. _~ 220,000) was more highly phosphorylated under initial rate conditions in D M D than in controls. The extent of peak II phosphorylation was greater in D M D erythrocytes and a Na + stimulated peak II phosphorylation effect (Avruch and Fairbanks 1974) was not found to account for the differences between D M D and controls. The phosphorylated state of spectrin proteins in the membrane was evaluated and no differences in D M D could be measured. The maximal transfer of phosphate from [7-3~P]ATP to spectrin peak lI accounts for approximately 5-103/o of the total phosphate content of spectrin.
INTRODUCTION Duchenne muscular dystrophy (DMD) is a rapidly progressive, crippling disease of young boys that is inherited as an X-linked recessive trait (Walton 1974). The direct analysis of muscle constituents has failed to yield significant information about the primary biochemical defect because of the presence of an extensive range of secondary effects such as increased connective tissue, fatty infiltration, and variable changes of degeneration and regeneration that are the responses of muscle to injuries This work was supported by RR-30 from the General Clinical Research Centers Program of the Division of Research Resources, National Institute of Health; 1 PO1 NS12213-01 NSPB from the National Institute of Neurological and Communicative Disorders and Stoke, National Institute of Health; and a Basil O'Connor Starter Research Grant No. 5-36 from the National Foundation March of Dimes to A.D.R.
186 of many different etiologies. Our previous studies of protein phosphorylation and electron spin resonance have demonstrated the usefulness of erythrocytes in exploring membrane abnormalities in the inherited muscular dystrophy, myotonic muscular dystrophy (MyD) (Roses and Appel 1973, 1975; Butterfield, Roses, Chesnut and Appel 1974; Butterfield, Roses, Cooper, Appel and Chesnut 1974; Hull and Roses 1976). The application of similar techniques to erythrocytes from patients with DMD demonstrated a significant increase in the rate of phosphorylation of peak I! (m.w. 220,000) and peak 11I (m.w. 90-100,000) (Roses, Herbstreith and Appel 1975). Phosphorylation of peak 11I is increased under initial rate conditions but peak il continues to be increased compared to controls following long incubations. The kinetic analysis of the endogenous phosphorylation of peak ii ira DMD as well as biochemical evaluation of the phosphorylated state of spectrin (peak 1 and peak 11), is reported in this communication. During the course of these experiments Avruch and Fairbanks (1974) suggested that at least 2 distinct protein kinase activities were present in the erythrocyte. They reported a Na +-stimulated phosphorytation of peak I1 that was an obvious possible explanation for the increased peak I! phosphorylation found in DMD. In order to evaluate possible protein kinase enzyme differences as well as possible substrate difference, the endogenous phosphorylation of erythrocyte membrane protein was also evaluated using the methods of Avruch and Fairbanks (1974). METHODS Patients are registered in the Duke Neuromuscular Research Clinic. Controls are unaffected family members, medical and paramedical personal, and their children. Age, sex, and race-matched controls are used whenever possible. All controls are designated for each patient before the experiment begins. Heparinized blood was obtained from patients and controls simultaneously and erythrocyte ghosts were prepared by a hypotonic lysis method which used 5 m M sodium phosphate buffer at pH 8.0 (5P8) (Fairbanks, Steck and Wallach 1971). All ghosts were freshly and simultaneously prepared and all assays of endogenous protein kinase were carried out immediately following ghost preparation as previously described (Roses and Appel 1975). The incubation mix contained 10 #moles of sodium acetate buffer, pH 6.5, 2.0 #moles of magnesium acetate, 0.06 #moles of ethylene glycol bis (aminoethyl)-N,N'-tetraacetic acid (EGTA), 1 nmole of [7-3~P]ATP (3-10 × 106 cpm) and approximately 200 #g ghost protein in a total volume of 0.20 ml. Reactions were performed in duplicate at 25 °C for the indicated time period. Six experiments were performed for 1, 5, 15, 30 and 60 min. All experiments were run for 1 and 15 min. The reaction was stopped and the membranes completely solubilized in 1 ~ SDS, 0.5 mM ethylene diarninetetraacetic acid (EDTA) and 0.6 mM mercapto-ethanol. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as previously described (Roses and Appel 1973, 1975). Peak 1I (as well as other other peaks) was determined by counting the solubilized Coomassie bluestained band from SDS-PAGE by previously described methods (Roses and Appel 1973, 1975)(Fig. 1).
187 A
1T
MOLECULARWEIGHT PEAK I ~ 240,000 1 PEAK1Z ~220,000 . "SPECTRIN" PEAK I ~ 90-100,000
+,_~ 80 c 60 c
TD
B
t
~_j- PEAK 1~ Phosphory/ahon used for DMD Study
II =40 ~_ 0
2O
E
r~
II 1 11 ]I .1-.4
1"i".5
]~
Onn
17,1-,2 17,3~.4 V
O0
5ZZ ~
0
,
"{7i11" TD
BAND NUMBER
Fig. 1. Top: densitometry recording of Coomassie blue-stained SDS-polyacrylamide gel electrophoresis. No differences were present in DMD or controls. Peak II is indicated. Bottom: bar graph illustrating typical pattern of phosphorylation after 15 rain incubation ill indicated gel slices. Peak II was measured in these experiments. Other proteins were calculated as control measurements of proteins electrophoresed on the same gels. No consistent difference between DMD and controls were noted in gel slices other than peak 11.
[7-aeP]ATP was prepared by a modification of the method of Glynn and Chappell (1964). In experiments measuring the a m o u n t o f [7-azp]ATP present after 15 rain incubations, reactions were stopped by the addition of 5 o/,o trichloracetic acid and the supernatant was chromatographed in the identical system used to prepare [7-a"P]ATP. Descending D E A E cellulose ( W h a t m a n DES1) paper chromatog r a p h y with a2p and [~,-a2p]ATP (treated similarly) as standards was performed in 0.3 M a m m o n i u m formate. The paper was cut and radioactivity counted in these experiments. For preparative procedures [7-a2p]ATP was eluted from the dried paper by 30)o~ triethylamine carbonate at p H 10.0, lyophilized and diluted to the appropriate specific activity.
