CREB: A Ca$^{2+}$-Regulated Transcription Factor Phosphorylated by Calmodulin-Dependent

Kinases
Author(s): Morgan Sheng, Margaret A. Thompson and Michael E. Greenberg
Source: Science, New Series, Vol. 252, No. 5011 (Jun. 7, 1991), pp. 1427-1430
Published by: American Association for the Advancement of Science
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cell surface molecule that can recognize this
domain.
The observation that only those CTLp's
and Thp's that have been signaled through
their antigen-specific surface receptors are
killed by exposure to an antibody recogniz-
ing all lymphocytes (anti-a3) may prove
useful for establishing tolerance in the adult
animal to a particular antigen.
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duced. Class I MHC reactive cells carryCD8 and are
predominately CTL's; class II reactive cells carry
CD4 and are predominately Th's. The MHC is
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CD4- CD8- fractions, which were subcultured
with fresh stimulator cells and a source of IL-2.
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Mak,Eur.J. Immunol.20, 2471 (1991).
10. When analyzed on a Becton-Dickenson FACScan
after staining for CD4 and CD8, only the appropri-
ate transfectants differed significantly from control.
These were 15 times (CD4 transfectant) and 40
times (CD8 transfectant) brighter than control.
When stained for H-2k, the lines were 10 times
(parental), 18 times (CD4+), and 16 times (CD8+)
as bright as control. All distributions were log
normal with, for the broadest distribution, maxi-
mum width at half height being defined by approx-
imately one-half and twice the peak intensity.
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tion 34, 113 (1982).
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13. As a negative control, anti-a3 does not recognize
(11) and did not inhibit C3H (H-2k) responder cells
in an MLR. Nor did it inhibit anti-CD3-induced
activation of C3H spleen cells.
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(1981).
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Ld. It inhibited H-2d responder cells, but only at
concentrations 2 3,g/ml. Ld expression is known to
be lower than that of Kd or Dd [W.-R. Lie et al.,
Nature 344, 439 (1990)].
7 JUNE 1991
This content downloaded from 146.201.200.226 on Fri, 24 Oct 2014 10:54:22 AM
All use subject to JSTOR Terms and Conditions
17. J. A. Bluestone, D. Pardoll, S. 0. Sharrow, B. J. demonstrating class II expression by mouse T cells
Fowlkes, Nature 326, 82 (1987). either taken from cultures or directlyfrom the animal
18. K. Haskins et al., J. Exp. Med. 160, 452 (1984). represent endogenous production or surface bound
19. H. M. Shapiro, PracticalFlow Cytometry(Liss, New molecules produced by other cells is controversial.
York, ed. 2, 1988), pp. 168-169. 27. F. Kiziroglu and R. G. Miller, J. Immunol. 146,
20. C. A. Smith, G. T. Williams, R. Kingston, E. J. 1104 (1991).
Jenkinson, J. T. T. Owen, Nature 337, 181 (1989). 28. E. Azuma and J. Kaplan, ibid. 141,2601 (1988); D.
21. The same may apply to B cells. In preliminary J. Cassell and J. Forman, ibid. 144, 4075 (1990).
experiments, spleen cells exposed to the B cell mito- 29. S. Muraoka and R. G. Miller, J. Exp. Med. 152, 54
gen lipopolysaccharide (LPS) were induced to die (1980); H. Nakamura and R. E. Gress, Transplan-
when anti-a3 was also added. tation49, 931 (1990); T. Takahashi, K. Mafune, T.
22. D. R. Kaplan, J. E. Hambor, M. L. Tycocinski, Proc. Maki, J. Exp. Med. 172, 901 (1990).
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26. N. Koch, B. Arnold, G. J. Hammerling, J. Heuer, E. 34. __, ibid., p. B23.
