Inhibition of Phorbol Ester Binding and Protein Kinase C Activity by. Heavy Metals*. (Received for publication, September 12, 1988). Louis A. Speizer, Michael J.
Vol. 264, No. 10, Issue of April 5,pp. 5581-5585,1989 Printed in U.S . A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Phorbol Ester Binding and Protein Kinase C Activity by Heavy Metals* (Received for publication, September 12, 1988)
Louis A. Speizer, Michael J. Watson, Joan R. Kanter, and Laurence L. Brunton From the Department of Pharmacology, Uniuersity of California at SunDiego School of Medicine, La Jolla, California 92093
Other laboratories have reported biphasic effects of heavy metals on protein kinase C activity: stimulation followed by inhibition at higher concentrations. We demonstrate that these earlier findings most likely resulted from a combination of the effect of the heavy metals to liberate Ca2+ from Ca2+-EGTA buffer systems and the direct inhibitory effects of the metals on protein kinase C. Simulations of such interactions substantiate this conclusion. When soluble protein kinase C is prepared without the addition of Ca2+ orchelator, heavy metals (Cd2+,Cu2+, Hg2+, Zn2+, in the 10 ~ L M range) inhibit the activity of,and the binding of regulatory ligands to, protein kinase C. Heavy metals inhibit the extent of['Hlphorbol dibutyrate binding without affecting the affinity of the interaction, an inhibition that is not surmounted by excess phospholipid. Heavy metals also inhibit the phospholipid-dependent catalyticactivity of protein kinase C in a manner that excess phosphatidylserine can overcome. The inhibition of enzyme activity by heavy metals cannot besurmounted by excess Ca2+orMg2+.The inhibitory effects of heavy metals are not confined to protein kinase C. Heavy metals also inhibit cyclic AMP binding to cyclic AMP-dependent protein kinase and the catalytic activityof that kinase, but in a distinctly different pattern.
EXPERIMENTALPROCEDURES
Methods"S49 cells were grownin suspension culture in Dulbecco's 10% horse serum (4). Membrane and modified Eagle's medium cytosolic fractions were prepared by a rapid freeze/thaw procedure in which nuclei were removed and discarded (5). The homogenization buffer contained 50 mM &glycerol phosphate, 30 mMMgC12, 10 pg/ ml leupeptin, and 0.3 mM phenylmethylsulfonyl fluoride (pH 7.4). Chelators were purposely omitted from the fractionation buffer. The activity of protein kinase C was defined as phosphate transferred from [Y-~'P]ATP histone to H1 in the presence of Ca2+(in the assay) and dependent on phospholipid (phosphatidylserine, 100 pg/ ml) diolein (3.3 pglml). Protein kinase C assays (containing, in 100p1 final volume, 10 mM MgCL, 500 p M CaCl', 0.8 mg/ml histone H1, 10 p~ ATP with ([y3'P]ATP, -1000 cpm/pmol) with or without the phospholipid mixture, in 20 mM Tris (pH 7.4)) were initiated by the addition of protein (5 pg ofS49 cytosolicprotein or-0.1 pg ofpartially purified rat brain protein kinase C), incubated at 30 'C for 3 min and terminated by spotting onto Whatman No. 3MM filters (6). The partially purified protein kinase C was prepared from rat cerebrum by homogenization as described above for S49 cells, addition of the cytosolic extract to DEAE, and collection of the protein retained a t 50 mM NaCl and eluting at 110 mM NaCl. The presence of chelator did not alter the elution pattern. CAMP-dependent protein kinase (-1 to 2 pg per tube) was added identically with protein kinase C except that 5 p~ CAMPwas included. Cyclic AMP-dependent protein kinase partially purified from rabbit skeletal muscle (peak 1 from a DEAE-cellulose column (7)) had an activity ratio (-cAMP/+cAMP) of 0.09. Phorbol ester receptors were quantified using [3H]PDBu1 in a filtration assay. The reaction mixture included 500 p M CaC12,10 mM MgC12, 100 pg/ml phosphatidylserine, and 60 nM [20-3H]phorbol 12,ls-dibutyrate ([3H]PDBu) in 20 mM Tris buffer (pH 7.4). Binding M or 3p~ phorbol12-myristate, The physiologic effects of heavy metals such as Cd", H e , occurring in the presence of 3 ~ L PDBu Cu2+,and Zn2+result from interactions with many cell con- 13-acetate was considered nonspecific and accounted for 4 0 % of the binding. stituents. Certain proteins are, however, unusually sensitive, total Protein was determined by the method of Bradford (8), with bovine frequently by virtue of critically placed sulfhydryl groups that serum albumin as a standard. Datawere analyzed using the program react with heavy metals. One