188 Incubations performed in 20 mM imidazole-chloride buffer at pH 7.4, followed the procedure of Avruch and Fairbanks (1974). Incubations were performed at 25 °C for 1 and 15 rain. Peak II phosphorylation was measured as described above. In order to measure the time required for maximum peak I1 phosphorylation, ghosts were incubated for 30 min in the usual incubation mix. Nine ml of cold 5P8 was added and the ghosts centrifuged at 15,000 × g for 10 min at 4 C . Following 2 additional washes in 5P8, the ghosts were reincubated with flesh [y-a2p]ATP for 15 mino Peak 1I phosphorylation was measured before the first 5P8 wash, before the second incubation and following the second incubation by the methods described above. Phosphoprotein phosphatase activity was measured by incubating 32p. labeled ghosts in the incubation medium with non-labeled ATP and by measuring protein radioactivity by procedures identical to protein kinase assays. Purified, delipidated spectrin was prepared as previously described (Roses Herbstreith, Metcalf and Appel 1976). Phosphate was assayed by the method of Friedel and Schanberg (1971). Protein was assayed according to Lowry, Rosebrough Farr and Randall (11951). H332PO4 was purchased from New England Nuclear and 3-Phosphoglycerat-kinase and Glycerinaldehyd-3-Phosphat-Dehydrogenase (Kaninchenmuskel) were purchased from Boehringer Mannheim Corporation Biochemical Division. All chemicals were reagent grade. Three methods of statistical evaluation are used to evaluate the data. The tstatistic for 2 means (Student t-test) grossly evaluates the difference between DMD and controls. An analysis of variance established that a significant error was introduced into the overall data by small differences in the calculation of the 18 separate high specific activity [y-32P]ATP preparations used during this period. The paired t statistic evaluates the significance of the difference between DMD and controls in each experiment, independent of the variation introduced by the different [yz:32p]ATP preparations. RESULTS Previous data demonstrated that peak II phosphorylation was increased in DMD erythrocyte membranes incubated under initial rate conditions (Roses et al. 1975). Fig. 2 illustrates the kinetics of the reaction. Increased peak I1 phosphorylation was still present following incubations. Thirty-four of 35 paired experiments using a 15 min incubation demonstrated a higher level of peak II phosphorylation in DMD than in the paired controls (Table 1). Twenty-six different DMD patients from 22 separate families over a 10-month period were examined. In 16 experiments it was possible to age match controls ~ 3 years of age; in the other experiments the average control age is 25 years. Studies of large numbers of controls, however, have demonstrated no significant difference in peak II phosphorylation in young males (3-14 years), adult males ( > 15 years), young females, adult females or non-Caucasian versus Caucasian controls (Roses, unpublished data). A 15 min incubation period was chosen because [7-a~P]ATP was not limiting within this period and phosphorylation levels became more variable over longer periods due to the partial extraction
189 ERYTHROCYTE MEMBRANE PROTEIN lq (MW~22qO00)
zI 100
I~-~-//
DUCHENNE M U S C U L A R
DYSTROPHY -~_
~
L
~//~///....
g~_o ~°204°
co.reo,
r6;
/z~ l
5
10
15
20 25 30 TIME (Minutes)
(~0
Fig. 2. Time course of erythrocyte spectrin peak 11 phosphorylation. Ghosts are incubated and the protein bands analyzed for 3•p incorporation as described in the text. Addition of fresh [7 azP]ATP after 30 rain did not increase the level of peak 11 phosphorylation in DMD or control ghosts.
TABLE I PHOSPHORYLATION OF ERYTHROCYTE PEAK 11 (mol wt 220,000) DMD
Control
(pm/mg ghost protein) (I 5 min incubation) Mean standard deviation standard error ofthe mean N P (Student's t-test)
71.3 ~ 27.0 { 4.56
Mean difference (DM D-control) standard deviation number of paired experiments P (paired t statistic)
± 17.3 :~ 15.5 35 .--i 0.001
54.0 ± 20.2 :i: 3.41 70 0.005
o f spectrin protein (including p e a k II) from the m e m b r a n e by the h y p o t o n i c , E G T A c o n t a i n i n g incubation solution. The leveling off o f p h o s p h o r y l a t i o n between 15 a n d 30 min did not a p p e a r to be limited by the availability o f [7-a2p]ATP. A f t e r 15 min the reaction was stopped a n d the s u p e r n a t a n t a n a l y z e d for [7-a2P]ATP. A p p r o x i m a t e l y 30',!,o o f the original [7-a2P]ATP r e m a i n e d in both D M D and control i n c u b a t i o n s (Table 2). This [7,a2P] A T P c o u l d be used to p h o s p h o r y l a t e fresh ghosts. The a d d i t i o n o f [7-a2p]ATP after 30 min did not increase the extent o f peak 11 p h o s p h o r y l a t i o n a n d the 15 rain incubation d a t a used in these m e a s u r e m e n t s represented a measure o f the a p p r o x i m a t e extent o f peak II p h o s p h o r y l a t i o n possible in D M D a n d control m e m b r a n e s . M e m b r a n e - a s s o c i a t e d p h o s p h o p r o t e i n p h o s p h a t a s e activity was not present
190 TABLE 2 [7-a2P]ATP REMAINING AFTER 15 MIN INCUBATION Radioactivity is expressed as the fraction of total counts applied to DEAE-cellulose paper. Approximately I5% of applied counts are present as background. Approximately 98 % counts applied are recovered from paper. Fractions are the mean of 3 separate experiments ~- standard deviation.