Kolsch, Immunogen.17,497 (1983); J. Heuer and E. 35. We thank V. Wallace for providing the CD4 and
Kolsch, J. Immunol.134, 4031 (1985); L. Graf, N. CD8 transfectants; F. Kiziroglu for discussion; and
Koch, V. Schirrmacher,Mol. Immunol. 22, 1371 B. H. Barber, E. A. McCulloch, and C. Paige for
(1985); A. B. Reske-Kunz, K. Reske, E. Rude, J. critically reading the manuscript. Supported by
Immunol. 136, 2033 (1986). These investigators MRC of Canada (MT-3017) and by an MRC
demonstratedproduction and expressionof low levels fellowship (S.S.).
of classII MHC by particularmouse T cell lines in the
absenceof other cells. Whether resultsof other studies 11 December 1990; accepted 15 March 1991
CREB: A Ca2+-Regulated Transcription Factor
Phosphorylated by Calmodulin-Dependent Kinases
MORGAN SHENG,*t MARGARET A. THoMPSON,t
MICHAEL E. GREENBERGt
The mechanism by which Ca2+ mediates gene induction in response to membrane
depolarization was investigated. The adenosine 3',5'-monophosphate (cAMP) re-
sponse element-binding protein (CREB) was shown to function as a Ca2+-regulated
transcriptionfactor and as a substratefor depolarization-activatedCa2+-calmodulin-
dependentprotein kinases (CaMkinases)I and II. CREB residue Ser'33was the major
site of phosphorylation by the CaM kinases in vitro and of phosphorylation after
membranedepolarizationin vivo. Mutation of Ser'33 impairedthe abilityof CREB to
respond to Ca2+. These results suggest that CaM kinases may transduce electrical
signals to the nucleus and that CREB fuinctionsto integrate Ca2' and cAMP signals.
D NA ELEMENTS THAT CONTAIN THE tein CREB on amino acid residue Ser'33
consensus sequence TGACGTCA (4). Phosphorylation of Ser'33 stimulates
were originally identified as cAMP the ability of CREB to activate gene tran-
response elements (CREs) in neuropeptide scription (6), suggesting that CREB may
genes (1), but recent studies in neuronal cell mediate transcriptional induction by Ca2"
lines have mapped inducibility by mem- as well as by cAMP. However, because
brane depolarization and increased intracel- multiple CRE-binding proteins exist (7, 8),
lular Ca2" concentrations to similar se- distinct members of this family may sepa-
quences in the cfos (2-4) and proenkephalin rately confer Ca2" and cAMP inducibility
genes (5). Depolarization of PC12 pheo- on the CRE (4). To determine whether
chromocytoma cells is correlated with rapid CREB functions as a Ca2"-activated tran-
phosphorylation of the CRE-binding pro- scription factor and to test if phosphoryla-
tion-of Ser133 is important for this activa-
Department of Microbiology and Molecular Genetics, tion, we have targeted CREB to a different
Harvard Medical School, Boston, MA 02115.
DNA regulatory sequence by fusing it to the
*Present address: Howard Hughes Medical Institute, DNA-binding and dimerization domain of
University of California School of Medicine, San Fran- the yeast transcriptionalactivator GAL4 (9)
cisco, CA 94143s
tThe first two authors contributed equally to this report.
(Fig. 1). The use of a reporter gene that
tTo whom correspondence should be addessed. contains GAL4 binding sites allowed a spe-
REPORTS 1427
cific functionalanalysisof CREB without
interferencefromendogenousCRE-binding
proteins.
When cotransfectedinto rat PC12 cells
with a humancfos reportergene that con-
tainedone GAL4 bindingsite (pAF42G1),
the GAL4-CREB fusion construct con-
ferredCa2+ inducibility(Fig. 1B). In the
absenceof stimulation,no transcriptswere
detected from transfectedor endogenous
c{fos genes. When the intracellularCa"2
concentrationwas increasedby K+ depolar-
ization or by treatment with the Ca2+
ionophoreA23187, the majorCa2"-induc-
ible transcriptcorrespondedto correctly
initiatedhumanc-fos (cfosH) mRNA. The
GAL4-CREB fusion protein conferred a
level of induction comparableto that ob-
served in depolarized PC12 cells trans-
fected with a reportergene containing a
CRE (Fig. 1B).
