the case of antibodies, by contrast, the
structural independence of V and C domains
(28), together with the much larger
separation expected between the V domains
and putative signal-transducing molecules
on the cell surface (30), effectively
rules out an allosteric mechanism to account
for B cell activation. In this respect, the
large protruding loop on the extemal face of
the C domain (and possibly of the C,
domain), comprising residues 219 to 232
(Fig. 1), could be particularly important in
contacting the extracellular portions ofCD3
molecules, thereby helping to couple the
antigen recognition and signal transduction
functions of the TCR-CD3 complex.
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Adv. Immunol. 54, 99 (1993).
5. S. Weber, A. Traunecker, F. Oliveri, W. Gerhard, K.
Karjalainen, Nature 356, 793 (1992).
6. We initially obtained crystals of a glycosylated form of
the 14.3.d 13 chain (26). As these diffracted only to
medium resolution, however, site-directed mutagenesis
was used to eliminate four out of the five potential
NH2-linked glycosylation sites. Asparagines at
positions 24, 74, and 121 were mutated to glutamine,
and Ser283, which is COOH-terminal to
Asn236, was mutated to valine. The prototype vector
containing immunoglobulin locus elements (31) was
used to drive expression ofthe mutated complementary
DNA corresponding to the rearranged V and
the first C region exon of the TCR P3 gene. Soluble a
chain was produced in myeloma cells and purified
from culture supernatants ( 10 mg/mI) by a single
affinity chromatography step with the C. monoclonal
antibody H57-597 (32). The unmutated NH2-linked
glycosylation site at position 186 was used only in a
few of the chains (<10%), as judged by SDS-polyacrylamide
gel electrophoresis. A homogeneous
product was obtained by treatment ofthe native protein
with neuraminidase and glycosidases.
7. Protein solution [5.8 mg/mI in 7.3 mM tris, 25 mM
Hepes (pH 7.5), 18.3 mM NaCI, 1.0% (w/v) polyethylene
glycol (PEG) 4000, 0.75% (saturated) ammonium
sulfate, and 0.01 4% agarose] was used to form a
sitting drop, which was equilibrated by vapor diffusion
over a buffer reservoir of higher concentration
(4% PEG 4000, 0.1 M Hepes, and 3% ammonium
sulfate at pH 7.25). Crystals (0.4 mm by 0.4 mm by
0.1 mm) usually grew within a period of about 3
weeks. They belong to the space group C2 with unit
cell parameters a = 100.6 A, b = 36.6 A, c = 71.5 A,
and 3= 1 13.40.
8. A data set was collected with a MARresearch (Hamburg,
Germany) imaging plate system mounted on the
wiggler line DW32 at the synchrotron at LURE (Laboratory
for the Utilization of Electromagnetic Radiation),
Orsay, France. A total of 60,827 reflections were obtained
from a single crystal to yield a set of 25,371
unique observations (Rs = 0.049), which was
95.6% complete at a resoution between 15.0 and 1.7
A. (For the outer resolution shell, 1.76-1.70 A, Rsym
was 0.399 with 95.1 % completeness and 34% having
I > 3c.) A preliminary model for the ,B chain was
obtained by molecular replacement with the program
AMoRe (33) with VL from HyHEL-1 0 (12) and CH1 as
search models. The structure was refined with the
program X-PLOR (34) during the preliminary stages,
then with the CCP4 (35) version of PROLSQ (36) for
the final steps. The R factor of the refined model is
0.198 for 21,745 reflections, with F > 3a in the resolution
range of 7.0 to 1.7 A (0.208 for all 23,577 reflections
in this resolution range). The rms deviations of
bonds and angles from the target values are 0.013 A
and 2.00, respectively. The final model of the 1 chain
included all 238 residues in the main chain trace ofthe
recombinant molecule and 107 solvent molecules.
9. F. Hochstenbach and M. B. Brenner, J. Clin. Immunol.
1 0,1 (1 990).
10. E. A. Kabat, T. T.Wu, H. M. Perry, K. S. Gottesman,
C. Foeller, Sequences of Proteins of Immunological
Interest (Public Health Services, National Institutes of
Health, Washington, DC, ed. 5,1991).
