©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Nitric Oxide-stimulated Guanine Nucleotide Exchange on p21(*)

(Received for publication, December 30, 1994; and in revised form, February 1, 1995)

Harry M. Lander (1)(§) Jason S. Ogiste (1) S. Frieda A. Pearce (2) Roberto Levi (1) Abraham Novogrodsky (3) (4)

From the  (1)Department of Pharmacology and (2)Medicine, Division of Hematology/Oncology, Cornell University Medical College, New York, New York 10021, the (3)Felsenstein Medical Research Center, Beilinson Campus, Petah-Tikva 49100, Israel, and the (4)Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The protooncogene p21, a monomeric G protein family member, plays a critical role in converting extracellular signals into intracellular biochemical events. Here, we report that nitric oxide (NO) activates p21 in human T cells as evidenced by an increase in GTP-bound p21. In vitro studies using pure recombinant p21 demonstrate that the activation is direct and reversible. Circular dichroism analysis reveals that NO induces a profound conformational change in p21 in association with GDP/GTP exchange. The mechanism of activation is due to S-nitrosylation of a critical cysteine residue which stimulates guanine nucleotide exchange. Furthermore, we demonstrate that p21 is essential for NO-induced downstream signaling, such as NF-kappaB activation, and that endogenous NO can activate p21 in the same cell. These studies identify p21 as a target of NO in T cells and suggest that NO activates p21 by an action which mimics that of guanine nucleotide exchange factors.


INTRODUCTION

Reactive oxygen species are known to mediate signal transduction events in lymphocytes (1, 2, 3) and have recently been implicated in the signaling process of a wide range of stimuli(4) . The reactive nitrogen species, nitric oxide (NO), (^1)plays critical roles in many diverse biological processes such as vasoregulation, host defense and synaptic plasticity(5, 6, 7, 8) . Recently, we identified a role for NO as a positive signaling molecule in the immune system(9, 10) . The protooncogene p21 has been identified as a key molecular switch involved in regulating T cell activation triggered by various mitogens(11, 12, 13) . Therefore, we hypothesized that the immune stimulatory properties of NO may be mediated through p21, and thus we examined whether NO could activate p21 in human T cells.


EXPERIMENTAL PROCEDURES

Materials

Nitric oxide and carbon monoxide gas were purchased from Matheson Gas, East Rutherford, NJ; [-P]GTP and PO(4) were from Amersham Corp.; p21 antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA; and all other biochemicals were from Sigma. Purified, recombinant p21 was kindly provided by Dr. Daniel Manor, Department of Pharmacology, Cornell University, Ithaca, NY.

Cell Culture

The human T cell line, Jurkat, was maintained in RPMI 1640 containing 10% heat-inactivated fetal calf serum in a 37 °C incubator with 5% CO(2). Human umbilical vein endothelial cells (HUVEC) were kindly provided by Dr. Steven S. Gross, Department of Pharmacology, Cornell University Medical College, New York, NY and PC12 mutant cells by Dr. G. Cooper, Dana-Farber Cancer Institute, Boston, MA.

Quantitation of p21-associated Nucleotides

The assay used to measure the GTP/GDP ratio on immunoprecipitated p21 was essentially that of Downward et al.(11) . Jurkat T cells (4 times 10^6 cells/ml) were labeled in phosphate-free RPMI 1640 containing 200 µCi/ml PO(4) for 16 h. NO, prepared as described previously(10) , was added directly to 1 ml of sample for the indicated time after which cells were analyzed for the percentage of GTP-bound p21. Anti-p21-agarose (clone Y13-259, Santa Cruz Biotechnology) was added for immunoprecipitation. After elution of guanine nucleotides, 5 µl of sample was spotted onto polyethyleneimine TLC plates, run for 3 h in 0.75 M KH(2)PO(4) (pH 3.4) and exposed to phosphorimaging screens overnight. Spots migrating with the same R(f) as GDP or GTP standards were quantified using a PhosphorImager (Molecular Dynamics). The percent of GTP bound to p21 was calculated using the following formula which takes into account the extra phosphate on GTP as compared with GDP: ( times GTP)/(GDP + times GTP). Control samples had non-immune IgG + protein A-agarose added rather than anti-p21, and no GDP or GTP spots were evident.