[7-:~2 P]ATP 3ep
DMD
Contcol
0.29 :k 0.02 0.57 =~ 0.06
0.30 .:L 0.02 0.55 :k 0.06
u n d e r these a s s a y c o n d i t i o n s . G h o s t s p h o s p h o r y l a t e d b y D,-3zP]ATP a n d then w a s h e d in 5P8 d o n o t significantly d e p h o s p h o r y l a t e p e a k II when i n c u b a t e d in the s t a n d a r d p r o t e i n kinase i n c u b a t i o n s o l u t i o n with 10 m M M g ~' a n d u n l a b e l e d A T P . D u r i n g the course o f these experiments. A v r u c h a n d F a i r b a n k s (1974) r e p o r t e d t h a t p h o s p h o r y l a t i o n o f p e a k I1 was s t i m u l a t e d b y N a +. E x p e r i m e n t s using thetr i m i d a z o l e buffer system a n d identical i n c u b a t i o n c o n d i t i o n s c o n f i r m e d this effect. H o w e v e r , the levels o f p h o s p h o r y l a t i o n in D M D a n d c o n t r o l s following 15 rain i n c u b a t i o n s were a p p r o x i m a t e l y 15~o o f the levels presented in T a b l e I (Table 3). The discrepancy between the two results c a n be explained by the different M g .... c o n c e n t r a t i o n s . A v r u c h a n d F a i r b a n k s d e m o n s t r a t e d N a + s t i m u l a t i o n in the presence o f I m M M g ~ f. The K m for M g ~+ is a p p r o x i m a t e l y 2 m M ( G u t h r o w , Allen a n d R a s mussen 1972; R u b i n , E r l i c h m a n a n d R o s e n 1972). W h e n 10 m M M g r was used with their i m i d a z o l e buffer system, the N a ~ specific effect was no l o n g e r a p p a r e n t (Table 3). In all e x p e r i m e n t s using the i m i d a z o l e buffer system, i n c l u d i n g those with I0 m M M g -H, the levels o f p e a k II p h o s p h o r y l a t i o n were less t h a n the s o d i u m a c e t a t e -
TABLE 3 PEAK 11 PHOSPHORYLATION - - EFFECT OF BUFFER, Mg ++ AND Na + Ghosts are prepared as described in Methods and incubated in the indicated buffer system for the indicated time. Ghosts are solubilized, electrophoresed and peak II is counted as described. Five experiments resulted in similar results. In this experiment 1090 counts/rain represent 1 pro/rag ghost protein/incubation time. The DMD patient is 7 years old, the control is 8 years old. Incubation solution buffer
50mMNaAc ÷ 0.6ram EGTA, pH6.5 20mMimidazole-Cl, pH 7.4 20raM imidazole-Cl, pH 7.4 + 50mMNaC1 20ram imidazole-Cl, pH 7.4 20raM imidazole-C1, pH 7.4 -k 50 mMNaCI
[Mg ~~]
DMD
Control
DMD
Control
(pm/mg/1 min incubation)
(pm/mg/15 min incubation)
10raM lmM lmM
12.1 1.36 2.21
8,41 1.15 2.11
79.6 6.83 10.82
47.5 4.83 7.92
10ram
11.9
7.88
47.9
34.1
7.49
42.6
32.7
10mM
9.27
191 EDTA buffer system but the increased peak II phosphorylation in DMD was still present. Experiments were performed to measure the phosphorylated state of spectrin as it exists in the membrane. Phosphate was measured in purified delipidized spectrin. Both DMD and control membranes demonstrated approximately 2 moles P/mole spectrin proteins. The maximal extent of phosphorylation with [y-aep]ATP represents approximately 0.1 moles a'-'P/mole spectrin protein. The sensitivity of the phosphate measurements did not allow measurement of any differences in the basal phosphorylated state of spectrin in D M D or controls that would account for the increased phosphorylation capability of DMD peak Ii. DISCUSSION Erythrocyte peak 11 phosphorylation is increased in DMD both at the initial rate and during extended periods of incubation. It is not possible to do formal kinetic analysis of this system because the substrate and enzyme cannot be separated from each other. Enzyme activity cannot be analyzed as substrate concentration is varied. Reconstituted systems with added lipid have not yet been developed. The increased peak II phosphorylation may be a substrate abnormality, an enzyme difference or an alteration in the state of the vectoral and geometric relationship of substrate to enzyme due to other membrane factors or constituents. Subtle biophysical and geometrical differences are difficult to evaluate with existing techniques but biochemical characteristics of the protein kinase and its substrates can be experimentally tested. Extraction of the protein kinase from the erythrocyte membrane is possible using 1 M NH4CL (Rubin et al. 1972). Kinetic evaluation of the extracted enzyme using a purified histone as substrate demonstrated no difference in MyD (Roses and Appel 1973) or DMD (Roses, unpublished data). The report of Na P-stimulated peak l l phosphorylation by Avruch and Fairbanks offered another approach to analyzing enzyme activity. Their system demonstrated the Na~-stimulating effect only at Mg ~ concentrations below the Km for Mg ~. Our conclusion was that the Na ~-stimulated peak II phosphorylation was apparent because suboptimal Mg' ~ concentrations were used. This was confirmed by finding only about 15°{~ of the possible peak II phosphorylation (Table 3). At optimal Mg ~ concentrations the Na~-stimulating effect was not apparent. The increased peak II phosphorylation in D M D was still present using the imidazole buffer system in these experiments. The possible leveling off of the phosphorylation of peak 11 was measured as a function of [7-a2P]ATP limitation and phosphoprotein phosphatase activity. Similar amounts (approximately 30~,/,) of the original [y aep]ATP were present following 15 rain incubations in DMD and controls. Addition of fresh [7-a2P]ATP to phosphorylated membranes did not increase the level of peak 1I phosphorylation, in addition, there was no phosphoprotein phosphatase activity over a 15 rain incubation. These experiments were subject to slight artifacts because long periods of incubation ( > 30 rain) caused slight extraction of the spectrin protein by the hypotonic, EGTA-containing incubation solution. The levels of peak II phosphorylation reached