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BrOnger, D. J. Leahy, T. R. Hynes, R. 0. Fox, J.
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U.S.A. 86, 5938 (1989).
13. M. M. Davis and P. J. Bjorkman, Nature 334, 395
(1988); J.-M. Claverie, A. Prochnicka-Chalufour, L.
Bougueleret, Immunol. Today 10, 10 (1989).
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I. A. Wilson, Science 257, 919 (1992); J. H.
Brown et al., Nature 364, 33 (1993).
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U.S.A. 87, 9138 (1990).
16. P. A. Patten et al., J. Immunol. 150, 2281 (1993).
17. J. Kang, C. A. Chambers, J. Pawling, C. Scott, N.
Hozumi, ibid. 152, 5305 (1994).
18. P. A. Cazenave et al., Cell 63, 717 (1990).
19. A. M. Pullen, T. Wade, P. Kappler, J. W. Kappler,
ibid. 61, 1365 (1990).
20. P. Patten et al., Nature 312, 40 (1984).
21. G. A. Bentley, G. Boulot, M. M.Riottot, R. J. Poijak,
ibid. 348, 254 (1990).
22. B. A. Fields et al., J. Mol. Biol. 239, 339 (1994).
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Biochemistry 12, 4620 (1973).
24. C. Saint-Ruf et al., Science 266, 1208 (1994).
25. A. M. Lesk and C. Chothia, Nature 335, 188 (1988).
26. G. Boulot, G. A. Bentley, K. Karjalainen, R. A. Mariuzza,
J. Mol. Biol. 235, 795 (1994).
27. G. Casorati, A. Traunecker, K. Karjalainen, Eur. J.
Immunol. 23, 586 (1993).
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29. K. Karjalainen, Curr. Opin. Immunol. 6, 9 (1994).
30. M. Reth, Annu. Rev. Immunol. 10, 97 (1992).
31. A. Traunecker, F. Oliveri, K. Karjalainen, Trends Biochem.
Sci. 9, 109 (1991).
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Pigeon, J. Immunol. 142, 2736 (1989).
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X-ray Crystallography and NMR (Yale Univ. Press,
New Haven, 1992).
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Cryst. D50, 760 (1994).
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37. We thank J. Montreuil, University of Lille, for aid in
characterizing the carbohydrate of the 13 chain.
Supported by grants from the Institut Pasteur and
CNRS. The Basel Institute for Immunology was
founded and is supported by F. Hoffmann-LaRoche,
Switzerland.
12 December 1994; accepted 23 February 1995
Regional Forest Fragmentation and the
Nesting Success of Migratory Birds
Scott K. Robinson,* Frank R. Thompson Ill,
Therese M. Donovan, Donald R. Whitehead, John Faaborg
Forest fragmentation, the disruption in the continuity of forest habitat, is hypothesized to
be a major cause of population decline for some species of forest birds because fragmentation
reduces nesting (reproductive) success. Nest predation and parasitism by
cowbirds increased with forest fragmentation in nine midwestern (United States) landscapes
that varied from 6 to 95 percent forest cover within a 1 0-kilometer radius of the
study areas. Observed reproductive rates were low enough for some species in the most
fragmented landscapes to suggest that their populations are sinks that depend for
perpetuation on immigration from reproductive source populations in landscapes with
more extensive forest cover. Conservation strategies should consider preservation and
restoration of large, unfragmented "core" areas in each region.
The conservation of neotropical migrant
bird species, which breed in North America
and winter in the tropics, has attracted
attention even though most are not yet
endangered (1, 2). Many neotropical migrants,
however, are suffering population
declines, the causes for which may include
the loss of breeding, wintering, and migraS.
K.Robinson, Illinois Natural History Survey, 607 East
Peabody Drive, Champaign, IL 61820, USA.
F. R. Thompson Ill, North Central Forest Experiment Station,
University of Missouri, Columbia, MO 6521 1, USA.
T. M. Donovan and J. Faaborg, Division of Biological
Sciences, University of Missouri, Columbia, MO 6521 1,
USA.
D. R. Whitehead, Department of Biology, Indiana University,
Bloomington, IN 47405, USA.
