Feb 25, 2018 - Growth Factor Receptor Kinuse Assay-Plasma membranes iso- lated from Rat-1 cells according to the procedure of Thom et al. (9).
THEJOURNAL OF BIOLOGICAL CHEHtSTRY Vol. 262 No. 6 Issue of February25 pp. 2431-2434 1957 0 1957 by ?he AAerican Society of Bioiogical Chernisd, Inc. Printed in U.S.A.
Communication Expression of pp6OV-"'" Alters the Ionic Permeability of the Plasma Membrane in Rat Cells* (Received for publication, October 31, 1986)
Jan van der ValkS, Ingrid Verlaan, Moolenaars Siegfried W. de Laat, and Wouter From the Hubrecht Laboratory, Uppsalalonn 8, 3584 CT Utrecht, The Netherlands
The transmembrane potential of Rous sarcoma virus (RSV)-infected Rat- 1 cells, expressing the pp60""'" protein kinase, is markedly less negative (by -30 mV) than that of their normal counterparts. By contrast, the membrane potential of Rat-1 cells infected with Kirsten sarcoma virus is virtually unaltered. The RSVinduced membrane depolarization is shown to be due to a severalfold increase in the cation permeability ratio ( P N J P Kof ) the plasma membrane. When cells infected with a temperature-sensitive mutant of RSV ( t s LA29), encoding a SFC protein with heat-labile kinase activity, are shifted from the nonpermissive to the permissive temperature, a rapid and sustained membrane depolarization is observed. Conversely, thermal inactivation of the t s LA29 pp60""" kinase activity rapidly restores the membrane potential to near normal levels. Addition of epidermal growth factor, platelet-derived growth factor, or insulin to uninfected cells fails to cause a detectable change in membrane potential. We conclude that, unlike growth factor receptor tyrosine kinases, pp60""" can induce, either directly or indirectly, a major change in the membrane permeability to monovalent cations.
events in growth factor-stimulated cells (2-5), but it is n o t known whether similar ionic changes may play a role in the action of pp60""". As a first step in testing this possibility, we have examined the ionic membrane properties of normal and RSV-infected rat fibroblasts (Rat-1 cells) by means of intracellularmicroelectrodes. We report here that, unlike growth factor receptor kinases, pp60""'" can induce a striking permanent depolarization of the plasma membrane. We present evidence that this action of pp60""", which is apparently not directly related to its mitogenic and transforming properties, is attributable an toalteration inthe membrane permeability to monovalent ions. This, in turn, suggests a possible function of the corresponding proto-oncogene product, pp60""". EXPERIMENTALPROCEDURES
Reagents-EGF was purchased from Collaborative Research, and PDGF wasgiven by Dr. C.-H. Heldin (Uppsala, Sweden). Bovine and 1,2-oleoyl-acetylinsulin, 12-0-tetradecanoylphorbol-13-acetate, glycerol were from Sigma. Antiserum from RSV-tumor-bearing rabbits was the kind gift of Dr. A. Barnekow (Giessen, Federal Republic of Germany). Cells-Rat-1 cells are a contact-inhibited,embryonal rodent fibroblast line. Normal and virally transformed Rat-1 cells were kindly provided by Dr. J. Wyke (ICRF, London). The cell lines were grown in HCOT-buffered DMEM containing 7.5% fetal calf serum. Soft Agar Growth-The percentage of cells able to form colonies in soft agar was determined by plating 10' cells in DMEM containing 7.5% fetal calf serum and 0.4% agar onto a base layer of 0.5% agar in 6-cm Petri dishes. Colonies larger than 8 cells were scored after 10 days by counting 20 random fields corresponding to 2.3% of the total dish area. Kinuse Assay-The pp60""" protein kinase activp ~ 6 O " -Protein *~ ity was detected in lysates of RSV-transformed Rat-1 cells by its ability to phosphorylate the heavy chain of IgG in immunoprecipitates. The procedures for lysing cells and immunoprecipitation are those of Barnekow and Bauer (6). Briefly, confluent cultures in 6-cm dishes were washed twice with ice-cold phosphate-buffered saline and lysed in extraction buffer (10 mM sodium phosphate, 40 mM NaF, 10 The productof the transforming src geneof Rous sarcoma mM EDTA, 1%Triton X-100, 5% Trasylol, pH 7.0). The extracts virus (RSV),' pp60""", i s a membrane-bound phosphoprotein were clarified by centrifugation (20,000 X g for 60 min). 