The Ca2"-dependentinductionof cfosH
by GAL4-CREBrequiredthe GAL4 bind-
ing site and was dependenton the CREB
moiety of the GAL4-CREBfusion protein
(Fig. 1B). The GAL4 DNA-binding do-
Fig. 1. Analysis of GAL4-CREB fiisions by
cotransfection assay. (A) Structure of GAL4-
CREB fusions and reporter genes (27). GAL4-
B 17 contains an acidic activating region (11)
fused to GAL4 (1 to 147), the DNA-binding
domain of GAL4 (9). GAL4-CREB contains the
complete CREB protein (341 amino acids) fused
to the COOH-terminus of GAL4 (1 to 147).
GAL4-CREBALZ lacks the COOH-terminal 29
amino acids of GAL4-CREB, including the leu-
cine repeat dimerization motif (LZ). GAL4-
CREBALZMI contains the Ser'33 to Ala muta-
tion. The constructs pAF42G1 (GI) and
pAF42G9 (G9) contain, respectively, one and
nine GAL4 binding sites inserted at position -42
in the core promoter construct pAF42 (GO),
which encodes the human c-fos gene (c-fos) (4).
In pAF42CRE (CRE), the consensus CRE is
inserted at position -42. (B) Ca2' and cAMP
inducibility of reporter
genes with GAL4-CREB
constructs. GAL4-CREB B U K+ F U K+ F A2 T U K+ F K+ A2 F U K+ F
fision constructs (5 jig)
were transiently cotrans-
fected into PC12 cells
(3, 4) with reporter con-
structs pAF42Go (GO) _. ...
or pAF42G, (GI) (20 .......
jig), as indicated. CRE ~~C-fos
g jP
z3SksN RB_0-!....
(20 jig) was transfected
3
alone because the CRIE
Caninteract with endog-
enous CREB. An
h-globin expression plas- c-fosR4 4n i-
mid (pSVot-1; 3 jig) was
used as a control for
transfection efficiency. Reporter OREG
Cells were left unstimu-
lated (U) or were stimu- Activator - GAL4-CREB
lated as indicated by 60
mM K+ and 10 mM Ca2+ (KC), A23187 (10 jig/mI) (Al), or 10 jiM forskolin(F) for 60 mm~
(0.3 jig/mil) (T) for 30 min. Correctly in~itiatedtranscriptsfrom
the endogenous rat gene (cf-osR),and the internal control gene (globin), were assayed by ribonuclease tides of 43 and 38 kD were purifiedby
(RNase) protection(3, 4) and areindicated.Bands abovecfosH representincorrectlyinitiatedtranscripts(28). DNA-affinitychromatography
1428
main either by itself (10) or fused to an construct(Figs. 1B and 2); however, the
acidic activatingregion (GAL4-B17) (11) magnitudesof Ca2+ and cAMP induction
was unableto conferCa2+ inducibility.As were reduced.Similarly,GAL4-CREBpro-
expected,the GAL4-CREBfusionalsocon- teins with other point mutationsor small
ferred cAMP-inducibleexpressionon the deletionswithin the CREB leucine zipper
GAL4 binding site (Fig. 1B) (12). The region that preventdimerization(14) also
levels of inducibilitywith Ca2+ and cAMP confer Ce2+ and cAMP inducibility, but with
were similar,suggesting that CREB func- reducedefficincy (10). The leucine zipper
tions as a transcriptionalregulatorthat is may be importantfor folding the CREB
responsiveto both second messengers.In dimerinto an optimalconformationfor tran-
contrast, GAL4-CREB did not mediate scriptionalactivation.Ahermatively, a portion
transcriptionalactivationby the proteinki- of the Ca2' and cAMPresponsesseen with
nase C activator12-0-tetradecanoylphor- GAL4-CREBmay be due to dimerization
bol-13-acetate(TPA) (Fig. 1B). with endogenousCREBproteins.
