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Kin) was cloned from cDNA by polymerase chain reaction (PCR) using the 18. Ren, R., Ye, Z.-S. & Baltimore, D. Abl protein-tyrosine kinase selects the Crk adapter as a substrate
using SH3-binding sites. Genes Dev. 8, 783-795 (1994).
forward primer CGGGATCCAGCGGCCAGTAGCATCTGAC and reverse 19. Shafman, T. et al. Defective induction of stress-activated protein kinase activity in ataxia-telangiecta-
primer CGGAATTCCTTTTCCACTTCGTCGTAG. Underlined sequences sia cells exposed to ionizing radiation. Cancer Res. 55, 3242-3245 (1995).
20. Zhang, N. et al. Correction of the ataxia-telangiectasa cellular phenotype with full-length ATM cDNA.
indicate the position of BamHI and EcoRI sites. The PCR product was digested
Proc. Natl Acad. Sci. USA (in the press).
with BamH2 and EcoRI and cloned into pGEX-5X-l. GST-Erk 2 (residues 163- 21. Rudolph, N. S. & Latt, S. A. Flow cytometric analysis of X-ray sensitivity in ataxia telangiectasia.
199; containing Y 185) was constructed by inserting a Bglll fragment of murine Mutat. Res. 211, 31-41 (1989).
22. Beamish, H. & Lavin, M. F. Radiosensitivity in ataxia-telangiectasia: anomalies in radiation-induced
Erk 2 cDNA into the BamHI site of pGEX-3X. GST-abl-SH3 and GST-Erk were cell cycle delay. Int. J. Radiat. Biol. 65, 175-184 (1994).
expressed in Escherichia coli and bound to glutathione-agarose beads 23 • These 23. Frangioni, J. V. & Neel, B. c;. Solubilization and purification of enzymatically active glutathione S-
transferase (pGEX) fusion proteins. Anal. Biochem. 210, 179-187 (1993).
beads (5 µg of GST fusion protein) were incubated with in vitro transcribed and
24. Gilad, S. et al. Ataxia-telangiectasia: founder effect among North African Jews. Hum. Mal. Genet. 5,
translated ATM protein in binding buffer (20 mM Tris-HCI, pH 7.4, 100 mM 2033-2037 (1996).
NaCl, 1 mM EDTA, 1 mM DTT, 0.1% NP40) for 1 hat 4 °C. A 5.7-kb partial 25. Watters, D. et al. Cellular localisation of the ataxia-telangiectasia (ATM) gene product and
discrimination between mutated and normal forms. Oncogene 14, 1911-1921 (1997).
ATM cDNA as described previously' was used as a template in an in vitro 26. Houldsworth, J. & Lavin, M. F. Effect of ionizing radiation on DNA synthesis in ataxia telangiectasia
coupled transcription/translation reaction (Promega) to produce 35 5-labelled cells. Nucleic Acids Res. 8, 3709-3720 (1980).
ATM protein. The sequence specifying the praline-rich region is present in this Acknowledgements. We thank A. Trezise for helpful discussion and advice on the manuscript, A. Farrell
cDNA. In two reactions either 10-fold or 100-fold molar excess of ATM peptide for technical assistance and A. Knight for typing the manuscript. We thank the National Health and
Medical Research Council of Australia, the Queensland Cancer Fund and the A-T Childrens Project for
(praline-rich region of ATM, residues 1,371-1,384) was added to compete support. T.Y. is supported by a Leukemia Society of America Scholar award, by a core grant, and by an
against the binding. Following binding, beads were washed and bound proteins appropriation from the Commonwealth of Pennsylvania.
were analysed by SDS-PAGE, followed by fluorography. Correspondence and requests for materials should be addressed to K.K.K. (e-mail: kumkumK@qimr.edu.au).
Kinase assays. Activation of c-Abl was determined with murine c-Crk as a
substrate and GST-Jun (residues 1-79) was used as a substrate to measure
"-
A B C i!"'"- D
.. .."
"-
i!"' "'"-<i.
"- "-
M "' '6. Q.
GFP.SF2/ASF
Alkaline
phosphatase
GFP-
S F2/ASF-
SF2/ASF_ - -
--
- --
. ...