0.2 mg of This soluble protein was treated with 5 pl of tumor-bearing rabbit serum with intrinsic tyrosine-specific protein kinase activity. kinase activity is shared with several growth factor receptors, for 60 min at 4 "C and precipitated with protein A-containing Sephsuch as the receptors for epidermal growth factor (EGF) and arose beads. The washed immunocomplexes were assayed for protein kinase activity in a 10-min reaction as described (7), with the followplatelet-derived growth factor (PDGF) (reviewed in Ref. I), ing modification (8) for the ts LA29-transformed cells. The immusuggesting that tyrosine-specificproteinphosphorylations nocomplexes werefirst incubated at 34 or 39.5 "C for 10 min in kinase may initiate a set of common biochemical events in RSV- reaction buffer (7) without [y-32P]ATP.The ATP was then added, infected and growth factor-stimulatedcells.Yet, it is currently and the phosphorylation reaction was run for 10 min at therespective not possible to relate increased protein-tyrosine kinase activ- temperatures. The protein A-bound immunocomplexes were then boiled in sodium dodecyl sulfate sample buffer and subjected to cells. ity to specific physiological alterations in responsive dodecyl sulfate gel electrophoresis and autoradiography. In addition to tyrosine phosphorylations, changes in ionic sodium Growth Factor Receptor Kinuse Assay-Plasma membranes isotransport across the plasma membrane and increases in cylated from Rat-1 cells according to the procedure of Thom et al. (9) toplasmic free Ca2+ and p H a r e a m o n g the first detectable were preincubated with EGF or PDGF (50 ng/ml) for 10 min at 0 "C. The phosphorylation reaction was started by adding EGF/PDGF* This work was supported by the Netherlands Cancer Foundation treated membranes to a mixture containing 20 mM HEPES (pH7.4), (Koningin Wilhelmina Fonds). The costs of publication of this article 10 mM MnC12,10 pM [y-32P]ATP(5000cpm/pmol), 0.1 mg/ml serum were defrayed in part by the payment of page charges. This article albumin, 50 pM Na3V04,and 10 mM P-nitrophenyl phosphate (total must therefore be hereby marked "advertisement" in accordance with volume of 60 pl). After 10 min at 0 "C, the reaction was stopped by addition of sample buffer. After boiling, the samples were subjected 18 U.S.C. Section 1734 solelyto indicate this fact. 3 Present address: Dept. of Behavioral Biology, Australian Na- to sodium dodecyl sulfate gel electrophoresis and autoradiography. Electrophysiology-Cells were grown to near confluency on circular tional University, Canberra, Australia. cover glasses (diameter 1.5 cm) and maintained in serum-free DMEM J To whom correspondence should be addressed. for at least 2 prior h to experimentation. Electrophysiologicalmethods I The abbreviations used are: RSV, Rous sarcoma virus; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; similar to those described previously were used (4,10). Cells were DMEM, Dulbecco's modified Eagle's medium; HEPES, 4-(2-hydrox- impaled under microscopic control with a single microelectrode (0.5 M KC1-filled, resistance 50-95 megaohm). The cultures were incuyethy1)-1-piperazineethanesulfonic acid.
Ionic Effects ofpp60"-""in Rat Cells
TABLE I Ionic parameter.s of normal and virally infected Rat-I cells Themeanmembranepotential (V,,,) was determined by direct intracellular recording in single cells. The cation permeability ratio (PNJPK) and the intracellular K ' concentration ([K'],) were determined from the graphs in Fig. 3 unless indicatedotherwise. The number"of_ cells is given in parentheses. Infecting virus V,,, (mean f S.E.) PN./P, [K+]& mV mM 155 f 10 -63.1 f 2.7 (40) 0.06 Control (uninfected) 178 f 10 -31.8 f 3.2 (40) 0.35 Wild-type RSV (R77) NDb -31.6 2 1.0 (30) 0.30" ts LA29 (34 "C) ND -53.4 & 0.9 (35) 0.09" ts LA29 (39.5 'C) 0.07" ND -60.5 & 2.0 (7) Kirsten sarcoma "
FIG. 1. pp60""" protein kinase activity in immunoprecipitates of RSV-infected Rat-1cells. The kinase activityis indicated
autoradiographically hy the extent of phosphorylation of the 53-kDa heavy chain of anti-pp6WnKIgG. Lune I, normal Rat-1 cells; lone 2, R77-infected cells; lone 3, ts LA29 cells a t 34 "C;lane 4, ts LA29 cells at 39.5 "C. See "Experimental Procedures" for details. bated either in serum-free HEPES-buffered DMEM( I ml) or in salt solution containing 145 mM NaCI, 5 mM KCI, 2 mM CaC12, 1 mM MgCI,, 10 mM HEPES (pH 7.4). and 10 mM glucose. In some test solutions the K+ and Na' concentrations were changed, keeping [KCI] + [NaCl] = 150 mM. The temperature of the bathing solution (3439.5 "C) was controlled by a Peltier element, allowing temperature shifts at a rate of about 3 "C/min.
Estimated from a formula derived by Woodbury, which expresses
V,,, as a function of extracellular Na+ and K' concentrations only (see Ref. 12). * ND, not determined.