BecauseGAIA-CREBcontains the leu- To determinewhetherphosphorylation of
cine zipper dimerizationmotif (13, 14), CREBis functionallyimportantfor depolar-
endogenousPC12 proteinsthatcontainleu- ization- and Ca2+-dependentactivationof
cine zippers might interact with GAL4- transciption,we mutatedthe majorsite of
CREBandcausethe observedtranscription- CREBphosphorylation in depolarizedPC12
al activation.To test this possibility,we cells(Ser133)to alanine.Thismutation(Ml)
constructeda GAL4-CREBmutantin which reduced by -80% the ability of GAL4-
the leucinezipperwas deletedbut the GALA CREBALZto mediatetranscriptional activa-
DNA-bindingand dimerizationdomainsre- tion by either membranedepolarizationor
mainedintact(GAL4-CREBALZ) (Fig. 1A). cAMP (Fig. 2). The Ml mutationdoes not
GALA-CREBALZ conferredboth Ca2+ and affectthe stabilityof CREB or its nudear
cAMP inducibilityon the GAL4 reporter localization(6). These resultssuggest that
Ca2+-dependent phosphorylation of Serl33is
importantforCREBactivationby membrane
A depolarization.However,mutationof Ser'33
GAL4-CREBt Ser13 312 341
1GAL(1-147) CREB 12
did not completely abolish the ability of
GAIA-CREBALZ to confereitherCa2+or
GAL4-B17 cAMP inducibility.The basisof this residual
GAL4-1 7 activityis unclear(15).
GAL4(1-147) B17 The transducingkinasethat couples the
TATA
depolarization-induced Ca2+ signal to the
Go -42
phosphorylationand activationof CREB
1 x GAL4site may be a Ca2+-calmodulin-dependent pro-
tein kinase(CaM kinase).CaM kinasesare
G1 -42 TATA
activatedwhen the intracellularCa2+ con-
9 x GAL4sites
centrationis increasedaftermembranede-
Gg -42 TATA polarization(16, 17), and inductionof cfos
by depolarizationis specificallyinhibitedby
1 xCRE calmodulinantagonists(18). CaM kinasesI
CRE 42 T
-42 and II havewide tissuedistributionsbut are
especially abundantin neural tissues (17,
19). CaM kinaseII has been implicatedin
:: diverseregulatoryprocessesin the nervous
system, induding long-term potentiation
(LTP) (20). The CaM kinase I substrates
synapsinI and proteinIII areboth neuron-
specificproteinsthat arephosphorylatedon
a singlecommonaminoacidresidueby both
cAMP-dependent proteinkinase(PKA)and
CaM kinase I (17, 21). The fact that a
commonresiduein CREB (Ser133) is phos-
cbi phorylatedin vivo in response to either
Ca2' or cAMP suggested the possibility
GGo G thatCaMkinaseI maymediateCa2+-depen-
dent phosphorylationof CREB.
GAL4-B17 GAL4-CREB GAL4-CREBALZ We tested whether purifiedCREB can
serveas a substratein vitrofor CaMkinaseI
or by TPA
the transfected reporter gene (c?os),
or II. Two major GRE-bindingpolypep-
fromextracts
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All use subject to JSTOR Terms and Conditions
 U K+ F U K+ F U K+ F Fig. 3. In vitro phos- A B C
phorylation of CREB Ca2+ + . + +
Jt
c-fos Hi3 ru:l and CREB B by purified CREB + + . . +
protein kinases. (A)
CaMkinaseII. (B) CaM
kinaseI. (C) PKA.Reac-
tions were performed -97
Globin- j ij with [32P]ATPat 300C
for5 min,andcontained
5 to 10 ng of affinity-
purified CREB (+
CREB)(29) or anequiv- CaMKUl
{ La -68

alent volume of buffer

c-fosH , _
Reporter
G0 G9 G9
GAL4-CREB ALZ ALZ ALZM1
Fig. 2. Effectof the Ser'33to Ala mutationon
GAL4-CREBALZ.Transienttransfectionassays
were performed as in Fig. 1 with GAL4-
CREBALZor GAL4-CREBALZM1 andreport-
er constructspAF42Goor pAF42G9as indicated.