192 in these studies did not appear to be due to an equilibrium between protein kinase and phosphoprotein phosphatase but rather the extent of substrate phosphorylation possible. The phosphorylated state of peak II could affect the extent of 32p labeling, if sites on the molecules are already phosphorytated in control membranes, then less 32p could be transferred to the protein. Measurements of P bonded to purified delipidated spectrin demonstrated approximately 2 moles P/mole spectrin protein. 32p labeling represents approximately 0.1 mole/mole spectrin. It was not possible to measure the phosphorytated state of spectrin with sufficient sensitivity to evaluate small differences. The relationship of increased erythrocyte peak II phosphorylation in DMD to the biochemical defect is unknown. It is possible that a highly phosphorylated component of peak II may account for the increase in phosphorylation, Purified delipidated a2P-labeled spectrin was subjected to isoelectric focusing electrophoresis and many labeled components were demonstrated (Roses et al. 1976). The same preparation stains only the spectrin doublet on SDS-PAGE. Preparative techniques for the separation of purified peak I and peak lI need to be developed in Order to facilitate study of substrate heterogeneity differences in DMD and controls. It is possible that the biochemical defect in the DMD erythrocyte may be an abnormal protein substrate of protein kinase. It is premature to attempt to relate peak II phosphorylation differences to the dystrophic process in muscle. Spectrin proteins have been referred to as myosin-like molecules because of the similarity in molecular weight to the heavy chains of myosin and their coextraction with actin-like protein by hypotonic EDTA solutions or water (Marchesi, Steers, Marchesi, and Tillak 1970; Clarke 1971 ; Trayer, Nozaki, Reynolds and Tanford 1971 ; Hartwig and Stossel 1975; Tilney and Detmers 1975). Isolation and characterization of the increased phosphorylated component or components of peak II in DMD may be a valuable tool with which to examine the relationship of spectrin to muscle proteins. It is apparent, however, that erythrocyte spectrin peak II phosphorylation is increased in DMD and the erythrocyte can be used to explore membrane abnormalities in this crippling, lethal disease. ACKNOWLEDGEMENTS We thank Mr. Michael Herbstreith and Mr. Bradley Metcalf for expert technical assistance, Mrs. Janet Worthington for expert secretarial assistance and Dr. Keith Hull, Mrs. Marcia Roses, L. P. T., Mrs. Megan Nicholson, M.A.P.A. (Australia), and Mrs. Cathy Kircher, L.P.T. whose volunteer services made the Duke Neuromuscular Research Clinic possible. REFERENCES Avruch, J. and G. Fairbanks (1974) Phosphorylation of endogenous substrates by erythrocyte membrane protein kinase -- I. A. monovalentcation-stimulated reaction, Bioehemi~try, 13: 5507.
193 Butterfield, D. A., A. D. Roses, D. B. Chesnut and S. H. Appel (1974) Electron spin resonance studies of erythrocytes from patients with muscular dystrophy, Proc. nat. ,4cad. Sci. (Wask.), 71 : 909. Butterfield, D. A., A. D. Roses, M. L. Cooper, S. H. Appel and D. B. Chesnut (1974) A comparative ESR study of the erythrocyte membrane in muscular dystrophy, Biochemistry, 13: 5078. Clarke, M. 0971) Isolation and characterization of a water-soluble protein from bovine erythrocyte membranes, Biockem. Biophys. Res. Commun., 45: 1063. Fairbanks, G., T. L. Steck and D. F. H. Wallach (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane, Biockemistry, 10: 2606. Friedel, R. O. and S. M. Schanberg (1971) Incorporation of in vivo of intracisternally injected 33Pi into phospholipids of rat brain, J. Neurockem., 18: 2191. Glynn, I. M. and J. B. Chappell (1964) A simple method for the preparation of 3eP-labeled adenosine triphosphate of high specific activity, J. Biockem., 90: 147. Guthrow, Jr., C. E., J. E. Allen and H. Rasmussen (1972) Phosphorylation of an endogenous membrane protein by an endogenous membraneassociated cyclic adenosine 3',5'-monophosphatedependent protein kinase in human erythrocyte ghosts, J. biol. Chem., 247: 8145. Hartwig, J. H. and T. P. Stossel (1975) Isolation and properties of actin, myosin, and a new actinbinding protein in rabbit alveolar macrophages, J. biol. Chem., 250: 5696. Hull, Jr., K. L. and A. D. Roses (1976) Stoichiometry of sodium and potassium transport in erythrocytes from patients with myotonic muscular dystrophy, J. Physiol. (Lond.), 254:169 181. Lowry, O. H., N. J. Rosebrough, A. C. Farr and R. J. Randall (1951) Protein measurement with the Folin phenol reagent, J. biol. Ckem., 193:265 275. Marchesi, S. L., E. Steers, V. T. Marchesi and T. W. Tillak (1970) Physical and chemical properties of a protein isolated from red cell membranes, Biochemistry, 9: 50. Roses, A. D. and S. H. Appel (1973) Erythrocyte protein phosphorylation, J. biol. Chem., 248: 1408. Roses, A. D. and S. H. Appel (1973) Protein kinase activity in erythrocyte ghosts of patients with myotonic muscular dystrophy, Proc. nat, Acad. Sci. (Wash.), 70: 1855. Roses, A. D. and S. H. Appel 0975) Phosphorylation of a component of the human erythrocyte membrane in myotonic muscular dystrophy, J. Membr. Biol., 20: 51. Roses, A. D., M. H. Herbstreith and S. H. Appel (1975) Membrane protein kinase alteration in Duchenne muscular dystrophy, Nature (Lond.), 254:350. Roses, A. D., M. H. Herbstreith, B. Metcalf and S. H. Appel (1976) Increased phosphorylated components oferythrocyte me :obrane spect rin band !I with reference to Duchenne muscular dystrophy, J. neurol. Sci., 30: 167. Rubin, C. S., J. Erlichman, and O. M. Rosen, (1972) Cyclic adenosine 3',5'-monophosphate-dependent protein kinase of human erythrocyte membranes, J. biol. Chem., 247: 6135. Tilney, L. G. and P. Detmers (1975) Actin in erythrocyte ghosts and its association with spectrin, J. Cell Biol., 66: 508. Trayer, H. R., Y. Nozaki, J. A. Reynolds and C. Tanford (1971) Polypeptide chains from human red blood cell membranes, J. Biol. Chem., 247: 4485. Walton. J. N. (Ed.) (1974) Disorders o/Vohmtary Muscle, 3rd edition, Churchill Livingstone, Edin burgh and London.
185
C~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
E R Y T H R O C Y T E SPECTRIN PEAK II P H O S P H O R Y L A T I O N IN D U C H E N N E MUSCULAR DYSTROPHY
ALLEN D. ROSES and STANLEY H. APPEL Division c~/ Neurology, Duke Medical Center, Durham, North Carolina 27710 (U.S.A.)
