Evidence for the presence of protein kinase C in barnacle muscle fibers

Comp. Biochem. Physiol. Vol. 88B, No. 2, pp. 687-690, 1987

0305-0491/87 $3.00 + 0.00 © 1987 Pergamon Journals Ltd

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EVIDENCE FOR THE PRESENCE OF PROTEIN KINASE C IN BARNACLE MUSCLE FIBERS E. EDWARD BITTAR and PEGGY R. GtRARD* Department of Physiology, University of Wisconsin, Madison, WI 53706, USA; Department of Pharmacology* Emory University, School of Medicine, Atlanta, GA 30322, USA

(Received9December 1986)

Abstract--1. The efflux of 22Na from single barnacle muscle fibers poisoned with ouabain (strophanthin G) is found to be very sensitive to the tumor-promoting agent, phorbol dibutyrate (PDBu). 2. Injection or external application of PDBu leads to stimulation of the ouabain-insensitive component of the Na efflux. This response is dose-dependent, the minimal effectiveconcentration being about 10-SM. 3. The observed stimulatory response is completely dependent on the presence of external Ca2+. 4. Biochemicalstudies including immunoblotanalysis reveal the presence in barnacle muscle of a protein kinase C with a mol. wt of 80,000, the activity of which is dependent on phosphatidylserine and Ca 2+. 5. Taken together, these results support the view that barnacle muscle fibers possess protein kinase C. They also raise the possibility that protein kinase C plays a role in modulating the ouabain-insensitive component of the Na et~ux.

INTRODUCTION There is evidence that the ouabain-insensitive component of the Na efflux into 10 mM-Mg2+-artifical sea-water from single barnacle muscle fibers is modulated by several, distinct mechanisms involving (i) adenylate cyclase, cAMP-dependent protein kinase (Bittar, Chambers and Schultz, 1976; Schultz and Bittar, 1978; Bittar et al., 1979); (ii) guanylate cyclase, cGMP-dependent protein kinase (Bittar and Sharp, 1979); (iii) a [Ca]i-dependent system (Schultz and Bittar, 1978; Mason-Sharp and Bittar, 1981; Bittar and Nwoga, 1982a, b) which may be associated with a putative calmodulin/Ca2+-dependentprotein kinase and/or a phospholipid/Ca2+-dependent protein kinase and (iv) a CO2-[HCOf]e-dependent system which does not involve cyclic nucleotides or Ca 2+ directly (Bittar, 1983). The possibility that phospholipid/Ca2+-dependent protein kinase C may play a role in modulating the ouabain-insensitive Na et~ux has now been explored by employing the tumorpromoting phorbol ester, 4-fl-phorbol-12, 13-dibutyrate (PDBu). As demonstrated by Nishizuka and coworkers (Nishizuka, 1986), protein kinase C is the specific receptor of phorbol esters. In the present report, it is shown that microinjection or external application of PDBu stimulates the ouabain-insensitive component of the Na efflux in single muscle fibers from the barnacle, Balanus nubilus and that this response is completely dependent on the presence of external Ca 2+. In addition, supporting biochemical evidence is brought forward which indicates that barnacle muscle possesses protein kinase C.

MATERIALS A N D M E T H O D S

Specimens of the barnacle Balanus nubilus were supplied by the Pacific Biomarine Laboratory in Venice,CA and kept in a 150-gaUonInstant Ocean aquarium containing artificial C.B.P. 88/21~T