*To whom correspondence should be addressed.
SCIENCE * VOL. 267 * 31 MARCH 1995
tion stopover habitats (3). A frequently
hypothesized cause for declines in populations
ofmigrant birds is the negative impact
of habitat fragmentation (4) on breeding
success (5). Habitat fragmentation may allow
higher rates of brood parasitism by
brown-headed cowbirds (Molothrus ater)
and nest predation (6, 7). Cowbirds lay
their eggs in the nests of other "host" species,
which then raise cowbirds at the expense
of their own young (8).
Populations of cowbirds and many nest
predators are higher in fragmented landscapes
where there is a mixture of feeding
habitats (agricultural and suburban) and
breeding habitats (forests and grasslands)
(8-10). In landscapes fragmented byagri-
1987
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cultural fields, levels of nest predation and
brood parasitism are so high that many
populations of forest birds in the fragmented
landscapes are likely to be population
"sinks" (l 1) in which local reproduction is
insufficient to compensate for adult mortality
(12). As landscapes become increasingly
fragmented, this reproductive dysfunction
could cause regional declinesof migrant
populations (7).
Previous studies of the effects of fragmentation
have focused primarily on reproductive
success as a function of local factors
such as habitat size or distance to the habitat
edge (5, 13). Many studies used artificial
nests (14), which may not reflect natural
predation levels ( 15), or relied on composite
data from natural nests of many host
species (6, 7,16, 17).
We tested the hypothesis that the reproductive
success of nine species of forest
birds was related to regional (midwestern
United States) patterns of forest fragmentation.
We measured nest predation and
brood parasitism in nine different landscapes
ranging from over 90% agricultural
to more than 90% forested. The study areas
were in Illinois, Indiana, Minnesota, Missouri,
and Wisconsin.
This study involved the coordinated efforts
of five teams of 5 to 25 researchers
(totaling over 100 assistants) who located
and monitored the fates of more than 5000
nests on nine study areas from 1989 to
1993. The study areas represented the entire
range of forest fragmentation available
in the Midwest (Fig. 1). Each study area
consisted of two to nine sites on which
nests were monitored every 2 to 7 days to
determine if they were parasitized and to
calculate the daily predation rate with the
Mayfield index (18). Data were pooled from
nests in all years and on all sites within a
study area (19). Comparing parasitism and
predation levels from different years could
result in errors, but these should be minimal
because most of the study area estimates are
means from several years with an overlap in
years among study areas. We include data
from nine species for which we have data
from at least four sites (20). All species
except the northern cardinal are neotropical
migrants.
We constructed a map of forest cover for
the entire region from 1: 250,000 scale digital
land use and land cover data derived
from thematic overlays (21). A spatial analysis
program (FRAGSTATS) (22) was used
to calculate the mean percent forest cover,
mean percent forest interior (forest >250 m
from an edge), and mean forest patch size
within a 10-km radius of the center of each
site. We then calculated means for the forest
statistics from all of the sites within a
study area (Fig. 2). For each species, we
calculated Pearson correlation coefficients
for the relation of the daily nest mortality
and percent nests parasitized with the percent
forest cover, percent forest interior,
and mean forest patch size. We tested the
hypothesis that brood parasitism and nest
predation were related to the forest cover
statistics for all species by combining the
probabilities from the species-specific correlations
(23). The forest cover statistics
were log-transformed when necessary. Percent
forest cover, percent forest interior,
and mean forest patch size were all highly
correlated [all correlations were significant
at the 0.001 level, correlation coefficient
(r) = 0.89 to 0.985]. We only present results
for percent forest cover because it had
the highest correlations with nesting success
for most species.
Cowbird parasitism was negatively correlated
with percent forest cover for all
species; correlations were significant [probability
(P) c 0.05] for five of the nine
species studied (Fig. 3). The combined
probabilities test (23) indicated that, overall,
nest parasitism was significantly negatively
related to the amount of forest cover
in the landscape (P . 0.01). Most wood
thrush nests in landscapes with less than
55% forest cover were parasitized. In some
landscapes, there were more cowbird eggs
than wood thrush eggs per nest (11). In
contrast, cowbird parasitism levels were so
low in the heavily forested landscapes that
cowbird parasitism is unlikely to be a significant
cause of reproductive failure (24).