(Received 23 January, 1976)
SUMMARY Duchenne muscular dystrophy (DMD) is a rapidly progressive crippling disease of young boys that is inherited as an X-linked recessive trait. Previous studies have demonstrated the usefulness oferythrocyte studies in exploring membrane abnormalities in inherited muscular dystrophy. Erythrocyte spectrin peak II protein (m.w. _~ 220,000) was more highly phosphorylated under initial rate conditions in D M D than in controls. The extent of peak II phosphorylation was greater in D M D erythrocytes and a Na + stimulated peak II phosphorylation effect (Avruch and Fairbanks 1974) was not found to account for the differences between D M D and controls. The phosphorylated state of spectrin proteins in the membrane was evaluated and no differences in D M D could be measured. The maximal transfer of phosphate from [7-3~P]ATP to spectrin peak lI accounts for approximately 5-103/o of the total phosphate content of spectrin.
INTRODUCTION Duchenne muscular dystrophy (DMD) is a rapidly progressive, crippling disease of young boys that is inherited as an X-linked recessive trait (Walton 1974). The direct analysis of muscle constituents has failed to yield significant information about the primary biochemical defect because of the presence of an extensive range of secondary effects such as increased connective tissue, fatty infiltration, and variable changes of degeneration and regeneration that are the responses of muscle to injuries This work was supported by RR-30 from the General Clinical Research Centers Program of the Division of Research Resources, National Institute of Health; 1 PO1 NS12213-01 NSPB from the National Institute of Neurological and Communicative Disorders and Stoke, National Institute of Health; and a Basil O'Connor Starter Research Grant No. 5-36 from the National Foundation March of Dimes to A.D.R.
186 of many different etiologies. Our previous studies of protein phosphorylation and electron spin resonance have demonstrated the usefulness of erythrocytes in exploring membrane abnormalities in the inherited muscular dystrophy, myotonic muscular dystrophy (MyD) (Roses and Appel 1973, 1975; Butterfield, Roses, Chesnut and Appel 1974; Butterfield, Roses, Cooper, Appel and Chesnut 1974; Hull and Roses 1976). The application of similar techniques to erythrocytes from patients with DMD demonstrated a significant increase in the rate of phosphorylation of peak I! (m.w. 220,000) and peak 11I (m.w. 90-100,000) (Roses, Herbstreith and Appel 1975). Phosphorylation of peak 11I is increased under initial rate conditions but peak il continues to be increased compared to controls following long incubations. The kinetic analysis of the endogenous phosphorylation of peak ii ira DMD as well as biochemical evaluation of the phosphorylated state of spectrin (peak 1 and peak 11), is reported in this communication. During the course of these experiments Avruch and Fairbanks (1974) suggested that at least 2 distinct protein kinase activities were present in the erythrocyte. They reported a Na +-stimulated phosphorytation of peak I1 that was an obvious possible explanation for the increased peak I! phosphorylation found in DMD. In order to evaluate possible protein kinase enzyme differences as well as possible substrate difference, the endogenous phosphorylation of erythrocyte membrane protein was also evaluated using the methods of Avruch and Fairbanks (1974). METHODS Patients are registered in the Duke Neuromuscular Research Clinic. Controls are unaffected family members, medical and paramedical personal, and their children. Age, sex, and race-matched controls are used whenever possible. All controls are designated for each patient before the experiment begins. Heparinized blood was obtained from patients and controls simultaneously and erythrocyte ghosts were prepared by a hypotonic lysis method which used 5 m M sodium phosphate buffer at pH 8.0 (5P8) (Fairbanks, Steck and Wallach 1971). All ghosts were freshly and simultaneously prepared and all assays of endogenous protein kinase were carried out immediately following ghost preparation as previously described (Roses and Appel 1975). The incubation mix contained 10 #moles of sodium acetate buffer, pH 6.5, 2.0 #moles of magnesium acetate, 0.06 #moles of ethylene glycol bis (aminoethyl)-N,N'-tetraacetic acid (EGTA), 1 nmole of [7-3~P]ATP (3-10 × 106 cpm) and approximately 200 #g ghost protein in a total volume of 0.20 ml. Reactions were performed in duplicate at 25 °C for the indicated time period. Six experiments were performed for 1, 5, 15, 30 and 60 min. All experiments were run for 1 and 15 min. The reaction was stopped and the membranes completely solubilized in 1 ~ SDS, 0.5 mM ethylene diarninetetraacetic acid (EDTA) and 0.6 mM mercapto-ethanol. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as previously described (Roses and Appel 1973, 1975). Peak 1I (as well as other other peaks) was determined by counting the solubilized Coomassie bluestained band from SDS-PAGE by previously described methods (Roses and Appel 1973, 1975)(Fig. 1).
187 A
1T
MOLECULARWEIGHT PEAK I ~ 240,000 1 PEAK1Z ~220,000 . "SPECTRIN" PEAK I ~ 90-100,000
+,_~ 80 c 60 c
TD
B
t
~_j- PEAK 1~ Phosphory/ahon used for DMD Study
II =40 ~_ 0
2O
E
r~
II 1 11 ]I .1-.4
1"i".5
]~
Onn
17,1-,2 17,3~.4 V
O0
5ZZ ~
0
,
"{7i11" TD
BAND NUMBER
Fig. 1. Top: densitometry recording of Coomassie blue-stained SDS-polyacrylamide gel electrophoresis. No differences were present in DMD or controls. Peak II is indicated. Bottom: bar graph illustrating typical pattern of phosphorylation after 15 rain incubation ill indicated gel slices. Peak II was measured in these experiments. Other proteins were calculated as control measurements of proteins electrophoresed on the same gels. No consistent difference between DMD and controls were noted in gel slices other than peak 11.
[7-aeP]ATP was prepared by a modification of the method of Glynn and Chappell (1964). In experiments measuring the a m o u n t o f [7-azp]ATP present after 15 rain incubations, reactions were stopped by the addition of 5 o/,o trichloracetic acid and the supernatant was chromatographed in the identical system used to prepare [7-a"P]ATP. Descending D E A E cellulose ( W h a t m a n DES1) paper chromatog r a p h y with a2p and [~,-a2p]ATP (treated similarly) as standards was performed in 0.3 M a m m o n i u m formate. The paper was cut and radioactivity counted in these experiments. For preparative procedures [7-a2p]ATP was eluted from the dried paper by 30)o~ triethylamine carbonate at p H 10.0, lyophilized and diluted to the appropriate specific activity.