such protein may be the Ca2+- Graphpad (ISI) which utilizes Marquardt's method for nonlinear least stimulated, phospholipid-dependent protein kinase (protein squares analysis. Multiple equilibria of Caz+,M e , and a heavy metal kinase C) that is regulated by Ca", M P , diacylglycerol/ in the presence of a chelator were calculated by a computer program phorbol esters, and phospholipid (1).Recent reports suggest kindly supplied by Dr. Adrian Gear, Dept. of Biochemistry, University of Virginia. This program uses equilibrium constants described by that Cd2+ and Zn2+ can biphasically activateandinhibit Fabiato and Fabiato (9) for Ca2+and M$+ interacting with EGTA. protein kinase C activity (2, 3). These data are at variance For heavy metal-EGTA interactions, we used the association conwith our own findings that the direct effect of heavy metals stants summarized by Bartfai (10). is inhibition of protein kinase C. The disparate results are Materials-[y-32P]ATP (39 Ci/mmol) was from Amersham. Phosexplained by the presence or absence of cation chelators in phatidylserine was obtained from Avanti Polar Lipids. [3H]PDBu multiple equilibria with Ca2+ and aheavy metal, an explana- was obtained from Du Pont at a specific activity of 12.5 Ci/mmol. tion that is relevant in studies of the effects of heavy metals Unlabeled phorbol esters were obtained from LC Services. ATP, histone H1, 1,2-diolein, Triton X-100, and other reagent grade comon otherCa2+-sensitiveenzymes. In the case of protein kinase pounds were obtained from Sigma. ~
~
~~
+
~~
C, the differential sensitivity of phorbol ester binding and enzyme activity to heavy metals helps to define functional domains of the protein.
* This work was supported by National Science Foundation Grant DCB 8600086, American Heart Association Grant 87-1086, and National Institutes of Health Grant 17682. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.
RESULTSANDDISCUSSION
Complicating Effects of Chelators-When protein kinase C is isolated and assayed in the absence of chelators, heavy
metals inhibit its activity. Contrary to earlier reports (2, 3), the metals do not stimulateactivity. For instance, Zn2+inhibThe abbreviations used are: PDBu, phorbol 12,13-dibutyrate; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaceticacid.
5581
Heavy Metals ~nhibitProtein Kinase C
5582
its the rat brain enzyme with an IC60 -10 p~ (Table I). In the presence of chelator, the situation is more complex,In the experimental protocol (1mM EGTA (pH 7.4), summarized in Table I), increasing thetotaleaz+ in the system causes activation of protein kinase C. Once the chelator is largely saturated such that free Ca" becomes significant to the enzyme, a notable activation occurs when free Ca" approaches 1p ~In. the presence of 300 pM Zn2+,this activation occurs at lower ea2+, then changes to inhibition as Ca" continues to rise. This result gives the impression that the heavy metal has activated the enzyme at low [Ca2+],when, in fact, Zn2+,with its higher affinity for the chelator ( K A M, compared to Ka -10" M for ea2+),has replaced ea2+ on the chelator, increasing free Ca2+(see calculated free Ca2+in Table I). Thus, the addition of heavy metal has the same effect as lowering the chelator concentration. At higher concentrations of eaz+(0.7 to 1.5 mM), ea2+competes sufficiently well for the chelator to free up some Zn2+,which has its potent inhibitory effect. Theoretical calculations of the influence of Zn2+on ea2+EGTA interactions substantiate this interpretation of these data. In a system containing 1 mM EGTA in a buffer at pH 7.4 with 10 mM M P , the addition of ea2+will cause little increase in free ea2+until the chelator is largely saturated (Fig. lA), ie. near 1 mM ea2+. IfCa" is titrated into this system in thepresence of 300 p~ Zn2+,with its higher affinity for EGTA, free ea2+rises dramatically at lower total concentrations of added Ca", the buffering capacity of the system having been effectively reduced roughly 300 p~ by the addition of Zn2+.The addition of ea2+in the presence of Zn2+also leads to a shift from chelated to free Zn2+ as ea2+,with its lower affinity for EGTA, becomes a significant competitor for the chelator (-1 mM Ca2+,see inset for Fig. lA).Conversely, the titration of Zn2+into a Ca2+-EGTAsystem (600 p M total ea2+, 1 mM EGTA, see Fig. 1B)will cause no substantial increase in the concentration of free metal until the EGTA binding capacity is saturated(1mM or -600 pM ea2+ and 400 p~ ZnZ+).In therange 400 p~ C [Zn2+jtOtai < 700 p ~addition , of Zn2+will lead to liberation of ea2+virtually stoichiometrically, with little increase in free Zn2+,as Zn2+binds preferentially to EGTA. With further additionof Zn2+,free [Zn"] begins to rise due to saturation of EGTA's capacity to bind divalent cation. Some investigators report abiphasic effect of heavy metals on proteinkinase C activity, activation followed by inhibition ( 2 , 3), using protocols similar to these simulations (Fig. 1,A and B ) . In accordance with the simulations, their resultslikely reflect liberation of Ca" from ea2+-EGTA