There were some exceptions to the
trends. Parasitism levels were consistently
higher in the four Illinois sites than in
comparably fragmented forests in the other
states. Cowbird abundance was also higher
in the Illinois landscapes (10). Several migrant
species were heavily parasitized in the
mostly forested Indiana landscape where
most other species were rarely parasitized.
These results suggest that local factors such
as the spatial distribution and kinds offorest
edges, the quality of cowbird feeding areas,
and preferences by cowbirds for particular
hosts also influence parasitism.
Levels of nest predation also declined
with increasing forest cover for all species.
Although only three of the nine species had
a significant (P . 0.05) negative correlation
with percent forest cover (Fig. 4), the combined
probabilities test for the overall effect
across all species was significant (P < 0.02).
Three ground-nesting warblers (the ovenbird
and the worm-eating and Kentucky warblers)
and two species that nest near the
ground in shrubs (hooded warbler and indigo
bunting) all had extremely high (6% or
higher) daily predation rates in the most
Fig. 1. Distribution of forest habitat
in the midwestern United
States and locations ofthe study
areas. Abbreviations: CAIL,
Cache River, Illinois; CNIL,
central Illinois; NCMO, northcentral
Missouri; NWIL, northwestern
Illinois; NWWI, northwestern
Wisconsin; SCMO,
south-central Missouri; SOIN,
southern Indiana; SWIL, southwestern
Illinois; and WCWI,
west-central Wisconsin.
Fig. 2. Forest cover statistics for the nine study
areas (landscapes) shown in Fig. 1 in the midwestern
United States. Abbreviations as in Fig. 1.
SCIENCE * VOL. 267 * 31 MARCH 1995
lninsilisi 1 '-M ROMIN~ 1 -1m
1988
onDecember5,2019http://science.sciencemag.org/Downloadedfrom
fragmented landscapes. Twelve of the 13
cases of daily predation rates exceeding 7%
(>80% of all nests lost to predators) were in
the four most fragmented landscapes.
Fragmentation at the landscape scale
thus affects the levels of parasitism and
predation on most migrant forest species in
the midwestern United States. Even the
indigo bunting, which prefers forest edges
(5), nests more successfully in less fragmented
landscapes. Cowbirds can commute up to
7 km between breeding and feeding areas
and therefore use widely scattered feeding
areas (25). The scale (10-km radius around
each study area) on which we measured
forest fragmentation is appropriate because
it is similar in size to the home ranges of
cowbirds. In heavily forested landscapes,
cowbird populations may be more limited
by the availability of foraging areas than by
host availability. In more fragmented landscapes,
on the other hand, the cowbird
populations may be more limited by the
0 20 40 60 80 100
Landscape forsted (%)
Fig. 3. Correlations between proportion of parasitized
nests and percent forest cover in nine
study areas (landscapes) in the midwestern United
States. Abbreviations: ACFL, acadian flycatcher;
INBU, indigo bunting; KEWA, Kentucky warbler;
NOCA, northern cardinal; OVEN, ovenbird;
REVI, red-eyed vireo; WEWA, worm-eating warbler;
and WOTH, wood thrush. For species
names, see (20). The statistics for each species
are as follows: ACFL (r = -0.66, P = 0.10), INBU
(r = -0.97, P 0.01), KEWA (r = -0.62, P =
0.19), NOCA (r = -0.69, P = 0.12), OVEN (r =
-0.76, P = 0.05), REVI (r = -0.94, P < 0.01),
WEWA (r = -0.97, P = 0.02), and WOTH (r =
-0.92, P < 0.01).
availability of hosts and may saturate the
available breeding habitat, which would result
in high levels of parasitism even in the
interior (>600 m from the forest edges) of
the largest (up to 2200 ha) tracts in Illinois
(10). Therefore, landscape-level factors
such as percent forest cover determine the
magnitude of local factors such as tract size
and distance from the forest edges, a result
consistent with continental analyses of parasitism
levels (25).