188 Incubations performed in 20 mM imidazole-chloride buffer at pH 7.4, followed the procedure of Avruch and Fairbanks (1974). Incubations were performed at 25 °C for 1 and 15 rain. Peak II phosphorylation was measured as described above. In order to measure the time required for maximum peak I1 phosphorylation, ghosts were incubated for 30 min in the usual incubation mix. Nine ml of cold 5P8 was added and the ghosts centrifuged at 15,000 × g for 10 min at 4 C . Following 2 additional washes in 5P8, the ghosts were reincubated with flesh [y-a2p]ATP for 15 mino Peak 1I phosphorylation was measured before the first 5P8 wash, before the second incubation and following the second incubation by the methods described above. Phosphoprotein phosphatase activity was measured by incubating 32p. labeled ghosts in the incubation medium with non-labeled ATP and by measuring protein radioactivity by procedures identical to protein kinase assays. Purified, delipidated spectrin was prepared as previously described (Roses Herbstreith, Metcalf and Appel 1976). Phosphate was assayed by the method of Friedel and Schanberg (1971). Protein was assayed according to Lowry, Rosebrough Farr and Randall (11951). H332PO4 was purchased from New England Nuclear and 3-Phosphoglycerat-kinase and Glycerinaldehyd-3-Phosphat-Dehydrogenase (Kaninchenmuskel) were purchased from Boehringer Mannheim Corporation Biochemical Division. All chemicals were reagent grade. Three methods of statistical evaluation are used to evaluate the data. The tstatistic for 2 means (Student t-test) grossly evaluates the difference between DMD and controls. An analysis of variance established that a significant error was introduced into the overall data by small differences in the calculation of the 18 separate high specific activity [y-32P]ATP preparations used during this period. The paired t statistic evaluates the significance of the difference between DMD and controls in each experiment, independent of the variation introduced by the different [yz:32p]ATP preparations. RESULTS Previous data demonstrated that peak II phosphorylation was increased in DMD erythrocyte membranes incubated under initial rate conditions (Roses et al. 1975). Fig. 2 illustrates the kinetics of the reaction. Increased peak I1 phosphorylation was still present following incubations. Thirty-four of 35 paired experiments using a 15 min incubation demonstrated a higher level of peak II phosphorylation in DMD than in the paired controls (Table 1). Twenty-six different DMD patients from 22 separate families over a 10-month period were examined. In 16 experiments it was possible to age match controls ~ 3 years of age; in the other experiments the average control age is 25 years. Studies of large numbers of controls, however, have demonstrated no significant difference in peak II phosphorylation in young males (3-14 years), adult males ( > 15 years), young females, adult females or non-Caucasian versus Caucasian controls (Roses, unpublished data). A 15 min incubation period was chosen because [7-a~P]ATP was not limiting within this period and phosphorylation levels became more variable over longer periods due to the partial extraction
189 ERYTHROCYTE MEMBRANE PROTEIN lq (MW~22qO00)
zI 100
I~-~-//
DUCHENNE M U S C U L A R
DYSTROPHY -~_
~
L
~//~///....
g~_o ~°204°
co.reo,
r6;
/z~ l
5
10
15
20 25 30 TIME (Minutes)
(~0
Fig. 2. Time course of erythrocyte spectrin peak 11 phosphorylation. Ghosts are incubated and the protein bands analyzed for 3•p incorporation as described in the text. Addition of fresh [7 azP]ATP after 30 rain did not increase the level of peak 11 phosphorylation in DMD or control ghosts.
TABLE I PHOSPHORYLATION OF ERYTHROCYTE PEAK 11 (mol wt 220,000) DMD
Control
(pm/mg ghost protein) (I 5 min incubation) Mean standard deviation standard error ofthe mean N P (Student's t-test)
71.3 ~ 27.0 { 4.56
Mean difference (DM D-control) standard deviation number of paired experiments P (paired t statistic)
± 17.3 :~ 15.5 35 .--i 0.001
54.0 ± 20.2 :i: 3.41 70 0.005
o f spectrin protein (including p e a k II) from the m e m b r a n e by the h y p o t o n i c , E G T A c o n t a i n i n g incubation solution. The leveling off o f p h o s p h o r y l a t i o n between 15 a n d 30 min did not a p p e a r to be limited by the availability o f [7-a2p]ATP. A f t e r 15 min the reaction was stopped a n d the s u p e r n a t a n t a n a l y z e d for [7-a2P]ATP. A p p r o x i m a t e l y 30',!,o o f the original [7-a2P]ATP r e m a i n e d in both D M D and control i n c u b a t i o n s (Table 2). This [7,a2P] A T P c o u l d be used to p h o s p h o r y l a t e fresh ghosts. The a d d i t i o n o f [7-a2p]ATP after 30 min did not increase the extent o f peak 11 p h o s p h o r y l a t i o n a n d the 15 rain incubation d a t a used in these m e a s u r e m e n t s represented a measure o f the a p p r o x i m a t e extent o f peak II p h o s p h o r y l a t i o n possible in D M D a n d control m e m b r a n e s . M e m b r a n e - a s s o c i a t e d p h o s p h o p r o t e i n p h o s p h a t a s e activity was not present
190 TABLE 2 [7-a2P]ATP REMAINING AFTER 15 MIN INCUBATION Radioactivity is expressed as the fraction of total counts applied to DEAE-cellulose paper. Approximately I5% of applied counts are present as background. Approximately 98 % counts applied are recovered from paper. Fractions are the mean of 3 separate experiments ~- standard deviation.