by the heavy metals, with subsequent stimulation of this ea2+dependent protein kinase activity. The inhibitory effects of higher concentrations of heavy metals are presumably due to their direct effects on protein kinase C as chelatorcapacity is exceeded and significant concentrations of free heavy metal result (Fig. 1B). Likewise, the report that heavy metals increase the sensitivity of protein kinase C for activation by ea2+(1,2) is a contrived condition resulting from lossof ea2+buffering capacity in the presence of heavy metal (Fig. LA). Inhibitory Effects of Heavy Metals on Protein Kinase CHaving resolved real and artifactual effects of heavy metals on protein kinase C activity, we began to probe the direct effects of heavy metals in more detail. These studiesrequired obtaining soluble protein kinase C in the absence of chelator. However, when ea2+ is not chelated during homogenization of tissue, protein kinase C is largely membrane-associated. We have circumvented this problem by homogenizing cells in low ea2+without chelators, but in the presence of M e (30 mM). Under these conditions, the enzyme is largely cytosolic (similar to thedistribution of protein kinase C in the presence of EGTA). Thus, we find that Mg2+ counteracts the effect of ea2+ tocause association of protein kinase C with the membrane (11) and that cells and tissue fractionated with low Ca2+/high M$+ are good sources of chelator-free protein kinase C. When protein kinase C is purified in the absence of chelating agents, heavy metals do not stimulate protein kinase C activity; rather, they only inhibit protein kinase C activity (Fig. 2 A ) . The IC50values for heavy metal inhibition of protein kinase c activity are Cd2+= 3 p M , Zn2+-20 pM, cu2+-30 p ~and , Mn" < 300 p ~Heavy . metals also inhibit the binding of phorbol esters to protein kinase C (Fig. 2 B ) . Cd2+,H e , Zn2+,and Cu2+ inhibit[3H]PDBu binding to S49 cytosol with ICbovalues -10 p ~whereas , Mn2+does not inhibitbinding a t concentrationup to 100 pM. Interestingly, the values for the inhibition of [3H]PDBubinding are identical, in contrast to their inhibition of protein kinase C activity which readily discriminates between inhibition by Cd" and Cu2+. The inhibition of protein kinase activity seems unlikely to be an effect of heavy metals on substrate (ATP and histone) or othernon-kinase components of the reaction mixture, since the cyclic AMP-dependent protein kinase and protein kinase C, assayed under identical conditions, have differential sensitivities to inhibition by heavy metals (Fig. 3). Cd2+inhibits protein kinase C activity with an ICs0 = 3 pM, whereas it inhibits protein kinase A with an IC50 = 100 pM. Cu2+inhibits protein kinase C activity with an ICs0 = 30 pM, whereas it
TABLEI Influence of zn2+on protein kinase C activity in thepresence or absence of chelators The activity (pmol/min/mg protein) of protein kinase C partially purified from rat cerebrum was assessed as described under "Experimental Procedures" with the concentrations of added divalent cations and EGTA noted above. Con~entrationsof free cations were calculated as described under"ExperimentalProcedures." Data are the mean f range of duplicate experiments or from a single representative experiment (right-hand column). 1 m M EGTA, Ca2+, varied 0 Zn2+ varied Ca",
Free 2+
PM
0
3697
0.3
600 700 800 1500
300 pM Zn2+
Protein
Protein kinase C activity
Added Ca2+ PM
33580.0002 0.45 0.77 500
0 EGTA, 500 p M caz+, varied Znz+
1 m M EGTA,
PM
* 154 600 f 262 3530 f 320 5883 1534 7177 f 182 5.6
*
Zn2+ PM
Protein kinase C activity
kinase C activity
PM
PM
02915 0.0010.0003 1.o 6050 0.01 14 0.11 700 101 0.73 800 795 1500
Added Zn2+
f 229 3 I 530 4639 f 370 3202 f 30649 1149 f100 617
0
10
7385 6399 3823 536 287
C
Kinase Protein Inhibit Metals Heavy
A 300
3
cc
1
;;:L.' :ci 0.0 420
W W
1001
1.0
0.5
t o t o 1 added Ca"'.