sea-water. The temperature of the aquarium water was maintained at about 12°C throughout. Dissection and cannulation Single fibers measuring 3-4 cm in length and I-2 mm in width were isolated by dissection from the depressor muscle bundles and then cannulated. A 50-80rag weight was attached to the tendon of the cannulated fiber, thereby keeping it in a vertical position while suspended in artificial sea-water (ASW). Solutions The experiments were done with ASW having the following composition (raM): NaCI, 465; KC1, 10; CaCI2, 10; MgCI2, 10; NaHCO 3, 10 and pH 7.8. Solutions of Ca2+-free ASW were prepared by raising NaC1 in equivalent amounts. Radioactivity measurements 22NaCl in aqueous solution obtained from AmershamSeafle, Arlington-Heights, IL, was dried down and redissolved in distilled water so that volumes of 0.4/~1 gave approx. 750,000epm. The effluent from the cannulated fiber following loading with 22Na by microinjection was collected every 5 min and the residual fiber activity was counted at the end of the experiment. A Beckman auto-gamma counter was used for the counting of samples. The data were fed into an Apple II computer programmed to calculate the fractional rate constant for 22Na effiux (i.e. efflux rate/fiber count + ~ efflux). An estimate of the size of the observed stimulating effect on Na efflux was computed by taking the difference between the maximum rate constant and the rate constant before the onset of stimulation. The results given in this paper are means + SE of mean. All flux experiments were carried out at an environmental temperature of 22-24°C. Purification of protein kinase C Barnacle muscle (2 g) was homogenized with 6 ml of ice-cold 50 mM Tris/C1 (pH 7.5) containing 2 mM EDTA, 3mM EGTA, 50mM mercaptoethanol, 0.3ram PMSF (phenylmethylsulfonyl fluoride), 0.4%, Triton X-100 for 90 sec in a Waring Blendor. The homogenate was then centrifuged at 30,000g for 30 rain and the supernatant was

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E. EDWARDBITTARand t~c_,oY R. GIRARD

filtered through glass wool. Next, the filtrate was applied to a DEAE-cellulose column, pre-equilibrated with elution buffer (50 mM Tris/Cl, pH 6.5 and 50 mM mercaptoethanol). Subsequent to washing the column with buffer, the enzyme was eluted with a linear gradient of NaC! (0-0.5 M; total volume, 0.21) added to the extraction buffer. The fractions containing the enzyme were pooled before assaying. Assay was carried out as described by Kuo et aL (1980) and Wise et al. (1982). Briefly, the reaction mixture contained 5 # mol of Tris-HC1 (pH 7.5), 2 #tool of MgC12, 5/~g of phosphatidyl serine (PS), with 0.I #mol or 0.01 #mol of CaCI2, 0.1 #mol of EGTA, 40#g of lysine-rich histone, I nmol of [?-32p]ATP and the enzyme protein (the final volume being 0.2 ml). The reaction was initiated by adding ATP and incubation was for a period of 5--10rain at 30°C. Immunoblot analysis was carried out as described by Girard et al. (1986). The homogenate (150#g) and the purified enzyme were subjected to SDS-PAGE and then transferred electrophoretically to a Zeta-Probe blotting membrane. The membrane was incubated with antisera against protein kinase C and then with nSI-protein A. The membrane was then autoradiographed to locate immunoreactive proteins. Agents

Ouabain, PDBu and PMSF (phenylmethylsulfonyl fluoride) were supplied by Sigma Chemical, St Louis, MO, USA. DMSO (dimethyl sulfoxide) was from Fisher Scientific, Fair Lawn, NJ. Zeta-Probe blotting membranes were purchased from Bio-Rad Laboratories, Richmond, CA USA. RESULTS

AND

DISCUSSION

In the first group of experiments, ouabainpoisoned fibers were injected with 10-2M PDBu in 1% DMSO. As illustrated in Fig. 1, injection of 1% DMSO into a fiber pretreated with 10-4M ouabain (a maximally inhibitory concentration) is without effect on the course of the ouabain-insensitive N a efflux (N -- 8). By contrast, injection of 10-2M PDBu causes a prompt and sharp rise in the Na efflux, which reaches a peak some 30 min later and then decays somewhat slowly. The magnitude of this response averages 555 _+ 83%, N = 4. Companion controls show that injection of 1% DMSO twice in succession is without effect (N = 4). In parallel experiments, unpoisoned fibers when injected with 1% DMSO show no change in the resting N a etflux (N = 4). By contrast, subsequent injection of 10-2M PDBu causes a temporary and prompt stimulatory response