Nest predators such as mammals, snakes,
and blue jays (Cyanocitta cristata) likely
have smaller home ranges than cowbirds
(26) and may therefore be more affected by
local than by landscape-level habitat conditions.
Small woodlots in agricultural landscapes,
for example, have high populations
of raccoons (Procyon lotor) (27). Censuses
in both Missouri and Wisconsin have
shown blue jay and crow (Corvus brachyrhynchos)
abundances to be much higher in
fragmented regions (28). High predation
rates of ground- and near-ground-nesting
birds in the most fragmented landscapes
may reflect the abundance of these predators
(29). We know very little, however,
0.1 |
\ _ EV1
0.08 \ NOCA
m0 0.080.06
OVEN
0.04
KEWA
0.02
00
*0 20 40 60 80 100
Landscape forested (%)
Fig. 4. Correlations between daily nest predation
rate and percent forest cover in nine study areas
(landscapes) in the midwestern United States.
SCTA, scarlet tanager. Other abbreviations are as
in Fig. 3. The statistics for each species are as
follows: ACFL (r = -0.12, P = 0.79), INBU (r =
-0.82, P = 0.05), KEWA (r = -0.67, P = 0.14),
NOCA (r = -0.47, P = 0.28), OVEN (r = -0.49, P
= 0.21), REVI (r = -0.55, P = 0.19), SCTA (r =
-0.49, P = 0.25), WEWA (r = -0.99, P = 0.01),
and WOTH (r = -0.74, P = 0.02).
about how fragmentation affects populations
of most nest predators.
The large differences between the levels
of parasitism and predation in fragmented
and unfragmented landscapes (Figs. 3 and
4) provide strong evidence that "sourcesink"
population models (12) may be applicable.
Parasitism levels of wood thrushes,
tanagers, and hooded warblers and predation
rates on ovenbirds and Kentucky warblers
were so high in the most fragmented
forests that they are likely population sinks
(28). Extensive forests of the Missouri
Ozarks, northern Wisconsin, and southcentral
Indiana have low levels of nest predation
and parasitism and may provide the
surplus of colonists necessary to maintain
populations in fragmented forests in southem
Wisconsin, Illinois, and northern Missouri
(30). Understanding source-sink population
dynamics, however, requires data on
the season-long productivity of females and
dispersal distances and a better understanding
of adult and juvenile mortality (24).
Nevertheless, the persistence of migratory
songbirds in areas of very low nesting success
provides strong evidence for sourcesink
metapopulation structure (11).
Our results suggest that a good regional
conservation strategy for migrant songbirds
in the Midwest is to identify, maintain, and
restore the large tracts that are most likely to
be population sources. Further loss or fragmentation
of habitats could lead to a collapse
of regional populations of some forest
birds (7, 30). Land managers should seek to
minimize cowbird foraging opportunities
within large, unfragmented sites (8, 10). In
more fragmented landscapes, the reduction
of cowbird parasitism may require trapping
and large-scale restoration efforts (8),
whereas reduction of local forest edges may
reduce nest predation (14) and increase
mating success (29, 31). As long as an adequate
number oflarge, unfragmented regions
remain in North America, it is unlikely that
fragmentation alone will drive populations
of migrant birds to extinction. Increasing
fragmentation oflandscapes, however, could
be contributing to the widespread population
declines of several species.
REFERENCES AND NOTES
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25 September 1992 (U.S. Department of Agriculture
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8. S. K. Robinson et al., in (2), pp. 93-102.
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and Management of Cowbirds, T. Cook, S. K.
Robinson, S.I. Rothstein, S. G. Sealy, J. N. M. Smith,
Eds. (Univ. of Texas Press, Austin, TX, in press).
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18. H. Mayfield, Wilson Bull. 87, 456 (1975). The Mayfield
index measures the daily predation rate by division
of the number of depredated nests by the total
number of "exposure days" during which
the nests were active and being monitored. Nest
predation rates in excess of 5% translate to overall
losses of 70% or more of all nests. We accumulated
more than 40,000 exposure days for the nine study
species.