GTPase Assay

The GTPase assay was performed exactly as we previously described(10) . Carbon monoxide was prepared as a saturated solution with a concentration of 1 mM(14) , in a similar manner as NO solutions(10) .

[^3H]GDP Release Assay

p21 (1 µM) was incubated for 30 min at 30 °C in 200 µl of GDP binding buffer (20 mM Tris, pH 7.4, 1 mM MgCl(2), 1 mM dithiothreitol, 100 mM NaCl, 0.1 mg/ml BSA, and 100 pmol of [^3H]GDP (10 Ci/mmol). Then, 20 µl of 5 mM GTP was added with any test reagents. After incubation for the required time at 30 °C, 40-µl aliquots were removed and bound [^3H]GDP was separated from free by washing with 20 mM Tris, pH 8.0, 100 mM NaCl, and 25 mM MgCl(2) on nitrocellulose filters. The filters were dried and counted in a liquid scintillation counter.

-S-GTP Binding Assay

The -S-GTP binding assay was performed exactly as described(15) , except that 100 nM -S-GTP was added to modify the specific activity of radiolabeled probe.

Assay of Nitrosothiol Content

Samples of p21 were exposed to NO (50 µM) for 10 min prior to analysis of nitrosothiol content by a colorometric method(16) . Samples containing either NO, p21, or IgG alone had no detectable nitrosothiol. S-Nitroso-N-acetylpenicillamine (10-1000 µM) was used as a standard.

Circular Dichroism Spectral Analysis

Circular dichroism spectral analysis was performed at 22 °C in 0.1-mm cuvettes in a Jasco 710 CD spectrophotometer. Samples were dissolved in 50 mM Tris buffer, pH 7.4, and measured with a 0.2-nm bandwidth, a 0.2 nm step scan, and varying scan speeds of 60-200 nm/min. NO (50 µM) was added to p21 (17 µM), and spectral characteristics measured over varying time periods indicated that p21 and the p21-NO complex remained stable for the duration of the data collection period. The spectra were analyzed using the LINCOMB program (Dr. G. D. Fasman, Brandeis University, Waltham, MA).

NF-kappaB Gel Shift Assay

Nuclei from PC12 cells were isolated and assayed for NF-kappaB binding activity as we described previously(9, 10) .


RESULTS AND DISCUSSION

Effects of NO on p21Enzymology

In its resting state, p21 has GDP bound. Upon in vivo activation, p21 releases GDP and binds GTP (17, 18, 19, 20) . To test if NO activates p21, we measured the ratio of GTP/GDP on p21 in Jurkat cells (a human T cell line) in which the nucleotide pools were labeled with PO(4). As seen in Fig. 1A, immunoprecipitation of p21 reveals a substantial increase in the percentage of GTP bound upon NO treatment. The curve is biphasic with a maximal effect at 10 nM, although higher concentrations of NO yielded levels of GTP-p21 that were above base line values. The kinetics of p21 activation (Fig. 1B) is of short duration and more similar to that seen upon growth factor receptor stimulation of neural cells than that of T cells(11, 21, 22) . The relatively short duration of the NO-induced signal may be due to the interaction of activated p21 with its effector and/or GTPase-activating proteins. Alternatively, this stimulation may be transient and therefore not lead to a fully activated phenotype.


Figure 1: Effect of NO on p21 activation in Jurkat T cells. The human Jurkat T cell line was stimulated with various amounts of NO for 10 min (A) or with NO (0.01 µM) for various times (B). At the appropriate times, cells were washed, p21 was immunoprecipitated, and the bound nucleotides analyzed. Data are expressed as the percentage of GTP-bound p21 of control. Control samples had a base line value of 12.4 ± 3.6% GTP bound. Data represent the mean and standard deviation from four to six experiments.