+
2.0
1.5
mH
f 0 0.0
-
0.2
2
00 80
60 40 0
Zn"'
0
v
1.0
20
v)
(3
0.6
I
T
0L 1 20
1
0.8 total added EoZ+, mM 0.4
A
h
Zn"'
-
5583
'
n Y
-7
-6 -5 log [HEAVY METAL].
-4
-3
M
B,
1
20
00
"lol
I
0
E
8 200 Ca"
4 L
1001
0L-
I
/
I
/
I
/ f - 0.6
0.2
3111
n
m
'-
O
u+$
Zn"
f
60
I
Iv)
m ..
o -
"
I
0.4 0.8 1.0 1.2 total added Zn2+, mM FIG. 1. Free metal concentrations in an EGTA buffer. Con0.0
80
c,
centrations of free Ca2+and ZnZ+were calculated as described under "Methods" and in Refs. 9 and 10.A, main figure, influence of 300 p M Zn2+on free Ca2+ as Ca2+ is added to a buffer system containing EGTA (1mM), M$+ (10mM) (pH 7.4). Inset, effect of added Ca2+to elevate free [Zn2+]at constant total ZnZ+= 300 pM. E, influence of varying [Zn"] in a system consisting of 1 mM EGTA, 10 mM M$+, and 0.6 mM total Ca2+(pH 7.4).
inhibits protein kinase A with an IC60 = 100 pM. A further contrast are the effects of heavy metals to modulate the binding of regulatory ligands to protein kinase C and CAMPdependent protein kinase. Although the heavy metals Cd2+, Cu2+,and Zn2+inhibit [3H]PDBu binding to protein kinase C, these cationsdo not inhibit the binding of [3H]cyclicAMP to protein kinase A (data notshown). Cyclic AMP-dependent protein kinase is not immune to theinhibitory effect of heavy metals: HgZ+ inhibits the binding of [3H]cyclicAMP with an ICs0 of 3 p ~ From . these experiments, we conclude that our measurements reflect direct effects of heavy metals on the protein kinases, not on other components of the reaction mixtures, and that protein kinase C and A have different susceptibilities to heavy metals. Heavy metals frequently interact with sulfhydryl moieties on proteins. Protein kinase C contains anumber of cysteinerich regions in both its regulatory and catalyticdomains (12). The sulfhydryl reagents @-mercaptoethanol and penicillamine both protect againstheavy metal inhibition of protein kinase C (Table11).This is consistent with heavy metals' interacting with a cysteine residue in proteinkinase C which is in a region that influences protein kinase C activity. Since protein kinase C is regulated by Caz+,M P , diacylglycerol/phorbol esters, and phospholipid, there are a number of potential sites at
0'
C
L
I
., -7
-6 -5 -4 -3 log [ H E A V MY E T A L I , M FIG. 2. Effects of heavy metals on protein kinaseC. Symbols: 0, Cu2+;0, Cd"; Zn2+;0, MnZ+;A, H e . A, activity of protein kinase C. Metals were included in standard reaction mixtures (see "Experimental Procedures"), and kinase assays were initiated by the addition of -5 pg of cytosol of S49 lymphoma cells prepared with 30 mM M e (and without chelator). Maximal activity averaged 580 pmol/min/mg of protein.Data are mean f S.E. of two to four independent experiments. B, binding of [3H]phorbol&butyrate. Metals were included in standard binding assay reactions (see "Experimental Procedures") to which wereadded -100 fig of S49 cell cytosol (prepared as for A ) .Data are mean f S.E. of two to four experiments. Maximal binding under these conditions averaged 4.7 pmol/mg of protein.