19. Study areas included northwestern Illinois (NWIL),
five woodlots (90 to 525 ha) (1992-1993 only) in Jo
Daviess and Carroll counties; central Illinois (CNIL),
three woodlots (65 to 300 ha) in Shelby, Champaign,
and Piatt counties; southwestern Illinois (SWIL), four
areas in 1000- to 3500-ha tracts in the Shawnee
National Forest and Trail of Tears State Forest in
Union and Alexander counties; Cache River, IL
(CAIL), six study sites in 25- to 1600-ha forests in the
Cache River Bioreserve of Johnson, Union, Pulaski,
and Alexander counties; southern Indiana (SOIN),
four study sites (ranging from 133 to 190 ha) in a
>40,000-ha forest tract in the vicinity of the Pleasant
Run Unit ofHoosier National Forest of Monroe,
Brown, Jackson, and Lawrence counties; northwestern
Wisconsin (NWWI), six study areas within
the Chequamegan National Forest, Bayfield County;
west-central Wisconsin (WCWI), six study areas (80to
160-ha patch size) along the St. Croix River in
Washington and Chisago counties, MN, and Polk
County, WI; south-central Missouri (SCMO), eight
study areas within the contiguous Ozark Forests in
Shannon, Reynolds, and Carter counties; north-central
Missouri (NCMO), nine study sites within Boone,
Callaway, and Randolph counties in tracts ranging
from 150 to 900 ha.
20. Study species included the acadian flycatcher
(ACFL) Empidonax virescens, wood thrush (WOTH)
Hylocichla mustelina, red-eyed vireo (REVI) Vireo olivaceus,
ovenbird (OVEN) Seiurus aurocapillus,
worm-eating warbler (WEWA) Helmitheros vermivorus,
Kentucky warbler (KEWA) Oporomis formosus,
scarlet tanager (SCTA) Piranga olivacea,
northern cardinal (NOCA) Cardinalis cardinalis, and
indigo bunting (INBU) Passerina cyanea. Sample sizes
per species ranged from 50 nests (SCTA) to over
500 (ACFL and WOTH).
21. The overlays are available from the U.S. Geological
1990
Survey, 507 National Center, Reston, VA 22092.
22. FRAGSTATS; Forest Science Department, Oregon
State University, Corvallis, OR.
23. R. R. Sokal and F. J. Rohif, Biometry (Freeman, New
York, 1981).
24. R. M. May and S. K. Robinson, Am. Nat. 126, 475
(1985).
25. J. P. Hoover and M. C. Brittingham, Wilson Bull. 105,
228 (1993).
26. S. I. Rothstein, J. Verner, E. Stevens, Ecology 65, 77
(1984); F. R. Thompson Ill, Auk, in press.
27. J. R. Bider, Ecol. Monogr. 38, 269 (1968).
28. T. M. Donovan, thesis, University of Missouri.
29. J. P. Gibbs and J. Faaborg, Conserv. Biol. 4, 193
(1990); M. A. Villard, P. R. Martin, C. G. Drummond,
Auk 110, 759 (1993); P. Porneluzi, J. C. Bednarz, L.
J. Goodrich, N. Zawada, J. P. Hoover, Conserv. Biol.
7, 618 (1993).
30. For source-sink models of the wood thrush and ovenbird
in this landscape, see T. M. Donovan, R. H.
Lamberson, F. M. Thompson Ill, J. Faaborg, Conserv.
Biol., in press; T. M. Donovan, F. R. Thompson
Ill, J. Faaborg, J. Probst, ibid., in press.
31. M. A. Van Horn, R. M. Gentry, J. Faaborg, Auk, in
press.
32. We would like to thank the many assistants who
made this project possible, especially the following:
in Illinois, S. Morse, S. Bailey, R. Jack, K. Bruner, J.
Hoover, C. Morse, S. Daniels, and R. Brumfield; in
Missouri, R. Clawson and P. Porneluzi; in Wisconsin,
J. Porath and A. R. Weisbrod; and in Indiana,
G. Greenberg, M. Koukol, D. Winslow, T. Ford, P.
Doran, C. Wilson, B. Geils, and B. Slusher. We also
thank D. Larsen and W. Dijak for assistance with
the landscape analyses and graphics. J. D. Brawn
kindly provided data from two woodlots in CNIL.