The activating effect of NO could be mediated either by direct interaction with p21 or indirectly through additional factors. Therefore, we mixed NO with pure recombinant p21in vitro and measured its intrinsic GTPase activity. Surprisingly, NO enhanced the GTPase activity of p21in vitro, indicating a direct effect (Fig. 2A). Co-incubation with catalase (1000 unitsml) or superoxide dismutase (30 units/ml) had no effect on the /ability of NO to enhance p21 GTPase activity (data not shown), indicating that the activating species is not hydroxyl radical, superoxide, or peroxynitrite. The amount of NO which gives a maximal response (75 µM) in vitro is much higher than that required in whole cells (0.01 µM, Fig. 1A). We have observed previously a similar differential sensitivity in leukocyte membranes treated with NO before or after isolation using a membrane GTPase assay(10) . This phenomenon may be due to a cellular component which makes NO more potent and is lacking in purified preparations. These are likely to be oxidizing conditions which favor formation of nitrosonium ion (NO) and facilitate nitrosothiol formation(23, 24, 25) . In contrast to NO, carbon monoxide, another gas claimed to be a signaling molecule in the brain (26, 27) , had no effect on p21 activity (Fig. 2A).


Figure 2: Effect of NO and carbon monoxide on p21 enzymatic activity in vitro. Pure recombinant p21 was incubated with various concentrations of NO or CO for 10 min (A) after which p21 GTPase activity was measured. p21 preloaded with [^3H]GDP, or GDP-loaded p21 mixed with -S-GTP, was treated with various amounts of NO for 10 min at 37 °C followed by assay of bound [^3H]GDP or -S-GTP (B). Data represent the mean and standard deviation from three to seven experiments.



The apparent increase in GTPase activity could be due to either: 1) an increase in GDP release from p21 (the rate-limiting step in the catalytic cycle) or 2) an increase in the intrinsic GTPase activity of p21. In vivo, GDP release is controlled by guanine nucleotide exchange factors (GEF), and represents activation, whereas GTP hydrolysis is controlled by GTPase-activating proteins and represents inactivation(17, 18, 19, 20) . To distinguish between these possibilities, we preloaded p21 with [^3H]GDP and tested whether NO increased GDP release. As seen in Fig. 2B, concentrations of NO which enhance GTPase activity (Fig. 2A) induced GDP release from p21. Furthermore, at concentrations of NO where [^3H]GDP was released, -S-GTP was bound (Fig. 2B). These data support our findings of an increase in the GTP/GDP ratio on p21 in whole cells (Fig. 1A) and verify the concept that NO is an activator of p21 by promoting guanine nucleotide exchange.

Studies on Mechanism

Hemoglobin (Hb) has a high affinity for NO and can thus prevent most of its biological actions by complexing with it(23, 28) . As expected, we found that Hb prevented NO from activating p21 when added prior to NO (Fig. 3A). Interestingly, when Hb was added 10 min after NO addition, p21 activation was reversed whereas Hb by itself had no effect on activity (Fig. 3A). A reversible activation of p21 by NO implies that either NO is acting allosterically or that it reversibly and covalently modifies an enzyme site. Of note, nitrosothiols are known to be reversibly nitrosated in the presence of Hb(23, 28) .


Figure 3: Effect of hemoglobin and the influence of blocking antibodies on p21 activity and nitrosothiol formation. Hb (0.4 mM) was added before or 10 min after NO (100 µM) addition (A). In antibody blocking experiments, 10 µg of the indicated antibody was incubated with 1 µM p21 for 30 min at 22 °C followed by NO (50 µM) addition and GTPase assay or nitrosothiol assay. Data represent the mean and standard deviation of three experiments. The asterisk denotes no detectable nitrosothiols (i.e. less than 0.2 mol/mol).



Release of GDP is effected by GEF's which bind to regions on p21 that can be blocked by the monoclonal antibody Y13-259 (29, 30) . To determine if NO induces GDP release in a manner similar to GEF's, we examined whether preincubation of p21 with Y13-259 prevents NO activation. As seen in Fig. 3B, antibody Y13-259 substantially prevented the ability of NO to activate p21. In contrast, monoclonal antibody Y13-238, which binds to a different region on the molecule(31) , had no effect (Fig. 3B). These data suggest that NO induces GDP release by interacting with a region of p21 which normally interacts with GEF's(32, 33) .