which heavy metals can interact to inhibit activity of this enzyme and its interactionwith modulators. We have tried to use these regulatory ligands systematically to determine whether they afford protection from the effects of heavy metals and whether particular functional regions of protein kinase C may be implicated in theactions of heavy metals. The inhibition of protein kinase C activity by heavy metals cannot be overcome by increasing concentrations of Caz+or M$+. For instance, the effect of 30 p~ Cu2+ (sufficient to reduce enzyme activity by 50%) is the same at 1 pM Ca2+(260 pmol/min/mg) and at 500 pM Ca2+(230 pmol/min/mg). Similarly, the stimulation of protein kinase C activity by M P is reduced by Cu2+in a manner that excess M$+ cannot surmount (data not shown). Experiments with partially purified protein kinase C from rat brain confirm these impressions: the IC50 for inhibition of enzyme activity by Zn2+(10.7 f 0.7 p ~ mean , & S.E., N = 3) is invariant with [Ca"] over the . ability of heavy metals to inhibit range 1p~ to 500 p ~ The binding of [3H]PDBu to protein kinase C is also noncompetitive with respect to phorbol ester (Fig. 4). Scatchard analysis
Heavy Metals Inhibit Protein Kinase C
PKA 300/
0
I
-6
-5
-4
-3
log [HEAVY METAL 2+1. M FIG.3. Differentialinhibition of protein kinase C and CAMP-dependent protein kinaseby heavy metals. Metals were included instandardproteinkinasereactions(see"Experimental Procedures") that were initiated by the additionof -5 pg of chelatorfree cytosol from S49 lymphoma cells, or -1 pg of partially purified CAMP-dependent protein kinase from rabbit skeletal muscle. Maximal activities were 560 pmol/min/mg of protein for protein kinaseC and 5600 pmol/min/mg of protein for CAMP-dependent protein kinase. Data are mean of duplicate experiments. Symbols: closed, protein kinase C; open, CAMP-dependent protein kinase; 0, 0, Cd"; 0, B. CU2+.
-5
-6
-4
-3
log [PHOSPHATIOYLSERINEI. gm/ml
\
T3
c
3 0
n
TABLE I1 Protection of protein kinase C from effects of heavy metals Data are protein kinaseC activities of S49 cell extracts, expressed as a percentage of the maximal (control) activity, which varied among the three experiments from 304 to 506 pmol/min/mg. Mercaptoethanol and penicillaminewere both used a t 1 mM. Penicillamine alone, a t 10 mM, was capable of fully inhibiting protein kinase C activity. Control Penicillamine
0
cu2+,100 p M Zn2+,30 p M
H e , 30 p M Cd2+,1 pM Cd2+,10 p M Cd2+,100 p M
100
78
2 29 5 74 8 0
116 106 92 74 61
90 71
k?-
-
0.0 0
1
2
3
a I
I
m
ln
e
n
I
L
W
-
-6
-5
-4
-3
FIG.5. Effects of phospholipid on Cu2+inhibition of protein kinase C activity and ['HIPDBu binding. A, concentration dependence for phosphatidylserine activation of protein kinase C from S49 cytosol (5 pg), in the absence(0)or presenceof 10 p M (0)or 30 p~ (B) Cu2+. B, concentrationdependence for phosphatidylserine enhanced bindingof [3H]PDBu toS49 cytosol (100pg), in the absence (0)or presence (0)or 10 p~ Cu".