Bandsthatrepresentcorrectlyinitiatedtranscripts
areindicatedas in Fig. 1.
of mouse L cells. The 43-kD protein has the
same molecular size as CREB purified from
brain or PC12 cells (22), comigrates with
CREB immunoprecipitated from PC12
cells, and is quantitatively immunoprecipi-
tated by a CREB-specific antiserum (Fig. 3)
(10). The 38-kD protein (termed CREB B)
is likely to be a distinct CREB-related pro-
tein (23).
Both CREB and CREB B are substrates
for CaM kinases I and II in vitro (Fig. 3).
After phosphorylation, both CREB and
CREB B retain their ability to bind the
CRE, as determined by specific retention on
a CRE-affinity column (10). We performed
two-dimensional tryptic phosphopeptide
mapping to identify the sites of CREB phos-
phorylation by CaM kinases I and II. The
major tryptic phosphopeptide (site 1) labeled
in vitro by CaM kinases I and II comigrated
with the CREB tryptic fragment phosphoryl-
ated in vivo in response to increasedCa2+ (4)
(Fig. 4). Tryptic peptide 1 contains Ser'33 as
the only Ser or Thr residue (6, 7). We there-
fore conclude that the major in vitro site of
CREB phosphorylation by CaM kinases I
and II is Ser'33. Whether CaM kinasesdirect-
ly phosphorylate CREB on Ser'33 in vivo
remains to be determined. The two-dimen-
sional tryptic maps revealed that in addition
to Ser133, CREB was consistently phospho-
rylated by CaM kinases I and II at several
minor sites (Fig. 4), the significanceof which
is unknown.
We found that CREB is a substrate for
PKA, as well as the CaM kinases, and that
the major site of phosphorylation by PKA is
also Ser'33 (Fig. 4) (22). The ability of both
GaM kinase II and PKA to phosphorylate
GREB in vitro on the same site has been
demonstrated independently by Dash and
(- CREB). CaMkinase a- *a CaMKi- " a * a -43
reactions (30) were per- a4 b S-* b b
formed in the presence
of Ca2 (+ Ca +) or in
the presence of excess
EGTA (- Ca24). La- -29
beledproductsweresep-
aratedon SDS-polyacrylamide gels and visualizedby autoradiography.
The positionsof CREB and
CREBB aremarkedby "a"and"b",respectively.The autophosphorylated productsof CaMkinaseI
(CaMKI) (37 to 42 kD) and CaMkinaseII (CaMKU) (51 to 53 kD and 60 kD) are indicated.A
diffuseband of -80 kD in the PKA lane is not consistentlyseen between differentexperiments.
Positionsof molecularsize markersareindicatedin kilodaltons.
co-workers (24). In addition, they have mumspecificitydeterminantfor CaMkinase
shown that phosphorylationof this site II (19), as well as to the PKA consensus
stimulatesthe abilityof CREB to activate recognition sequence (-Arg-Arg-X-Ser-)
in an in vitroRNA polymerase
transcription (25). Overlapin the phosphorylationsitesof
II-dependent transcription system. The CaMkinaseII and PKA has been described
phosphorylationin vitro of a commonreg- for othersubstrates(26). Moreover,the Ser
ulatory site (Ser'33) by Ca2+- and cAMP- residuein synapsinI that is phosphorylated
regulatedprotein kinases provides a bio- by both CaM kinaseI and PKA (21) lies
chemical explanationfor how Ca2" and within a sequence (Arg-Arg-Arg-Leu-Ser-
cAMP signalingpathwaysmight converge Asp) thatis similarto the CREBSer'33site.