Because p21 does not contain a heme group, and Hb reversed the activation by NO, we examined the possibility that the mechanism of activation was via formation of a nitrosothiol (Fig. 3B). We found that NO induced the formation of only one nitrosothiol on p21, even though p21 contains 5 Cys residues. Importantly, the antibody Y13-259, which prevented activation of p21 by NO, also prevented nitrosothiol formation, whereas the control antibody Y13-238, which did not prevent activation, also did not prevent nitrosothiol formation (Fig. 3B). Furthermore, Hg, a known thiol-reactive reagent, enhanced p21 GTPase activity (data not shown). These data strongly suggest that formation of a nitrosothiol on p21 by NO is responsible for activation.

To examine if NO causes a conformational alteration in p21, we used circular dichroism (CD) spectral analysis to assess secondary structure. Treatment of p21 with NO led to a dramatic change in its CD spectral properties, and computer analysis revealed a profound reduction in alpha-helical content (from 60 to 36%) and a concomitant increase in beta-sheet content (from 18 to 44%). The basal spectra we obtained for untreated p21 were identical to that published previously(34) . These data suggest that NO induces a conformational change in p21 concomitant upon nitrosothiol formation, leading to enhanced nucleotide exchange.

Downstream Signaling

Previous data from our laboratory have broadened the spectrum of NO action to include an immune stimulatory component(9, 10) . In those studies a G protein inhibitor, GDPbetaS, blocked NO-induced NF-kappaB transcription factor activation in human lymphocytes. Although those findings implicated G proteins as transducers of NO signals, exactly which G proteins were involved was unclear. Here, we examined whether NO could activate NF-kappaB in cells harboring a mutant, inactive p21(35) . As seen in Fig. 4, upper panel, wild type parental cells responded to sodium nitroprusside by translocating NF-kappaB to the nucleus. In contrast, the p21-defective cells did not respond to sodium nitroprusside. Thus, p21 is necessary for NO-induced cellular activation signals. Others have found that PC12 cells expressing a dominant negative mutation in p21 no longer respond to tumor necrosis factor-alpha and UV light by activating NF-kappaB(36) . These free radical generators (4) were proposed to act upstream of p21. Our data suggest that reactive nitrogen species, and therefore possibly reactive oxygen species, directly activate p21, leading to subsequent downstream events such as NF-kappaB translocation.


Figure 4: Role of p21 in NO signaling. Upper panel, parental PC12 wild type (wt) or ras-negative (mt) cells were serum-starved overnight and then treated with the indicated concentration of sodium nitroprusside (SNP), an NO-generating compound, for 4 h followed by nucleus isolation and assay for NF-kappaB binding activity. The arrow denotes migration of the NF-kappaB/DNA complex. Lower panel, HUVECs were either untreated or treated 16 h with lipopolysaccharide (LPS, 30 µg/ml) and interferon- (IFN-, 50 ng/ml) or N-methyl-L-arginine (NMA) (3 mM) in phosphate-free RPMI 1640 containing 100 µCi/ml PO(4). Then, indicated wells were treated with L-arginine (ARG, 10 mM) for 15 min and samples assayed for GTP-bound p21.



The physiological relevance of our findings are strengthened by our demonstration that endogenous NO activates p21 (Fig. 4, lower panel). We induced nitric oxide synthesis in HUVEC with LPS and IFN-(37) . We found that in the presence of the nitric oxide synthase inhibitor N-methyl-L-arginine, providing a pulse of the substrate, L-arginine (to avoid the desensitization effect found in Fig. 1B), led to recovery of an activated form of p21 (Fig. 4, lower panel). Thus, an autocrine loop may exist within cells, whereby endogenous NO activates cellular p21, leading to cell activation and nitric oxide synthase transcriptional regulation. NO is a highly reactive gas which can be generated in high local concentrations at sites of inflammation. Thus, our findings suggest that activation of p21 by NO is likely to be a major mechanism of amplification of leukocyte-induced local tissue damage.