2 3 4
•
- ~ ~
indicated SF2/ ASF construct. Splice site selection was analysed as described'. In
analysis of GFP- SF2/ ASF expression 14h post-transfection using anti-SF2/ ASF control cells, cryptic splice site 2 is used predom inantly (lane 1). Upon expression
antibody. Lanes 1 and 2, control cells; lanes 3 and 4, tra nsfected cells; lanes 2 and of either SF2/ ASF (lane 2) or GFP-SF2/ ASF (lane 3), a sw itch to the most proximal
4, alka line phosphatase-treated lysates. Both endogenous SF2/ ASF and GFP- 5' splice site 3 occu rred. GFP alone did not cause this shift (lane 4). M, electro-
SF2/ ASF were phosphorylated in vivo . B, GFP- SF2/ASF colocalized with phoresis marker. D, GFP- SF2/ASF accumulated at the site of transcription of
endogenous pre-mRNA splicing factors in nuclear speckles. GFP- SF2/ ASF (a ) exogenous transcripts. BHK cells were cotransfected w ith GFP- SF2/ ASF and [3-
localized exclusively to the nucleus and co localized w ith splicing factor U2-B" (b ; tropomyosin m inigene, cells were fixed at 6 h post-transfection; [3-tropomyosin
anti-mouse-Texas red secondary anti body) and several splicing factors conta in- RNA was detected by fluorescence in situ hybridization; [3-tropomyosin RNA (a)
ing the Sm epitope (c; anti-human-Cy5 secondary antibody). Sca le bar. 5 µ,m. C, and GFP- SF2/ ASF (b ) co loca lized at sites of plasm id transcription (arrows) Scale
GFP-SF2/ASF was active in alternative splice-site selection in vivo . Hel a cells bar, 7µ,m.
were cotransfected w ith a [3-thalassemia reporter substrate (bottom) and the
0
Figure 2 Time-lapse fluorescence microscopy of GFP-
SF2/ASF iri living BHK cells. Images w ere taken at 1.5- • / • / • / /
min intervals and the time is indicated in minutes in the
bottom left-hand corner of the image. a, The position of
most speckles w ithin the nucleus did not change
significantly over time (arrow s). Some speckles fused
(arrowhead ) or changed their nuclea r position. b, Distinct
/
O'
' ' /
15'
' ' /
30'
' ' /
45'
' '
changes in shape and peripheral extensions (arrows)
'b
were observed in virtually all speckles. c , What appeared
- - - -
~
to be particles were frequently seen to dissociate from or
associate with speckles. The spectrum of pseudoco-
lours represents the intensities of the fluorescence O' 4.5' 11' 13.5' 18' 22.5' 'ZT' 31.5' 35' 39.5'-
signal measured for each pixel (blue, lowest; red, C
brightest). Speckles appear in red against a green
background representing the nucleoplasmic pool of
O'
GFP- SF2/ASF. Scale bars: a , 6.5 µ,m; b. 750 nm; c, 1.5' 3' 4.5' 8' 7.5' 9' 10.5' -
500 nm. 0 greylevel 255
O'
20' _ _ __ 30'
O greylevel 255
Figure 3 Speckle movements are sens itive to inhibitors of RNA polymerase II (o:- Figure 4 GFP-SF2/ ASF responds to tran sc riptional activation of genes by
amanitin), protein kinases (stau ros porin e), and Ser/Thr phosphatases 1 and 2A red istribution to sites of tran scri ption. Transcription of BK-virus (a ) or CMV-IE
(okadaic acid). a , Addition of 50 µg ml - ' o:-amanitin for 2 h caused a marked genes (b) were triggered by ad dition of 50 µg ml - 1 cAMP or 50 µg ml - ' cyclohex-
rounding up of speckles and completely prevented peri pheral movement. b, c , imide, respectively. Images of speckles after induction of transcri ption were taken
Staurosporin e for 2 h (b) or okadaic acid for 1 h (c) had no signifi cant effect on at the in dicated time points. After acquisition of the fin al image, the ce ll w as fixed
ove rall nuclea r morph ology. d, Stau rosporine prevented all dyn amic, peripheral and the position of the in duced RN A detected by fluorescence in situ hybridiza-
movements. e, Okadaic acid caused a grad ual increa se in the nucleoplasmic tion. Pre-mRN A splicing facto rs form ed trails from existing speckl es in the
pool of GFP-SF2/ AS F. Images were ta ken at th e indicated timepoints afte r 60 min direction of the induced genes. Note that two neighbouring speckles (arrow-
(d) or 15 min (e) of incubation. Images are pseudoco loured; speckles appear in heads in b) respond to gene activation by formi ng peripheral exten sions. Images
red. Times (min) are given in the bottom left-hand co rn er. Scale bars: a. d , 500 nm ; are pseudocoloured; speckles appear in red. Times (min) are given in th e bottom
b, c 5 µm ; e, 720 nm. left-hand co rn er. Arrow, pos ition of the RN A signal. Sca le bars: 1.5 µm .