Characterization of Virally Transformed Rat-1 Cells-Infection of Rat-1fibroblasts with RSV (strain B77) or with Kirsten sarcoma virus, encoding the p21'"" protein,* induces the ability to form colonies in semisolid agar medium, with virtuallynochange in the mean cell doubling time.'? The temperature-sensitive RSV mutantts LA29, whose lesion maps in the carboxyl-terminalregion of pp60""" ( l l ) , induces colony formation a t 35 "Ccomparable to thatinduced by wildtype B77 virus; however, the ' I colony-forming ability is completely lost a t a nonpermissive temperature of 39.5 The protein kinase activity of pp60'~"" was monitored after immunoprecipitation fromRSV-infected Rat-1 cell lysates, and representative results are shown in Fig. 1. As a control, uninfected cells were used in parallel. 'The ts mutant LA29 shows a large decrease in kinase activity within 10-20 min of shifting the temperature from 34 to 39.5 "C. Conversely, the kinase activity rapidly increases upon a shift from 39.5 to 34 "C; no significant change in kinase activity was observed in the wild-typevirus-infected cells following temperature shifts in either direction (not shown). These findings are in agreement with those of Stoker et al. (8),who were the first to show that ts LA29 encodes a mutant src protein with heatlabile kinase activity both in uitro and in uiuo. Membrane Potential Measurements-Nearly confluent Rat-1 cells have a mean resting transmembrane potential of -63.1 mV, as measured by direct intracellular recording in single cells (Table I). Infection of Rat-1 cells with Kirsten sarcoma virus does not inducea significant change in the mean membranepotential; however, infection of the cells with RSV causes a substantial reduction in resting membrane potential by more than 30 mV (Table I). Rat-1 cells infected with the temperature-sensitive mutant ts LA29 show a similarly reduced membrane potentiala t 34 "C and a clear increase in potential to normal cell values a t a nonpermissive temper-
'Increased ros gene expression was confirmed by immunoblotting
using monoclonal antibody 259 (A. Feijen and P. T. van der Saag, unpublished data). The percentage of cells able to form colonies is as follows: normal Rat-l,
- 60 - 70 0
15 Time at 35
FIG.2. Effect of temperature shift on membrane potential in t s LA29-infected Rat-1 cells. A , typical microelectrode recordings (upper panel) showing gradual membrane hyperpolarization in ts LA29-infectedcells and lack of potential changes in wild-type (B77)-transformed cells and in control cells. Lower panel shows temperature timecourse in incubation medium. B, cells grown to near confluency a t 39.5 "C were shifted to 35 "C for the indicated times before the membrane potentialwas measured in a t least eight different cells. 0, t s LA29-infected cells; 0, control cells. Error bars represent S.E.
ature of 39.5 "C (Table I). Kinetic analysisof the temperature shift-induced effects demonstrates that the membrane polarizations occur rapidly (within 25 min) and arefully reversible (Fig. 2, A and R ) , while wild-type RSV-infected and control cells maintain their membrane potential a t either temperature. Thus, the membrane depolarization observed in RSVinfected cells parallels the pp60""" protein kinase activity as measured in an immunocomplex assay (Fig. 1).
of pp60"'""in Rat Cells
Ionic Basis of the Membrane Potential-To examine the as monitored by receptor autophosphorylation and by the origin of the membrane potential in normal and RSV-infected appearance of a 35-36 kDa phosphoprotein in response to Rat-1 cells, the cultures were incubated in media of varying EGF (Fig. 4). external K' concentration ([K'],), andthecorresponding Despite the presence of a functional ligand-dependent remembrane potential (V,) wasmeasured. Fig. 3 shows the ceptor kinase,growth factors fail to elicit a detectable electrodependence of V,,, on [K'l0 for both normal andB77-infected physiological response in an impaled cell within 10-15 min cells. The smooth curves are drawn according to the theoret-(not shown). The effects of EGF, PDGF (20 ng/ml),and ical relationship insulin ( 5 pg/ml) onmembranepotential were also determined by successive impalements of a t least 10different cells [K+lo + p[Na+I, between 0 and 60 min after growth factor addition.We found V , = 61 log [K'li that there is no significant difference in membrane potential where p = P N a / P ~i.e. , the ratioof the membrane permeability ( d . 0 mV) between quiescent and growth factor-treated cells during the first hourof receptor stimulation. It thus appears to Na+ and K+, respectively, [Na+], is the extracellular Na' concentration, and [K']i is thecytoplasmic K' concentration. that the membrane depolarization observed with pp60""" is not a common physiological effect of membrane-bound proEquation 1 is asimplifiedform of the Goldman equation (reviewed in Ref. 12), here written for the case that p[Na+Ii tein tyrosine kinases. We finally tested whether activatorsof