The present findings identify p21 as a target of NO in T cells. We propose that NO, via formation of a nitrosothiol, leads to a conformational change which enhances guanine nucleotideexchange, thus mimicking endogenous GEF's. The ability of NO to activate p21, a non-heme enzyme, represents a novel signal transduction pathway. Furthermore, because nitrosothiol formation on a critical cysteine is apparently responsible for its activation, p21 may be ideally suited to transduce other oxidative stress signals from both endogenous and exogenous sources in many cell types. In fact, others have found that redox agents modulate binding of GTP to p21 immunoprecipitates(38) . Furthermore, the activation of p21 or other monomeric G proteins by NO may not be restricted to T cells and conceivably could mediate many signals transmitted by NO, such as Ca-independent vesicle exocytosis (39) and synaptic plasticity in the brain(8, 40) . Heterotrimeric G proteins may also be a target of NO as the G subunits are highly homologous to p21(18) . For example G proteins are known to mediate olfaction, and NO has recently been implicated in this sytem(41) .


FOOTNOTES

*
This work was supported by United States Public Health Service Grants HL34215, HL46403, and HL07423. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Cornell University Medical College, New York, NY 10021. Tel.: 212-746-6462; Fax: 212-746-8789.

(^1)
The abbreviations used are: NO, nitric oxide; HUVEC, human umbilical vein endothelial cells; GEF, guanine nucleotide exchange factors.


ACKNOWLEDGEMENTS

We thank Dr. D. Manor, Cornell University, Ithaca, NY for generously providing the purified recombinant p21; Dr. G. Cooper, Dana-Farber Cancer Institute, Boston, MA, for the mutant PC12 cell line; and Dr. Steven S. Gross for reviewing the manuscript.