Funding was provided by the Illinois Departments
of Energy and Natural Resources and Conservation
(NWIL and SWIL), the National Fish and Wildlife
Foundation (SWIL, SOIN, and SCMO), the U.S.
Forest Service, North Central Forest Experiment
Station (SWIL, NCMO, WCWI, and NWWI), the Illinois
Chapter of The Nature Conservancy (CAIL),
the U.S. Army Construction Engineering Research
Laboratory (CNIL), the Indiana Department of Natural
Resources (SOIN), the Martin Foundation
(SOIN), the David G. Frey Memorial Fund (SOIN),
the Conservation and Research Foundation, Wild
Birds Unlimited, the Indiana Academy of Science
(SOIN), many Audubon chapters (SOIN), Minnesota
Department of Natural Resources Nongame
Wildlife Research Fund (WCWI), Missouri Department
of Conservation (SCMO), and the Global
Change Program (BBIRD) of the National Biological
Service (NCMO and SCMO).
24 October 1994; accepted 30 January 1995
Requirement of Serine Phosphorylation for
Formation of STAT-Promoter Complexes
Xiaokui Zhang, John Blenis, Heng-Chun Li, Chris Schindler,
Selina Chen-Kiang*
Members of the interleukin-6 family of cytokines bind to and activate receptors that
contain a common subunit, gpl 30. This leads to the activation of Stat3 and Statl, two
cytoplasmic signal transducers and activators of transcription (STATs), by tyrosine phosphorylation.
Serine phosphorylation of Stat3 was constitutive and was enhanced by
signaling through gpl30. In cells of lymphoid and neuronal origins, inhibition of serine
phosphorylation prevented the formation of complexes of DNA with Stat3-Stat3 but not
with Stat3-Statl or Stati -Stati dimers. In vitro serine dephosphorylation of Stat3 also
inhibited DNA binding of Stat3-Stat3. The requirement of serine phosphorylation for
Stat3-Stat3DNA complex formation was inversely correlated with the affinity of Stat3Stat3
for the binding site. Thus, serine phosphorylation appears to enhance or to be
required for the formation of stable Stat3-Stat3.DNA complexes.
The Janus kinase (Jak)-STAT pathway
transduces the signals of many cytokines
and peptide growth factors (1). Ligand
binding rapidly triggers tyrosine phosphorylation
of STATs (2, 3) by receptor-associated
Jak family tyrosine kinases (4, 5).
The activated STATs dimerize and translocate
into the nucleus, where they directly
activate target genes by binding to specific
promoter sequences (1). The pleiotropic cytokines
interleukin-6 (IL-6), ciliary neuroX.
Zhang and S. Chen-Kiang, Brookdale Center for Molecular
Biology, Mount Sinai School of Medicine, New
York, NY 10029, USA.
J. Blenis, Department of Cell Biology, Harvard Medical
School, Boston, MA 02115, USA.
H.-C. Li, Department of Biochemistry, Mount Sinai
School of Medicine, New York, NY 10029, USA.
C. Schindler, Department of Medicine, College of Physicians
and Surgeons, Columbia University, New York, NY
10032, USA.
*To whom correspondence should be addressed.
SCIENCE * VOL. 267 * 31 MARCH 1995
trophic factor (CNTF), leukemia inhibitory
factor (LIF), oncostatin M, and IL-11 transduce
signals through gpl30, a common
component of the receptor complexes (6,
7). IL-6 primarily activates Stat3 [also
known as acute-phase response factor
(APRF)] (8, 9) in the liver (9, 10), but it
also activates Statl (1 1) in human hepatoblastoma
HepG2 cells (8, 12, 13). Signaling
by CNTF activates Statl and two Statlrelated
proteins in a human neuroblastoma
cell line SK-N-MC (14). Activated Stat3
and Statl form three distinct protein-DNA
complexes that contain either Statl homodimers,
Statl-Stat3 heterodimers, or
Stat3 homodimers when they bind to the
Stat-binding site present in the c-fos promoter
(8, 12, 13,15). Studies of the activation
of Statl by interferon y (IFN-,y)
suggest that Statl can form stable dimers by
interactions between the Src homology 2
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