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(averaging 92 _ 13%, N = 4) which reaches a peak rather slowly (i.e. 30-50 rain later). External application of 10-4 M ouabain shortly after the onset of peak stimulation leads to a marked fall in the N a effiux, indicating that the membrane Na+-K+-ATPase system is unaffected by PDBu (N = 4). In the second group of experiments, PDBu was applied externally to fibers pretreated with 10-4M ouabain. The results obtained show that PDBu always stimulates the ouabain-insensitive N a efllux, the minimal effective concentration being about 10-SM. This reponse is often preceded by a 5-10 rain latency period and reaches a maximum (averaging 567 _+ 12%, N = 4 when 10-5M PDBu is used) rather slowly. The third group of experiments was designed to determine whether the response to injection or external application of PDBu depends on externl Ca 2+. Hence ouabain-poisoned fibers were injected with 10-2M PDBu in 1% DMSO, followed by sudden removal and then restoration of external Ca 2+. The results show that omission of external Ca 2+ following the onset of peak stimulation by PDBu results in a clear-cut, fully reversible step-down in the Na efflux (N = 5). Companion control fibers (N = 3) show little or no change in the behavior of the resting residual efflux foUowing omission and then restoration of external Ca ~+. Another striking feature of these results is that the response following restoration of external Ca 2+ fails to decay. The reason for this is far from clear. Next, experiments were done in which external Ca 2+ was omitted prior to external application of 10-SM PDBu. As illustrated in Fig. 2, the ouabaininsensitive Na efflux is unaltered by sudden removal of external Ca 2+, followed by external application of 10-SM PDBu. However, restoration of external Ca 2+ results in a prompt and marked stimulatory response, the magnitude of which averages 864 _ 99% (N = 5), a value not very different from that found in companion controls, viz 957 _+ 92%, N - - 5 (P being >0.5). Again, it is not without some significance that the stimulatory response fails to decay (N = 5). Companion controls show decay of the response in four of the five fibers. Collectively, the above results support the view that the location of the primary site of action of PDBu is myoplasmic and that the stimulatory response caused by PDBu depends completely on the

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Fig. 1. Marked stimulation of the ouabain-insensitive Na etfinx by injecting 10-2M phorbol dibutyate in 1% DMSO. Also shown is the lack of effect of injecting 1% DMSO beforehand (rate constant for "Na effiux plot).

Protein ldnase C

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Fig. 2. Lack of effect on the ouabain-insensitive Na efltux of sudden omission of external Ca 2+ (at t ffi 60 rain) and external application of 10-5M phorbol dibutyrate (at t ffi 80 min). Prompt and marked stimulation follows restoration of external Ca 2+ (at t = 130 rain). Table 1. Effects of PS, Ca 2+, TPA and diolein on protein kinase C activity Protein kinase activity (pmol p/min) Addition None TPA (25 nM) Diolein (0.8 rag/0.2 ml)

- C a 2+ (EGTA, 3 mM)

PS (0.5 #g/0.2ml) CaCI2 (0.05 mM)

PS (5/~g0.2ml) CaCI2 (0.5 raM)

3.1 3.6 3.5

4.5 13.8 12.6

10.2 13.5 11.8

presence of external Ca 2+. Such a result can be accounted for by supposing that activation of protein kinase C by PDBu leads to increased phosphorylation of the Na+-Ca 2+ exchanger in the sarcolemma and/or the Ca 2+ channel protein. In the latter case, one would expect activation of the Ca 2+channel to produce a fall in myoplasmic pCa, thus leading to stimulation o f the ouabain-insensitive N a et~ux (Schultz and Bittar, 1978; Bittar and Nwoga, 1982a, b; Bittar, 1983) via not only activation of cAMP-dependent protein kinase by newly formed cAMP (Mason-Sharp and Bittar, 1981), but also via calmodulin/Ca2+-dependent protein kinase (Bittar and Nwoga, 1982a, b) and PL/Ca2+-dependent protein kinase C. This is rendered likely in the fight of evidence that a raised internal free Ca 2+ concentration augments the membrane binding action of Immunoblot (extract, 350 ~g protetn)

80 K (PK-C)

(PK-C

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phorbol ester on protein kinase C (Wolf et al., 1985; May et al., 1985). The biochemical characteristics of barnacle muscle protein kinase C are similar to those in other species and tissues. As shown in Table 1, the activity of the enzyme is dependent on Ca 2+ and PS and diolein can serve as a substitute for PS. TPA (12-O-tetradecanoyl phorbol 13-acetate) is able to increase the activity of the enzyme in spite of a 10-fold reduction in Ca :+ concentration. This finding is not surprising, since phorbol esters activate protein kinase C partly by reducing the Km for Ca 2+ (Nishizuka, 1986). Immunoblot analysis confirms the presence of protein kinase C in barnacle muscle. This is shown in Fig. 3 where it can be seen that the enzyme is a protein with mol. wt = 80,000 which is associated with a mol. wt = 50,000 fragment. As pointed out by Girard et al., 1986), the mol. wt = 50,000 species is most likely protein kinase M produced as the result of the action of Ca2+-dependent proteases on the native tool. wt = 80,000 enzyme. The observation that protein kinase C is found in barnacle muscle is in keeping with the view that the enzyme is ubiquitous in the Animal Kingdom (Kuo et al., 1980).