to activateCREB in vivo. The finding that a regulatory site in
Analysis of the amino acids that flank CREBis phosphorylated by both Ca2"-and
Ser'33in CREB (7) revealedthat this resi- cAMP-dependentprotein kinases in vitro
due lies within a stretchof aminoacidsthe providesan explanationfor the observation
sequenceof whichis consistentwith it being that CREB can mediate a transcriptional
a recognitionsite for CaM kinasesI and II response to either Ca2+ or cAMP. Thus, the
and PKA.The localpeptidesequence(-Ser- increasinglycomplex role of the CRE in
Arg-Arg-Pro-Ser'33-Tyr-Arg-) conformsto gene regulationmay be a reflectionof its
the motif -Arg-X-X-Ser(Thr)- (whereX rep- importanceas a target of multiple signal
resentsany aminoacid), which is the mini- transductionpathwaysin the cell.
Fig. 4. Sites of CREB A B C
phosphorvlation bv
CaM kinase I, CaM ki-
nase II, and PKA. Two- 1 9 1
dimensional tryptic
phosphopeptide maps
(4, 3 1) are shown.
CREB was phosphoryl-
atedinvitro by (A) CaM .
kinase I, (B) CaM kinase
II, or (C) PKA, as indicated. (D) CREB B D E
phosphorvlated by CaM kinase 11(32). (E) CREB
immunoprecipitated from 32P-labeled PC12 cells
that had been subjected to K+-induced depolar- a.
ization for 5 min (4). The major phosphopeptide
(marked "1") comigrates with the CREB trvptic 2
fragment phosphorylated in vivo in response to
Ca2" in mixing experiments (10) and corresponds
to phosphorylation of Ser'33 (4, 6). The track of
faint spots trailing beneath phosphopeptide 1
varies in intensity between experiments and probably represents peptides phosphorvlated at the same
site that have not completely migrated to the major spot 1 position during chromatography. Origins are
marked by a black dot. Electrophoresis at pH 1.9 was performed from left (+) to right (-), and
chromatography was performed in the ascending direction.

7 JUNE 1991 REPORTS 1429

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14. V. J. Dwarki, M. Montminy, I. M. Verma, EMBO
J. 9, 225 (1990).
15. The slight residual activity of GAL-CREBALZMI
is in apparent disagreement with a previous report
(6) but may reflect the high sensitivity of the cfos
mRNA induction assay.
16. L. C. Griffithand H. Schulman, J. Biol. Chem. 263,
9542 (1988).
17. A. C. Nairn and P. Greengard, ibid. 262, 7273
(1987).
18. J. I. Morgan and T. Curran, Nature 322, 552
(1986).
19. R. J. Colbran et al., Biochem.J. 258, 313 (1989).
20. M. B. Kennedy, Cell 59, 777 (1989).
21. A. J. Czernik, D. T. Pang, P. Greengard, Proc. Natl.
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22. Z. Zhu, G. M. Andrisani, D. A. Pot, J. E. Dixon, J.
Biol. Chem. 264, 6550 (1989); M. R. Montminy
and L. M. Bilezikjian, Nature 328, 175 (1987); K.
K. Yamamoto, G. A. Gonzalez, W. H. Biggs, M. R.
Montminy, ibid. 334, 494 (1988).
23. Several observations suggest that CREB B is a
distinct CRE-binding protein rather than a degra-
dation product of CREB: it copurifies with CREB
through multiple steps as a constant proportion of
the total protein; it is poorly immunoprecipitated by
an antiserum to CREB that is specific for an internal
CREB peptide (10); and its tryptic phosphopeptide
map is distinct from that of CREB (Fig. 4).