[7-:~2 P]ATP 3ep
DMD
Contcol
0.29 :k 0.02 0.57 =~ 0.06
0.30 .:L 0.02 0.55 :k 0.06
u n d e r these a s s a y c o n d i t i o n s . G h o s t s p h o s p h o r y l a t e d b y D,-3zP]ATP a n d then w a s h e d in 5P8 d o n o t significantly d e p h o s p h o r y l a t e p e a k II when i n c u b a t e d in the s t a n d a r d p r o t e i n kinase i n c u b a t i o n s o l u t i o n with 10 m M M g ~' a n d u n l a b e l e d A T P . D u r i n g the course o f these experiments. A v r u c h a n d F a i r b a n k s (1974) r e p o r t e d t h a t p h o s p h o r y l a t i o n o f p e a k I1 was s t i m u l a t e d b y N a +. E x p e r i m e n t s using thetr i m i d a z o l e buffer system a n d identical i n c u b a t i o n c o n d i t i o n s c o n f i r m e d this effect. H o w e v e r , the levels o f p h o s p h o r y l a t i o n in D M D a n d c o n t r o l s following 15 rain i n c u b a t i o n s were a p p r o x i m a t e l y 15~o o f the levels presented in T a b l e I (Table 3). The discrepancy between the two results c a n be explained by the different M g .... c o n c e n t r a t i o n s . A v r u c h a n d F a i r b a n k s d e m o n s t r a t e d N a + s t i m u l a t i o n in the presence o f I m M M g ~ f. The K m for M g ~+ is a p p r o x i m a t e l y 2 m M ( G u t h r o w , Allen a n d R a s mussen 1972; R u b i n , E r l i c h m a n a n d R o s e n 1972). W h e n 10 m M M g r was used with their i m i d a z o l e buffer system, the N a ~ specific effect was no l o n g e r a p p a r e n t (Table 3). In all e x p e r i m e n t s using the i m i d a z o l e buffer system, i n c l u d i n g those with I0 m M M g -H, the levels o f p e a k II p h o s p h o r y l a t i o n were less t h a n the s o d i u m a c e t a t e -
TABLE 3 PEAK 11 PHOSPHORYLATION - - EFFECT OF BUFFER, Mg ++ AND Na + Ghosts are prepared as described in Methods and incubated in the indicated buffer system for the indicated time. Ghosts are solubilized, electrophoresed and peak II is counted as described. Five experiments resulted in similar results. In this experiment 1090 counts/rain represent 1 pro/rag ghost protein/incubation time. The DMD patient is 7 years old, the control is 8 years old. Incubation solution buffer
50mMNaAc ÷ 0.6ram EGTA, pH6.5 20mMimidazole-Cl, pH 7.4 20raM imidazole-Cl, pH 7.4 + 50mMNaC1 20ram imidazole-Cl, pH 7.4 20raM imidazole-C1, pH 7.4 -k 50 mMNaCI
[Mg ~~]
DMD
Control
DMD
Control
(pm/mg/1 min incubation)
(pm/mg/15 min incubation)
10raM lmM lmM
12.1 1.36 2.21
8,41 1.15 2.11
79.6 6.83 10.82
47.5 4.83 7.92
10ram
11.9
7.88
47.9
34.1
7.49
42.6
32.7
10mM
9.27
191 EDTA buffer system but the increased peak II phosphorylation in DMD was still present. Experiments were performed to measure the phosphorylated state of spectrin as it exists in the membrane. Phosphate was measured in purified delipidized spectrin. Both DMD and control membranes demonstrated approximately 2 moles P/mole spectrin proteins. The maximal extent of phosphorylation with [y-aep]ATP represents approximately 0.1 moles a'-'P/mole spectrin protein. The sensitivity of the phosphate measurements did not allow measurement of any differences in the basal phosphorylated state of spectrin in D M D or controls that would account for the increased phosphorylation capability of DMD peak Ii. DISCUSSION Erythrocyte peak 11 phosphorylation is increased in DMD both at the initial rate and during extended periods of incubation. It is not possible to do formal kinetic analysis of this system because the substrate and enzyme cannot be separated from each other. Enzyme activity cannot be analyzed as substrate concentration is varied. Reconstituted systems with added lipid have not yet been developed. The increased peak II phosphorylation may be a substrate abnormality, an enzyme difference or an alteration in the state of the vectoral and geometric relationship of substrate to enzyme due to other membrane factors or constituents. Subtle biophysical and geometrical differences are difficult to evaluate with existing techniques but biochemical characteristics of the protein kinase and its substrates can be experimentally tested. Extraction of the protein kinase from the erythrocyte membrane is possible using 1 M NH4CL (Rubin et al. 1972). Kinetic evaluation of the extracted enzyme using a purified histone as substrate demonstrated no difference in MyD (Roses and Appel 1973) or DMD (Roses, unpublished data). The report of Na P-stimulated peak l l phosphorylation by Avruch and Fairbanks offered another approach to analyzing enzyme activity. Their system demonstrated the Na~-stimulating effect only at Mg ~ concentrations below the Km for Mg ~. Our conclusion was that the Na ~-stimulated peak II phosphorylation was apparent because suboptimal Mg' ~ concentrations were used. This was confirmed by finding only about 15°{~ of the possible peak II phosphorylation (Table 3). At optimal Mg ~ concentrations the Na~-stimulating effect was not apparent. The increased peak II phosphorylation in D M D was still present using the imidazole buffer system in these experiments. The possible leveling off of the phosphorylation of peak 11 was measured as a function of [7-a2P]ATP limitation and phosphoprotein phosphatase activity. Similar amounts (approximately 30~,/,) of the original [y aep]ATP were present following 15 rain incubations in DMD and controls. Addition of fresh [7-a2P]ATP to phosphorylated membranes did not increase the level of peak 1I phosphorylation, in addition, there was no phosphoprotein phosphatase activity over a 15 rain incubation. These experiments were subject to slight artifacts because long periods of incubation ( > 30 rain) caused slight extraction of the spectrin protein by the hypotonic, EGTA-containing incubation solution. The levels of peak II phosphorylation reached