Dimeric association and 'torso' and the C termini as 'legs', as if perching on a hypothetical
membrane surface perpendicular to the diad axis. Each DlD2
segmental variability in fragment is a fixed entity at this resolution, as is each D3D4
dimer. There is, however, appreciable variability in the DlD2 to
the structure of human CD4 D3D4 junctions, which ranges up to 10.4° in orientational differ-
ence (Fig. ld). The angle at the DlD2 to D3D4 junction is 140°
(type-C molecule 1) as defined by the angle between the 'Cys-axes'
Hao Wu, Peter D. Kwong & Wayne A. Hendrickson*
(line through midpoints of canonical disulphide bonds or equiva-
Department of Biochemistry and Molecular Biophysics and * Howard Hughes lents in neighbouring domains), and the DlD2 Cys-axis is 53° from
Medical Institute, Columbia University, New York, New York 10032, USA the molecular diad axis and presumed membrane normal. The
refined D3D4 component is very similar to the rat D3D4 model
CD4 is a co-receptor in the cellular immune response. It increases ( 1.07 and 0.96 Ar.m.s. deviations for Ca position in D3 and D4 with
the avidity of association between a T cell and an antigen-presenting 5.4° relative difference in interdomain orientation).
cell by interacting with non-polymorphic portions of the complex The interface between D2 and D3 is formed almost exclusively by
between class II major histocompatibility complex (MHC) and T-cell the AB loop of D2 and the FG loop of D3, both of which are more
receptor (TCR) molecules, and it contributes directly to signal extended than their counterparts in D4 or Dl, plus the connecting
transduction through its cytoplasmic association with the lym- strand itself (Fig. la). There is 450 A.2 of non-hydrated surface area
phocyte kinase Lek (ref. I). CD4 also serves as the high-affinity buried from each domain into this interface, which is similar to the
receptor for cellular attachment and entry of the human immuno- interfacial areas between Dl and D2 and between D3 and D4. Four
deficiency virus (HIV)2. The extracellular portion of CD4 com- hydrophobic residues (Leu 109, Leu 177, Leu200 and Leu283)
prises four immunoglobulin-like domains (D1-D4). This part of dominate the interface. There is also an apparent hydrogen bond
human CD4 (residues 1-369) has been characterized as a recom- between Gln 112 and Gly 281. All of these are conserved residues.
binant soluble protein (sCD4) 3·4, and crystal structures have been The last strand of D2 (G) continues directly into the first strand of
described for the human D1D2 fragment 5•6 and for the rat D3D4 D3 (A), but in a tortuous manner because of kinks in the A strand of
fragment7. We have now determined the structures of intact sCD4 D3 that switch the pairing from strand B initially to strand G later, as
in three crystal lattices. These structures have a hinge-like variability in most immunoglobulin-variable domains. The hinge flexibility at
at the D1D2 to D3D4 junction that might be important in immune the junction is likely to be at residues Leu 177 and Ala 178 because
recognition and HIV fusion, and a common dimeric association Val 176 and Phe 179 are core elements of the D2 and D3 J3-sandwiches,
through D4 domains. Dynamic light scattering measurements respectively. The junctional variability among the six sCD4 copies
and chemical crosslinking of sCD4 corroborate dimerization at (Fig. ld) is accommodated with little adjustment at the mostly
high protein concentration. We suggest that such dimers may have hydrophobic interface; indeed, much larger movements toward
relevance as mediators of signal transduction in T cells. more acute junction angles can be modelled without much steric
Crystals of sCD4 were grown as described previously4 except we hindrance. Such junctional flexibUity is compatible with the results
started from the selenomethionyl protein. Moreover, the extent of of the limited proteolysis experiments done on sCD44.
measurable diffraction was increased through cryoprotection. The The interface between protomers in the sCD4 dimer involves the
structure of a tetragonal type-C crystal was determined by mole- D4 domains exclusively, and the buried surface (500 A2 from each
cular replacement8, confirmed by selenium Bijvoet-difference non-hydrated protomer) is comparatively small, which suggests a
Fourier analyses, and refined with tight restraints at 3.9 A resolu- relatively weak interaction. Although the operation that relates the
tion. This model was then used to solve the structures of seleno- protomers is non-crystallographic, it is within the bounds of error
methionyl sCD4 in a new monoclinic type-F lattice and of native for a pure 2-fold axis (x = 178.9° and tx = 0.37 A after the type-C
sCD4 in the trigonal type-A lattice. These structures were refined refinement). Both the CC' and FG loops ofD4 protrude prominently
from rigid-body components only (Table 1). Although the results from the C'CFG face of the J3-sandwich 7 and the diad interface
were obtained at modest resolution, they can be interpreted with involves these features in a manner reminiscent of clasped hands.