24. P. K. Dash, K. A. Karl, M. A. Colicos, R. Prywes, E.
R. Kandel, Proc. Natl. Acad. Sci. U.S.A., in press.
25. B. E. Kemp, D. J. Graves, E. Benjamini, E. G.
Krebs, J. Biol. Chem. 252, 4888 (1977).
26. T. R. Soderling, L. M. Schworer, M. R. El-Magh-
rabi, S. J. Pilkis, Biochem. Biophys. Res. Commun.
139, 1017 (1986); L. M. Schworer, M. R. El-
Maghrabi, S. J. Pilkis, T. R. Soderling, J. Biol.
Chem. 260, 13018 (1985); A. P. Doskeland et al.,
Eur.J. Biochem. 145, 31 (1984).
27. The complete coding region of the rat CREB cDNA
(7) was amplified by the polymerase chain reaction
(PCR) and fused in frame to GAL4 (1 to 147)
encoded in the plasmid pSG424 (9) to form GAL4-
CREB. The Ser'33 to Ala mutation (M1) was
subcloned (as an Aat II-Kpn I restriction fragment)
into GAL4-CREB from the plasmid RSV-
CREBfM1 (6) to generate GAL4-CREBM1.
GAL4-CREBALZ and GAL4-CREBALZM1 were
constructed by amplifying the corresponding re-
gions from GAL4-CREB and GAL4-CREBM1 by
PCR.
28. The two bands slightly above cfosH that are also
detected in the ribonuclease (RNase) protection
assay represent transcriptsinitiating from within the
vector and from a cryptic mRNA start site in the
TATA region of the cfEos construct (4).
29. CREB was purified by DNA-affinity chromatogra-
1430
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All use subject to JSTOR Terms and Conditions
phy [J. T. Kadonaga and R. Tjian, Proc. Natt. Acad. and 1.5 mM CaCl2. In vitro phosphorylation by
Sci. U.S.A. 83, 5889 (1986)] on columns (40 ,ug PKA catalyticsubunit (20 to 40 ,ug/ml) (Sigma) was
of DNA per milliliter) prepared from oligonucleo- performed in 10 mM Hepes (pH 7.2), 10 mM
tides of the following sequences: CRE column, MgCI2, 1 mM DIT, 10 ,uM ATP, and 1.5 ,uM
5'-CCCGTGACGTCAACAC-3' and 5'-TGACG- [Y_32P]ATP.
TCACGGGGTGT-3'; AP-1 column, 5'-CCCGT- 31. K. Luo, T. R. Hurley, B. M. Sefton, Oncogene 5,
GAGTCAACAC-3' and 5'-TGACTCACGGGG- 921 (1990).
TGT-3' (consensus elements in italics). Nuclear 32. Two-dimensional tryptic analysis of CREB B phos-
extracts (200 mg of protein) were prepared from phorylation in vitro by CaM kinase I, CaM kinase II,
2 x 1011 mouse L cells by the chloroquine DNA and PKA reveals that the major phosphopeptide
intercalation method [H. Schroter, P. E. Shaw, A. labeled by these enzymes comigrates exactly with
Nordheim, Nucleic Acids Res. 15, 10145 (1987)]. phosphopeptide 1 labeled in CREB (Fig. 4) (10),
We purified CREB by sequential passage through a suggesting that CREB and CREB B may possess a
serum response element column [R. Treisman, conserved phosphorylation site. Hai et al. (8) have
EMBOJ. 6, 2711 (1987)], two CRE columns, one identified a protein, ATFI, that contains a sequence
AP-1 column to rerove AP-1 proteins, and a final that is identical to the CREB peptide that contains
CRE column. CRE-binding activity was followed Ser'33. ATFI may therefore be identical to CREB
by gel shift assay (4). Purified CREB (-0.5 ,ug, as B.
estimated by in vitro DNA-binding activity) was 33. We thank H. Schulman and N. Waxham for purified
concentrated in a Centricon ultrafiltration device CaM kinase II; A. Nairn for CaM kinase I; I.