REFERENCES

  1. Novogrodsky, A., Ravid, A., Rubin, A. L., and Stenzel, K. H. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1171-1174 [Abstract]
  2. Whitacre, C. M., and Cathcart, M. K. (1992) Cell Immunol. 144, 287-295 [Medline] [Order article via Infotrieve]
  3. Israel, N., Gougerot-Pocidalo, M.-A., Aillet, F., and Verelizier, J.-L. (1992) J. Immunol. 149, 3386-3393 [Abstract/Free Full Text]
  4. Schreck, R., Rieber, P., and Baeuerle, P. A. (1991) EMBO J. 10, 2247-2258 [Abstract]
  5. Nathan, C. (1992) FASEB J. 6, 3051-3064 [Abstract/Free Full Text]
  6. Dinerman, J. L., Lowenstein, C. J., and Snyder, S. H. (1993) Circ. Res. 73, 217-222 [Medline] [Order article via Infotrieve]
  7. Karupiah, G., Xie, Q.-W., Buller, M. L., Nathan, C., Duarte, C., and MacMicking, J. D. (1993) Science 261, 1445-1448 [Medline] [Order article via Infotrieve]
  8. Schuman, E. M., and Madison, D. V. (1991) Science 254, 1503-1506 [Medline] [Order article via Infotrieve]
  9. Lander, H. M., Sehajpal, P., Levine, D. M., and Novogrodsky, A. (1993) J. Immunol. 150, 1509-1516 [Abstract/Free Full Text]
  10. Lander, H. M., Sehajpal, P. K., and Novogrodsky, A. (1993) J. Immunol. 151, 7182-7187 [Abstract/Free Full Text]
  11. Downward, J., Graves, J. D., Warne, P. H., Rayter, S., and Cantrell, D. (1990) Nature 346, 719-723 [CrossRef][Medline] [Order article via Infotrieve]
  12. Graves, J. D., Downward, J., Rayter, S., Warne, P., Tutt, A. L., and Cantrell, D. A. (1991) J. Immunol. 146, 3709-3712 [Abstract/Free Full Text]
  13. Downward, J., Graves, J., and Cantrell, D. (1992) Immunol. Today 13, 89-92 [Medline] [Order article via Infotrieve]
  14. Budavari, S. (1989) The Merck Index, 11th Ed., p. 275, Merck & Co. Inc., Rahway, NJ
  15. Carty, D. J., and Iyengar, R. (1994) Methods Enzymol. 237, 38-44 [Medline] [Order article via Infotrieve]
  16. Saville, B. (1958) Analyst (Lond.) 83, 670-672
  17. Satoh, T., Nakafuku, M., and Kaziro, Y. (1992) J. Biol. Chem. 267, 24149-24152 [Free Full Text]
  18. Bokoch, G. M., and Der, C. J. (1993) FASEB J. 7, 750-759 [Abstract/Free Full Text]
  19. Bourne, H., Sanders, D. A., and McCormick, F. (1991) Nature 349, 117-127 [CrossRef][Medline] [Order article via Infotrieve]
  20. Boguski, M. S., and McCormick, F. (1993) Nature 366, 643-654 [CrossRef][Medline] [Order article via Infotrieve]
  21. Muroya, K., Hattori, S., and Nakamura, S. (1992) Oncogene 7, 277-281 [Medline] [Order article via Infotrieve]
  22. Li, B.-Q., Kaplan, D., Kung, H.-F., and Kamata, T. (1992) Science 256, 1456-1459 [Medline] [Order article via Infotrieve]
  23. Stamler, J. S., Singel, D. J., and Loscalzo, J. (1992) Science 258, 1898-1902 [Medline] [Order article via Infotrieve]
  24. Stamler, J. S., Simon, D. I., Osborne, J. A., Mullins, M. E., Jaraki, O., Michel, T., Singel, D. J., and Loscalzo, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 444-448 [Abstract]
  25. Lipton, S. A., Choi, Y.-B., Pan, Z.-H., Lei, S. Z., Chen, H.-S. V., Sucher, N. J., Loscalzo, J., Singel, D. J., and Stamler, J. S. (1993) Nature 364, 626-632 [CrossRef][Medline] [Order article via Infotrieve]
  26. Zhuo, M., Small, S. A., Kandel, E. R., and Hawkins, R. D. (1993) Science 260, 1946-1950 [Medline] [Order article via Infotrieve]
  27. Verma, A., Hirsch, D. J., Glatt, C. E., Ronnett, G. V., and Snyder, S. H. (1993) Science 259, 381-384 [Medline] [Order article via Infotrieve]
  28. Stamler, J. S. (1994) Cell 78, 931-936 [Medline] [Order article via Infotrieve]
  29. Sigal, I. S., Gibbs, J. B., D'Alonzo, J. S., and Skolnick, E. M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4725-4729 [Abstract]
  30. Hattori, S., Clanton, D. J., Satch, T., Nakumura, S., Kaziro, Y., Kawakita, M., and Shih, T. Y. (1987) Mol. Cell. Biol. 7, 1999-2002 [Medline] [Order article via Infotrieve]
  31. Furth, M. E., Davis, L. J., Fleurdelys, B., and Skolnick, E. M. (1982) J. Virol. 43, 294-304 [Medline] [Order article via Infotrieve]
  32. Quilliam, L., Kato, K., Rabun, K. M., Hisaka, M. M., Huff, S. Y., Campbell-Burk, S., and Der, C. J. (1994) Mol. Cell. Biol. 14, 1113-1121 [Abstract]
  33. Mosteller, R. D., Han, J., and Broek, D. (1994) Mol. Cell. Biol. 14, 1104-1112 [Abstract]
  34. Pingoud, A., Wehrmann, M., Pieper, U., Gast, F.-U., Urbanke, C., Alves, J., Feuerstein, J., and Wittinghofer, A. (1988) Biochemistry 27, 4735-4740 [Medline] [Order article via Infotrieve]
  35. Szeberenyi, J., Cai, H., and Cooper, G. M. (1990) Mol. Cell. Biol. 10, 5324-5332 [Medline] [Order article via Infotrieve]
  36. Devary, Y., Rosette, C., DiDonato, J. A., and Karin, M. (1993) Science 261, 1442-1445 [Medline] [Order article via Infotrieve]
  37. Rosenkranz-Weiss, P., Sessa, W. C., Milstien, S., Kaufman, S., Watson, C. A., and Pober, J. S. (1994) J. Clin. Invest. 93, 2236-2243 [Medline] [Order article via Infotrieve]
  38. Wilkinson, F. E., Paulik, M., and Morre, D. J. (1993) Biochem. Biophys. Res. Commun. 190, 229-235 [CrossRef][Medline] [Order article via Infotrieve]
  39. Meffert, M. K., Premack, B. A., and Schulman, H. (1994) Neuron 12, 1235-1244 [Medline] [Order article via Infotrieve]
  40. Schmidt, H. H. H. W., and Walter, U. (1994) Cell 78, 919-925 [Medline] [Order article via Infotrieve]
  41. Gelperin, A. (1994) Nature 369, 61-63 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.