Mercaptoethanol
1.2
1.0
0
log [PHOSPHATIDYLSERINEI. gm/ml
Additions Cation
m
4
5
BOUND (pmoles PDB)
FIG.4. Scatchard analysis of ['HIPDBu binding to 549 cytosol in the presence (0)or absence (0) of 10 PM Cu2+. The binding assay was conducted as described under "Experimental Procedures" with [3H]PDBu varying between 0.1 nM and 60 nM. In the representative experimentshown, the concentrationof phosphatidylserine was 30 pglml. The same results were achieved with phosphatidylserine at 300 pg/ml.
of [3H]PDBu binding to protein kinase C demonstrates that 10 p~ Cu2' inhibits the maximal extent of binding by 50% without any effect on theK d of [3H]PDBu binding to protein kinase c ( K d -3 nM). These data suggest that the divalent cation and diacylglycerol sites are not those susceptible to heavy metals. However, the phospholipid dependence for protein kinase C activationisaltered by Cu2+(Fig. 5 A ) , which inhibits phospholipid-dependent activity. Increasingconcentrations of phosphatidylserine overcome this effect of Cuz+. In the absence of Cu2+,the for phosphatidylserine activation of protein kinase C is 3 pg/ml. In thepresence of 10 p M cuz+, the ECSO forphosphatidylserine activation is increased to 30 pg/ml. As Fig. 5A indicates, protein kinase C is most susceptible to inhibition by heavy metals in the relative absence of phospholipid. In the absence of phospholipid, protein kinase C can be stimulated by fatty acids (13, 14);this stimulation, too, can be inhibited by Zn2+ (when proteinkinase C is stimulated by 300 PM oleic acid, the IC50 for Zn2+ is 8 pM). Thus, the phospholipid environment of protein kinase C can markedly influence its susceptibility to inhibition by heavy metals. The data further suggest that cellular protein kinase C, if activated by fatty acids released by the actions of phos-
Kinase Protein Inhibit Metals Heavy pholipases, might be especially susceptible to this sort of inhibition. In distinctionto itssurmountable effects on enzyme activity with respect to phospholipid, Cuz+ inhibits the binding of [3H]PDBubinding to protein kinase C noncompetitiuely with respect to phospholipid (Fig. 5B). Due to thegreater sensitivity of the activity assay compared to the binding assay, 20 times more protein is needed in the binding reaction. Thus, quantitative comparison between kinase and binding data should be made cautiously. Taken together, the data of Fig. 5A and B suggest that there aremultiple sites at which heavy metals interact with protein kinase C. In summary, the primary influence of heavy metals on the functioning of protein kinase C is inhibition. Examination of protein kinase C activity in the absence of chelators makes this point clearly and distinguishes this direct effect from effects of heavy metals to alter equilibria among EGTA and divalent cations in the reaction mixture. Knowledge that these metals do not act like tumor promoters and do not activate protein kinase C may beof importance in comprehending their toxic effects and their effects on enzyme induction. Our current experiments are directed toward assessing the influence of heavy metals on the functions of protein kinase C and CAMP-dependent protein kinase in the intact cell. From the current data, we predict that cellular responses mediated via products of phospholipases C and Az and by CAMP will be
C
5585
markedly attenuated by heavy metals. Acknowledgments-We appreciate the technical assistance of Mitchell Shultz and the secretarial assistance of Susan George. REFERENCES 1. Nishizuka, Y. (1986) Science 233,305-312 2. Mazzei, G. J., Girard, P. R., and Kuo, J. F. (1984) FEBS. Lett. 173,124-128 3. Murakami, K., Whiteley, M. K., and Routtenberg, A. (1987) J. Biol. Chem. 262,13902-13906 4. Kanter, J. R., and Brunton, L.L. (1981) J. Cyclic Nucleotide Res. 7,259-268 5. Bell, J. D., Buxton, I. L. O., and Brunton, L. (1985) L. J. Biol. Chem. 260,2625-2628 6. Corbin, J. D., and Reimann, E. M. (1974) Methods Enzymol. 38, 287-290 7. Beavo, J. A,, Bechtel, P. J., and Krebs, E. G. (1974) Methods Enzymol. 38, 299-308 8. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 9. Fabiato, A., and Fabiato, F. (1979) J. Physiol. 75,463-505 10. Bartfai, T. (1979) Adu. Cyclic Nucleotide Res. 10, 219-242 11. Brunton, L. L., Watson, M. J., Shultz, M., Trejo, J., and Speizer, L.A. (1987) Fed. Proc. 46, 2066 12. Parker, P. J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M. D., and Ullrich, A. (1986) Science 233,853-859 13. McPhail, L. C., Clayton, C. C., and Snyderman, R. (1984) Science 224,622-625 14. Speizer, L.A., Watson, M. J., and Brunton, L. L. (1988) FASEB J. 2, A595