192 in these studies did not appear to be due to an equilibrium between protein kinase and phosphoprotein phosphatase but rather the extent of substrate phosphorylation possible. The phosphorylated state of peak II could affect the extent of 32p labeling, if sites on the molecules are already phosphorytated in control membranes, then less 32p could be transferred to the protein. Measurements of P bonded to purified delipidated spectrin demonstrated approximately 2 moles P/mole spectrin protein. 32p labeling represents approximately 0.1 mole/mole spectrin. It was not possible to measure the phosphorytated state of spectrin with sufficient sensitivity to evaluate small differences. The relationship of increased erythrocyte peak II phosphorylation in DMD to the biochemical defect is unknown. It is possible that a highly phosphorylated component of peak II may account for the increase in phosphorylation, Purified delipidated a2P-labeled spectrin was subjected to isoelectric focusing electrophoresis and many labeled components were demonstrated (Roses et al. 1976). The same preparation stains only the spectrin doublet on SDS-PAGE. Preparative techniques for the separation of purified peak I and peak lI need to be developed in Order to facilitate study of substrate heterogeneity differences in DMD and controls. It is possible that the biochemical defect in the DMD erythrocyte may be an abnormal protein substrate of protein kinase. It is premature to attempt to relate peak II phosphorylation differences to the dystrophic process in muscle. Spectrin proteins have been referred to as myosin-like molecules because of the similarity in molecular weight to the heavy chains of myosin and their coextraction with actin-like protein by hypotonic EDTA solutions or water (Marchesi, Steers, Marchesi, and Tillak 1970; Clarke 1971 ; Trayer, Nozaki, Reynolds and Tanford 1971 ; Hartwig and Stossel 1975; Tilney and Detmers 1975). Isolation and characterization of the increased phosphorylated component or components of peak II in DMD may be a valuable tool with which to examine the relationship of spectrin to muscle proteins. It is apparent, however, that erythrocyte spectrin peak II phosphorylation is increased in DMD and the erythrocyte can be used to explore membrane abnormalities in this crippling, lethal disease. ACKNOWLEDGEMENTS We thank Mr. Michael Herbstreith and Mr. Bradley Metcalf for expert technical assistance, Mrs. Janet Worthington for expert secretarial assistance and Dr. Keith Hull, Mrs. Marcia Roses, L. P. T., Mrs. Megan Nicholson, M.A.P.A. (Australia), and Mrs. Cathy Kircher, L.P.T. whose volunteer services made the Duke Neuromuscular Research Clinic possible. REFERENCES Avruch, J. and G. Fairbanks (1974) Phosphorylation of endogenous substrates by erythrocyte membrane protein kinase -- I. A. monovalentcation-stimulated reaction, Bioehemi~try, 13: 5507.
193 Butterfield, D. A., A. D. Roses, D. B. Chesnut and S. H. Appel (1974) Electron spin resonance studies of erythrocytes from patients with muscular dystrophy, Proc. nat. ,4cad. Sci. (Wask.), 71 : 909. Butterfield, D. A., A. D. Roses, M. L. Cooper, S. H. Appel and D. B. Chesnut (1974) A comparative ESR study of the erythrocyte membrane in muscular dystrophy, Biochemistry, 13: 5078. Clarke, M. 0971) Isolation and characterization of a water-soluble protein from bovine erythrocyte membranes, Biockem. Biophys. Res. Commun., 45: 1063. Fairbanks, G., T. L. Steck and D. F. H. Wallach (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane, Biockemistry, 10: 2606. Friedel, R. O. and S. M. Schanberg (1971) Incorporation of in vivo of intracisternally injected 33Pi into phospholipids of rat brain, J. Neurockem., 18: 2191. Glynn, I. M. and J. B. Chappell (1964) A simple method for the preparation of 3eP-labeled adenosine triphosphate of high specific activity, J. Biockem., 90: 147. Guthrow, Jr., C. E., J. E. Allen and H. Rasmussen (1972) Phosphorylation of an endogenous membrane protein by an endogenous membraneassociated cyclic adenosine 3',5'-monophosphatedependent protein kinase in human erythrocyte ghosts, J. biol. Chem., 247: 8145. Hartwig, J. H. and T. P. Stossel (1975) Isolation and properties of actin, myosin, and a new actinbinding protein in rabbit alveolar macrophages, J. biol. Chem., 250: 5696. Hull, Jr., K. L. and A. D. Roses (1976) Stoichiometry of sodium and potassium transport in erythrocytes from patients with myotonic muscular dystrophy, J. Physiol. (Lond.), 254:169 181. Lowry, O. H., N. J. Rosebrough, A. C. Farr and R. J. Randall (1951) Protein measurement with the Folin phenol reagent, J. biol. Ckem., 193:265 275. Marchesi, S. L., E. Steers, V. T. Marchesi and T. W. Tillak (1970) Physical and chemical properties of a protein isolated from red cell membranes, Biochemistry, 9: 50. Roses, A. D. and S. H. Appel (1973) Erythrocyte protein phosphorylation, J. biol. Chem., 248: 1408. Roses, A. D. and S. H. Appel (1973) Protein kinase activity in erythrocyte ghosts of patients with myotonic muscular dystrophy, Proc. nat, Acad. Sci. (Wash.), 70: 1855. Roses, A. D. and S. H. Appel 0975) Phosphorylation of a component of the human erythrocyte membrane in myotonic muscular dystrophy, J. Membr. Biol., 20: 51. Roses, A. D., M. H. Herbstreith and S. H. Appel (1975) Membrane protein kinase alteration in Duchenne muscular dystrophy, Nature (Lond.), 254:350. Roses, A. D., M. H. Herbstreith, B. Metcalf and S. H. Appel (1976) Increased phosphorylated components oferythrocyte me :obrane spect rin band !I with reference to Duchenne muscular dystrophy, J. neurol. Sci., 30: 167. Rubin, C. S., J. Erlichman, and O. M. Rosen, (1972) Cyclic adenosine 3',5'-monophosphate-dependent protein kinase of human erythrocyte membranes, J. biol. Chem., 247: 6135. Tilney, L. G. and P. Detmers (1975) Actin in erythrocyte ghosts and its association with spectrin, J. Cell Biol., 66: 508. Trayer, H. R., Y. Nozaki, J. A. Reynolds and C. Tanford (1971) Polypeptide chains from human red blood cell membranes, J. Biol. Chem., 247: 4485. Walton. J. N. (Ed.) (1974) Disorders o/Vohmtary Muscle, 3rd edition, Churchill Livingstone, Edin burgh and London.