(Amicon, Danvers, MA) and dialyzed overnight Sadowski for pSG424; M. Carey for pG9E4T; and
against 10 mM Hepes (pH 7.4) and 10% glycerol. M. Montminy for antiserum W39 to CREB and
30. In vitro phosphorylation of CREB by purified rat RSV-CREBfM1. Supported by NIH grant RO1 CA
brain CaM kinase II (1 to 5 ,ug/ml) was performed 43855, grant ROI NS 28829 from the National
in 50 mM Pipes (pH 7), 10 mM MgCG2, 1 mM Institute of Neurological Disorders and Stroke, an
dithiothreitol (DIT), calmodulin (5 ,ug/ml), bovine American Cancer Society Faculty Research Award
serum albumin (100 ,ug/ml), 10 ,uM adenosine (FRA-379) (M.E.G.), and a Scholar's Award from
triphosphate (ATP), 1.5 ,uM [y-32P]ATP, and ei- the McKnight Endowment Fund for Neuroscience
ther 0.3 mM CaCl2 or 0.2 mM EGTA. In vitro (M.E.G.).
phosphorylation by CaM kinase I was performed as
described (17) with 1 mM EGTA or 1 mM EGTA 20 December 1990; accepted 22 March 1991
Recognition of Self Antigens by Skin-Derived T Cells
Sy Antigen Receptors
with Invariant
WENDY L. HAvRAN, YUEH-HSIU CHIEN, JAMES P. ALLISON
Thy-l+ dendritic epidermal T cells (dECs) express invariant 'yi antigen receptors and
are found in intimate contact with keratinocytes in murine epidermis-thus raising the
possibility that keratinocytes express a ligand for the antigen receptor of these T cells.
Thy-I' dECs were stimulated to produce lymphokines by interaction with kerati-
nocytes in vitro. This stimulation was mediated through the dEC antigen receptor and
did not appear to be restricted by the major histocompatibility complex. Thus, dECs
can recognize self antigens and may participate in immune surveillance for cellular
damage rather than for foreign antigens.
IN MICE, MOST T CELLS IN THE LYM- in the adult mouse (13). Similarly,the intra-
phoid tissues express diverse antigen epithelial T cells of the female reproductive
receptors that consist of acand i chains tract and the tongue express yb TCRs com-
and recognize antigens bound to self major posed of an invariantV,4/J,,1-C,,l chain and
histocompatibility complex (MHC) pro- an invariant8 chain identical to that found in
teins. A minority of lymphoid T cells express the dECs (14). The absenceof clonal diversity
clonally diverse y6 T cell receptors (TCRs) in the TCRs of these epithelium-associatedT
(1) and recognize a wide array of antigens, cells suggests that they might perform immu-
including MHC class I, II, and lb gene nological fimctions that are distinct from
products, as well as bacterial heat shock those of T cells with clonally diverse antigen
proteins (2-7). In contrast to the lymphoid receptors. These nondiverse T cells might
organs, severalepithelialtissues contain most- recognize damage-induced self antigens in a
ly y T cells. The epidermiscontains a unique mechanism of trauma signal surveillance(11,
population of cells, the Thy- I' dECs (8-10), 15). Here, we provide evidence in support of
which express invariant y6 TCRs composed this concept by demonstrating that dECs can
of V,3/J,1-C,l and V,1/D,2/Jz2-C8 chains specifically recognize self antigens produced
(11, 12) and have not been found elsewhere by skin-derived keratinocytes.This informa-
tion and the physical interactionsobserved in
W. L. Havran and J. P. Allison, Department of Molec- situ lend support to a potential role of dECs
ular and Cell Biology, and Cancer Research Laboratory, in surveillanceof skin keratinocytesin vivo for
University of California, Berkeley, CA 94720.
Y.-H. Chien, Department of Microbiology and Immu-
damage induced by stress, infection, or other
nology, Stanford University, Stanford, CA 94305. means.
SCIENCE, VOL. 252