Acknowledgements--Warmest thanks

to Professor J. F. K u o for advice and his comments on the first draft o f the MS. T h a n k s are also due to M r K. U e n o for technical assistance. This work received support from N S F and from N I H t h r o u g h a General Research Support G r a n t to the University o f Wisconsin Medical School. It also received support from N I H (HL15696 to J. F. Kuo) and a National Research Service Award to P.R.G.

REFERENCES

Bittar E. E. (1983) The barnacle muscle fibre as a model system for the investigation of the ouabain-insensitive sodium efl]ux and hormonal actions. Prog. Neurobiol. 20, Fig. 3. Immunoblot tracing showing protein kinase C a n d its immunoreactive fragment in barnacle muscle

homogenate.

1-54.

Bittar E. E., Chambers (3. and Schultz R. 0976) Mode of stimulation by adenosine Y, Y-cyclic monophosphate of

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E. EDWARD BITTARand ~._,oY R. GIRARD

the sodium efltux in barnacle muscle fibres. J. Physiol. 257, 561-579. Bittar E. E., Demaille J., Fischer E. H. and Schultz R. (1979) Mode of stimulation by injection of cyclic AMP and external acidification of the sodium efflux in barnacle muscle fibres. J. Physiol. 296, 277-289. Bittar E. E. and Nwoga J. (1982a) Stimulation by injected guanosine triphosphate of the sodium etflux in barnacle muscle fibres. J. Physiol. 322, 389-397. Bittar E. E. and Nwoga J. (1982b) Some further observations on the stimulation by high external potassium of the sodium efflux in barnacle muscle fibres. Pflugers Archs 395, 318-325. Bittar E. E. and Sharp D. M. (1979) Stimulation by cyclic GMP of sodium efltux in barnacle muscle fibres. J. Physiol. 293, 135-151. Girard P. R., Mazzei G. J. and Kuo J. F. (1986) Immunological quantitation of phospholipid/Ca2+-dependent protein kinase and its fragments. J. biol. Chem. 261, 370-375. Kuo J. F., Anderson R. G. G., Wise B. C., Mackerlova L., Salomonsson I., Brackett N. L., Katoh N., Shoji M. and Wrenn R. W. (1980) Calcium-dependent protein kinase: Widespread occurrence in various tissues and phyla of the animal kingdom and comparison of effects of phospholipid, calraodulin, and trifluorperazine. Proc. hath. Acad. Sci. 77, 7039-7043.

Mason-Sharp D. and Bittar E. E. (1981) Stimulation by high external potassium of the sodium efflux in barnacle muscle fibers. J. Membr. Biol. 58, 213-226. May W. S., Sahyoun N., WolfM. and Cuatrecasas P. (1985) Role of intracellular calcium mobilization in the regulation of protein kinase c-mediated membrane processes. Nature 317, 548-550. Nishizuka Y. (1986) Studies and perspectives of protein kinase C. Science 233, 305-312. Nwoga J. (1987) Concerning stimulation by high external potassium and calcium injection of the ouabalninsensitive sodium efflux in barnacle muscle fibers. Comp. Biochem. Physiol. 86A, 733-737. Schultz R. and Bittar E. E. (1978) Studies of the mode of stimulation by external acidification and raising the internal free calcium concentration of the sodium eiflux in barnacle muscle fibres. Pflugers Archs 374, 31-38. Wise B. C., Raynor R. L. and Kuo J. F. (1982) Phospholipid-sensitive Ca2+-dependent protein kinase from heart--I. Purification and ~general properties. J. biol. Chem. 257, 8481-8488. Wolf M., Levine H., May W. S., Cuatrecasas P. and Sahyoun N. (1985) A model for intraceUular translocation of protein kinase C involving synergism between Ca 2+ and phorbol esters. Nature 317, 546-548.